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The Role of Voltage-Gated Calcium Channels in Pancreatic ß-Cell Physiology and Pathophysiology

The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, SE-171 76 Stockholm, Sweden

Correspondence: Address all correspondence and requests for reprints to: Shao-Nian Yang, Ph.D., or Per-Olof Berggren, Ph.D., The Rolf Luft Research Center for Diabetes and Endocrinology L1:03, Karolinska University Hospital Solna, SE-171 76 Stockholm, Sweden. E-mail: shao-nian.yang{at}ki.se or per-olof.berggren{at}ki.se

	Abstract

Top Abstract I. Introduction II. General Aspects of... III. Role of CaV... IV. Role of CaV... V. ß-Cell CaV Channel... VI. Future Perspectives References

 
Voltage-gated calcium (Ca_V) channels are ubiquitously expressed in various cell types throughout the body. In principle, the molecular identity, biophysical profile, and pharmacological property of Ca_V channels are independent of the cell type where they reside, whereas these channels execute unique functions in different cell types, such as muscle contraction, neurotransmitter release, and hormone secretion. At least six Ca_V1 subunits, including Ca_V1.2, Ca_V1.3, Ca_V2.1, Ca_V2.2, Ca_V2.3, and Ca_V3.1, have been identified in pancreatic ß-cells. These pore-forming subunits complex with certain auxiliary subunits to conduct L-, P/Q-, N-, R-, and T-type Ca_V currents, respectively. ß-Cell Ca_V channels take center stage in insulin secretion and play an important role in ß-cell physiology and pathophysiology. Ca_V3 channels become expressed in diabetes-prone mouse ß-cells. Point mutation in the human Ca_V1.2 gene results in excessive insulin secretion. Trinucleotide expansion in the human Ca_V1.3 and Ca_V2.1 gene is revealed in a subgroup of patients with type 2 diabetes. ß-Cell Ca_V channels are regulated by a wide range of mechanisms, either shared by other cell types or specific to ß-cells, to always guarantee a satisfactory concentration of Ca²⁺. Inappropriate regulation of ß-cell Ca_V channels causes ß-cell dysfunction and even death manifested in both type 1 and type 2 diabetes. This review summarizes current knowledge of Ca_V channels in ß-cell physiology and pathophysiology.

I. Introduction

II. General Aspects of Ca_V Channels

A. Types of Ca_V channels

B. Molecular knowledge of Ca_V channels

III. Role of Ca_V Channels in ß-Cell Physiology

A. Types of ß-cell Ca_V channels

B. Molecular components of ß-cell Ca_V channels

C. Ca_V channel regulation in insulin secretion

D. Effect of Ca_V channels on ß-cell development, survival, and growth

E. Novel molecular networks of ß-cell Ca_V channels

IV. Role of Ca_V Channels in ß-Cell Pathophysiology

A. Disorder of ß-cell Ca_V channels in diabetic milieu

B. Phenotypic switch of ß-cell Ca_V channels in diabetes

C. Ca_V channel mutation and inadequate insulin secretion

D. Ca_V channel gene polymorphism and diabetes

E. Ca_V channels and ß-cell death

F. Ca_V1 channels and type 1 diabetic serum-induced ß-cell apoptosis

V. ß-Cell Ca_V Channel Regulation

A. ß-Cell Ca_V channel regulation by serine/threonine protein kinases and phosphatases

B. ß-Cell Ca_V channel regulation by Ca²⁺/calmodulin

C. ß-Cell Ca_V channel regulation by G protein-coupled receptors

D. ß-Cell Ca_V channel regulation by tyrosine kinase receptors

E. ß-Cell Ca_V channel regulation by phosphorylated inositol compounds

F. ß-Cell Ca_V channel regulation by glucose

G. ß-Cell Ca_V channel regulation by FFA

H. ß-Cell Ca_V channel regulation by nitric oxide

I. ß-Cell Ca_V channel regulation by Ras-related G proteins

J. ß-Cell Ca_V channel regulation by temperature

VI. Future Perspectives

	I. Introduction

Top Abstract I. Introduction II. General Aspects of... III. Role of CaV... IV. Role of CaV... V. ß-Cell CaV Channel... VI. Future Perspectives References

 
VOLTAGE-GATED CALCIUM (Ca_V) channels are ubiquitously expressed and critical for life. Phylogenetically, Ca_V channels appear in both prokaryotes and eukaryotes, being more diversified in higher forms of life (1). In the mammalian body, Ca_V channels not only distribute in all types of excitable cells but also exist in some nonexcitable cells (2). Knowledge of Ca_V channels has been expanding quickly with the development and application of advanced methodologies, such as patch-clamp methods, molecular biological techniques, x-ray crystallography, and confocal microscopy. Nucleotide and amino acid sequences, biophysical and pharmacological features, subcellular distributions and functions of Ca_V channels have been extensively investigated by combining these techniques. Ca_V channels function as Ca²⁺-conducting pores in the plasma membrane. In response to membrane depolarization, Ca_V channels undergo an extremely rapid conformational switch from an impermeable state to a highly permeable pore. The highly permeable pore allows extracellular Ca²⁺ to rapidly enter the cytoplasm, where Ca²⁺ serves as a second messenger to couple electrical signaling to Ca²⁺-dependent protein-protein interactions and enzymatic responses (3). Therefore, the Ca_V channel-mediated Ca²⁺ influx controls a diverse range of cellular processes including exocytosis, endocytosis, muscle contraction, synaptic transmission, and metabolism (3, 4). It triggers life at fertilization and controls proliferation, differentiation, and development through the regulation of protein phosphorylation, gene expression, and the cell cycle (3, 4). A pathophysiologically exaggerated (superactivated) Ca_V channel-mediated Ca²⁺ influx causes cell death through initiation of apoptosis and necrosis (4, 5, 6, 7). The malfunction of Ca_V channels, resulting from their mutation, altered expression, and impairment by autoantibodies, leads to a series of clinical signs and symptoms, referred to as calcium channelopathies or calcium channel diseases (8, 9, 10).

Pancreatic ß-cells, as a major cellular component of the islets of Langerhans, are electrically excitable and exquisitely sensitive to glucose. In response to blood glucose, these cells secrete insulin and thus play a unique role in glucose homeostasis. ß-Cell Ca_V channels take center stage in this process (2, 11). In addition to controlling insulin secretion, ß-cell Ca_V channels are also involved in ß-cell development, survival, and growth (12, 13, 14, 15). The ß-cells from diabetes-prone or diabetic animal models display qualitative and quantitative alteration of Ca_V channels (16, 17, 18, 19, 20). Point mutation of human Ca_V1.2 channels makes the ß-cell secrete insulin excessively (21). ß-Cell Ca_V channel activity and density are regulated by numerous mechanisms, e.g., protein phosphorylation, Ca²⁺/calmodulin, G protein-coupled receptors, and phosphorylated inositol compounds (2). Within the physiological range, up-regulation of ß-cell Ca_V channel activity and/or density results in enhanced insulin exocytosis and more efficient glucose homeostasis (2). Moreover, down-regulation of Ca_V channel activity and/or density causes less insulin secretion and glucose intolerance, being associated with a group of type 2 diabetic patients (20, 22, 23). Therefore, up-regulation of ß-cell Ca_V channel activity and/or density is a potential way to treat diabetics characterized by a low capacity of insulin exocytosis. However, hyperactivation of ß-cell Ca_V channels leads to ß-cell death (24, 25).

This review focuses on the role of Ca_V channels in pancreatic ß-cell physiology and pathophysiology.

	II. General Aspects of CaV Channels

Top Abstract I. Introduction II. General Aspects of... III. Role of CaV... IV. Role of CaV... V. ß-Cell CaV Channel... VI. Future Perspectives References

 
A. Types of Ca_V channels
Initially, Ca_V currents were named Ca²⁺-dependent slow inward currents when the classical voltage clamp method and ion substitution were the only available techniques (26). Later on, Hagiwara et al. (27) identified two types of Ca_V currents with distinct activation and inactivation properties from fertilized starfish eggs. The channels conducting these two types of Ca_V currents were termed channel I [low-voltage activated (LVA)] and channel II [high-voltage activated (HVA)] (27). Following the establishment of advanced technology, such as patch-clamp, biochemical, and molecular biological techniques, as well as the discovery of selective Ca_V channel blockers, diverse Ca_V currents, Ca_V channel proteins, and genes have been identified (2, 3, 28, 29). However, researchers in different fields did not devote efforts toward the establishment of a common nomenclature of Ca_V channels. They used to employ their own terminologies to describe these same entities. Electrophysiologists characterized and classified Ca_V currents according to phenomenological parameters including biophysical and pharmacological properties. Therefore, they created the phenomenological nomenclature, the earliest systematic designation of Ca_V currents, such as L-, P/Q-, N-, R-, and T-type Ca_V currents (30). The L-, P/Q-, N-, and R-type Ca_V currents have high thresholds for activation and are referred to as HVA Ca²⁺ currents (30). T-type Ca_V currents were designated LVA Ca²⁺ currents because a small depolarization makes them become activated (30). Following tradition in biochemistry, biochemists used Greek letter-based nomenclature to illustrate Ca_V channel subunits as ₁, ß, , and ₂ subunits (31, 32). In the field of molecular biology, the alphabetic system, e.g., class A, class B, class C, etc., was applied for Ca_V channel gene products (33). The gene nomenclature, such as CACNA1 and CACNB, was employed to depict Ca_V channel genes (34). These multiple nomenclatures of Ca_V channels have given rise to obvious communication problems. To avoid the problems, a group of leading scientists in the area of Ca_V channel research recommended a comprehensive nomenclature of Ca_V channels based on sequence analysis in 2000 (29). Primary structure analysis of Ca_V1 subunits categorizes Ca_V channels into three families, Ca_V1, Ca_V2, and Ca_V3, consisting of closely related members (29). This structure-based nomenclature has been widely accepted to describe Ca_V channel proteins and mRNAs. However, it is hardly applied to the description of Ca_V currents recorded from native cells expressing complex mixtures of Ca_V channel subunits. For example, both Ca_V1.2 and Ca_V1.3 subunits conduct the ß-cell L-type Ca_V current. There is no way to discriminate the current flowing through Ca_V1.2 from that through Ca_V1.3 subunits. In this case, L-type may be the best description. In this article, we employ the phenomenological nomenclature to describe Ca_V currents and the structural nomenclature to depict Ca_V channel proteins and mRNAs. Table 1 summarizes the biophysical characteristics, pharmacological properties, localizations, and functions of Ca_V channels mediating different types of Ca_V currents. It is noteworthy that the selectivity of different Ca_V channel blockers should be understood as a relative term because more and more nonselective effects of these blockers have been reported.

2. Ca_V2 channels.
The Ca_V2 channel family includes three members, Ca_V2.1, Ca_V2.2, and Ca_V 2.3 channels (2, 3, 29). Ca_V2.1 channels mediate P/Q-type Ca_V currents. Biophysical features of P/Q-type Ca_V currents conducted by Ca_V2.1 channels are very similar to those of N-type Ca_V currents mediated by Ca_V2.2 channels. P/Q-type Ca_V currents are indistinguishable from N-type Ca_V currents without peptide blockers (38). P-type Ca_V currents were first identified using -agatoxin IVA (-Aga IVA) in cerebellar Purkinje cells (39). Subsequently, Q-type Ca_V currents were revealed in cerebellar granule cells (38). Both the P-type and the Q-type Ca_V currents are blocked by -Aga IVA. However, the P-type Ca_V current is more sensitive to this blocker and does not inactivate during 0.1-sec depolarizations. The Q-type Ca_V current is inhibited by higher concentrations of -Aga IVA and displays approximately 35% inactivation during 0.1-sec depolarizing pulses (38). It seems that one can discriminate the P-type from the Q-type on the basis of differences in their inactivation rate and sensitivity to -Aga IVA. However, molecular studies revealed that these two types of Ca_V currents are conducted by the same pore-forming subunit Ca_V2.1 (2, 3, 29). Hence, they are now combined as P/Q-type Ca_V currents (2, 3, 29). Ca_V2.1 channels play a key role in neurotransmitter release (Table 1) (2, 3).

Ca_V2.2 channels conduct N-type Ca_V currents, which fall in between the T- and L-type Ca_V currents in terms of biophysical properties. They display smaller unitary Ba²⁺ conductance, lower activation threshold, and faster inactivation rate than L-type Ca_V currents, but larger unitary Ba²⁺ conductance, higher activation threshold, and slower inactivation rate than T-type Ca_V currents (30). Apparently, such currents are neither T nor L. Additionally, they were only found in neurons in the earliest studies on Ca_V channels. Therefore, these currents were classified as a distinct type named N-type. N-type Ca_V currents are blocked by the peptide blocker -conotoxin GVIA (-CTX GVIA), but are insensitive to the organic Ca_V channel antagonists DHPs, phenylalkylamines, and benzothiazepines (30). Ca_V2.2 channels mainly mediate Ca²⁺ influx in active zones to trigger neurotransmitter release (Table 1) (2, 3).

Ca_V2.3 channels were discovered in cerebellar granule cells subjected to combined treatment with the multiple Ca_V channel blockers nimodipine, -CTX GVIA, and -Aga IVA. The treated cerebellar granule cells showed a residual Ca_V current resistant to these Ca_V channel blockers (38). Therefore, this residual current resistant to all available Ca_V channel blockers was termed the R-type. The R-type Ca_V current is now no longer resistant to "everything." A novel toxin, SNX-482, with high affinity for Ca_V2.3 channels has been purified (40). However, it is worthwhile to note that SNX-482 is capable of blocking other types of Ca_V channels as well (11, 41). Therefore, caution should be exercised when using this Ca_V channel blocker to dissect native R-type Ca_V currents in cells containing multiple types of Ca_V channels. The R-type current is characterized by faster inactivation rate in comparison with other HVA Ca²⁺ channels. The Ca_V2.3 channel is a key player in the generation of Ca²⁺-dependent action potentials and plays a role in neurotransmitter release (Table 1) (2, 3).

3. Ca_V3 channels.
Ca_V3 channels are categorized into Ca_V3.1, Ca_V3.2, and Ca_V3.3 channels according to their pore-forming subunits (2, 3, 29). These channel-mediated Ca_V currents display low threshold for activation. Additionally, these currents are also characterized by a tiny unitary Ba²⁺ conductance and a transient kinetics of inactivation. Therefore, they are termed T-type Ca_V currents (30). Like Ca_V1 channels, Ca_V3 channels distribute in a wide range of cell types including neurons, muscle cells, endocrine cells, sperm, and even nonexcitable cells (2, 3, 42). They are mainly involved in repetitive firing in excitable cells, play an important role in fertilization, and may mediate steady Ca²⁺ influx near resting membrane potentials in nonexcitable cells (Table 1) (2, 3, 30, 42).

B. Molecular knowledge of Ca_V channels
1. Molecular composition of Ca_V channels.
During the past two decades, great progress has been made toward elucidating the molecular entities responsible for Ca_V currents. The Catterall group (31) has pioneered the field of Ca_V channel biochemistry. Initially, this group found that the skeletal muscle Ca_V channel consists of the Ca_V1, Ca_Vß, and Ca_V subunits (31). Shortly thereafter, this group and several other groups demonstrated that the skeletal muscle Ca_V channel is composed of not only the Ca_V1, Ca_Vß, and Ca_V subunits but also the Ca_V2 subunit (32, 43, 44, 45, 46, 47). Accordingly, the Catterall group proposed a widely accepted model of the subunit structure of Ca_V channels on the basis of the systematic biochemical analysis of Ca_V1.1 channels (3, 32). In their experiments, the molecular mass, DHP binding site, PKA phosphorylation site, glycosylation, hydrophobicity, disulfide-bridge heterodimer, and noncovalent association of Ca_V1.1 channel subunits were carefully characterized. These biochemical properties demonstrated that hydrophobic Ca_V1.1 subunits noncovalently associate with a disulfide-linked Ca_V2 dimer, an intracellular phosphorylated Ca_Vß subunit, and a transmembrane Ca_V subunit. In this complex, only the Ca_V1.1 subunit is large enough to contain four homologous transmembrane domains forming a Ca²⁺ conducting pore (3, 32). Other subunits, Ca_Vß, Ca_V, and Ca_V2 subunits, modulate Ca_V channels in many important ways (28, 48, 49, 50, 51). Hence, these subunits are referred to as auxiliary subunits. The successful purification and characterization of Ca_V channel subunits laid a solid foundation for the first-ever cloning of a Ca_V channel gene, Ca_V1.1 gene. In cloning this gene, the Numa group first isolated tryptic peptides derived from the purified Ca_V1.1 subunit. Subsequently, they determined the sequence of these peptides and made oligo probes according to these peptide sequences. Finally, they screened rabbit skeletal muscle cDNA libraries with these probes and fished out the Ca_V1.1 gene (52). Thereafter, numerous Ca_V channel genes and their products have been extensively characterized (3, 29). The molecular composition of Ca_V channels has thereby become clearer (Fig. 1).

View larger version (53K): [in this window] [in a new window]   FIG. 1. A structural model of CaV channels. A, CaV channel subunit assembly in the plasma membrane. Pore-forming subunits CaV1 complex with auxiliary subunits CaVß, CaV, and CaV2 to form functional CaV channels in the plasma membrane. B, Predicted topology and nomenclature of CaV channel subunits. The CaV1 subunit has four homologous transmembrane domains (I to IV), each containing six transmembrane segments and a membrane-associated loop between transmembrane segments 5 and 6, three intracellular linkers, and intracellular N and C termini. The CaVß subunit is entirely cytosolic. The CaV subunit comprises a conserved four-transmembrane domain, and intracellular N and C termini. The CaV2 subunit is a two-peptide dimer linked by disulfide bonds. The CaV2 is an entirely extracellular polypeptide, and the CaV possesses a single transmembrane domain with a very short intracellular C terminus and long extracellular portion. So far, 10 CaV1 subunits, four CaVß subunits, four CaV2 subunits, and eight CaV subunits have been identified.

a. Ca_V1 subunits.

Molecular characterization demonstrates that the Ca_V1 subunit is the primary determinant of Ca_V channel biophysics. The Ca_V1 subunit has four homologous repeats or domains (I to IV), each containing six transmembrane segments (S1 to S6) and a membrane-associated pore loop (P-loop) between the S5 and S6 segments, three intracellular linkers (L_I-II, L_II-III, and L_III-IV), and N- and C-termini (Fig. 1). This four homologous repeat structure endows the Ca_V1 subunit with a Ca²⁺-conducting pore controlled by voltage sensors and activation and inactivation gates. The S5 and S6 segments along with the P-loops form the pore lining of Ca_V channels (3). There are four glutamic acid residues situated in the four P-loops of HVA Ca²⁺ channels (54). In LVA Ca²⁺ channels, two glutamic and two aspartic acid residues are localized in the corresponding positions (42, 55, 56, 57, 58). Partial or full substitution of these glutamic acid residues in HVA Ca²⁺ channels results in drastic changes in ion permeation (54, 59, 60, 61). This strongly indicates that these four glutamic acid residues are responsible for selective filtration of Ca²⁺. It is suggested that these four glutamic acid residues form a Ca²⁺ selectivity filter, with an inner cavity of about 6 Å in diameter (62). Two of them are deprotonated, while the other two are not ionized. They interact with Ca²⁺ with different binding affinities. It has been proposed that the higher affinity site is located at the cytoplasmic side and occupied by a Ca²⁺. When an incoming Ca²⁺ binds to the low-affinity site, cationic repulsive forces push the Ca²⁺ away from the high-affinity site into the cell. It has been generally accepted that the size and dynamic interaction of the Ca²⁺ selectivity filter with Ca²⁺ together determine the Ca²⁺ selectivity of Ca_V channels (63).

The voltage-dependent allosteric change of the Ca²⁺-conducting pore is a fundamental process of Ca_V channels. To be able to experience voltage alteration, the Ca_V channel must be equipped with electrically charged elements as voltage sensors. In essence, the voltage sensors of voltage-gated ion channels are charged amino acid residues in pore-forming subunits. These charged residues rapidly move in response to cell membrane depolarization (3, 64). Comparison of amino acid sequence of the S4 segments in voltage-gated ion channels shows that cationic arginine or lysine residues appear at every third position. This characteristic module is highly conserved in all known voltage-gated ion channels (64). Evidence has clearly demonstrated that the S4 segments are the principal voltage sensors in Ca_V channels, although the S1, S2, and S3 segments are also involved (3, 64).

The depolarization-evoked movement of the voltage sensors leads to the conformational change and/or physical reposition of two intrinsic structures, activation and inactivation gates. To date, the exact location of the Ca_V channel activation gate is not known due to the lack of crystallographic evidence. Recently, crystallographic analysis along with electrophysiological recording points to a K⁺ channel activation gate near the cytoplasmic end of the S6 segments (65, 66). Electrophysiological analysis of the Cd²⁺ block of Ca_V currents implies that the Ca_V channel is closed at both ends of the Ca²⁺-conducting pore (67). However, the structural basis of the Ca_V channel activation gate remains to be explored. During membrane depolarization, opened Ca_V channels become less permeable. This process is termed inactivation. In general, Ca_V channels undergo two different types of inactivation, Ca²⁺-dependent inactivation and voltage-dependent inactivation (68). The Ca²⁺-dependent inactivation is mediated by calmodulin and is involved in sites on the C terminus and probably also a site on the N terminus of Ca_V1 subunits (68, 69, 70). The voltage-dependent inactivation is distinguished into fast and slow voltage-dependent inactivation on the basis of their kinetics. The voltage-dependent inactivation is an intrinsic property of Ca_V1 subunits although it is modulated by other auxiliary subunits (68). Molecular structures underlying Ca_V channel inactivation, i.e., Ca_V channel inactivation gates, have been investigated extensively. Inactivation analysis of point-mutated Ca_V1 subunits and chimeric Ca_V1 subunits has demonstrated that the Ca_V1 subunit is equipped with multiple structural determinants of the voltage-dependent inactivation. These inactivation determinants are mainly localized in the L_I-II, S5, and S6 segments and their adjacent P-loops (68).

A striking biochemical feature of the Ca_V1 subunit is that this subunit contains multiple potential protein phosphorylation sites. For example, NetPhos 2.0 predicts that the rabbit Ca_V1.1 subunit carries 27 serine, seven threonine, and seven tyrosine residues with high phosphorylation potentials (Fig. 2). Early studies showed that purified Ca_V1.1 subunit can be phosphorylated by multiple protein kinases, such as PKA, protein kinase C (PKC), protein kinase G (PKG), calcium/calmodulin-dependent kinase II (CaMKII), and casein kinase II (43, 71, 72, 73). However, only a few exact residues phosphorylated by distinct protein kinases have been identified. In vitro experiments have demonstrated that PKA phosphorylates S687, S1617, S1757, S1772, and S1854 of the purified rabbit Ca_V1.1 subunit (Fig. 2) (74, 75, 76). PKA phosphorylation at S1757 and S1854 of the rabbit Ca_V1.1 subunit has also been demonstrated in intact cells (77). Furthermore, both in vitro and in vivo experiments have demonstrated that the rabbit Ca_V1.2 subunit S1928 is a substrate of PKA (78). In vitro phosphorylation at S1627 and S1700 of the bovine Ca_V1.2 by PKA has also been shown (79). It is worthwhile to note that there are species differences in the availability of PKA phosphorylation sites. The Ca_V1.1 subunit S687 cannot be phosphorylated by PKA in intact rabbit skeletal muscle (77). However, phosphorylation of the corresponding serine by PKA can be detected in intact rat skeletal muscle (80). The purified rabbit Ca_V1.2 subunit can be phosphorylated at S1928 by PKA, but the corresponding site of the bovine Ca_V1.2 subunit cannot (78, 79). PKC phosphorylation sites at the Ca_V1.2 subunit have not been biochemically identified. Electrophysiological analysis reveals that the Ca_V1.2 channel mutated at either T27 or T31 can no longer respond to PKC activation. This indicates that the Ca_V1.2 T27 and T31 must be phosphorylated by this protein kinase (81, 82). Furthermore, all known Ca_V1 subunits carry potential N-glycosylation sites (52, 56, 57, 58, 83, 84, 85, 86, 87, 88, 89). However, actual N-glycosylation has not been detected in Ca_V1.1 and Ca_V1.2 subunits (32, 90). Interestingly, a short form of the Ca_V2.1 subunit, containing approximately the first half of the full-length Ca_V2.1 subunit, is glycosylated (91).

View larger version (38K): [in this window] [in a new window]   FIG. 2. Potential phosphorylation sites predicted by NetPhos 2.0 (plot) and in vitro phosphorylated serine residues by PKA (inset) in the rabbit CaV1.1 subunit (Swiss-Prot: P07293).

View larger version (25K): [in this window] [in a new window]   FIG. 3. Human CaVß subunit splice variants. We hereby suggest a new nomenclature for CaVß subunit splice variants. This nomenclature avoids confusion created by naming schemes just indicating the order of CaVß subunit isoform discovery and importantly provides concise information on gene structure of these isoforms. The designation in this nomenclature starts with four families of CaVß subunits, i.e., CaVß1, CaVß2, CaVß3, and CaVß4, with the following subscript brackets containing expressed alternative exon(s) indicated by exon number(s), deleted exon(s) denoted by the sign "" with exon number(s), and truncated exon described by exon number with the sign "*". All known isoforms of human CaVß subunits are listed here. Individual CaVß gene structures are depicted by filled squares labeled with exon numbers, whereas distinct CaVß subunit splice variants are illustrated by open squares labeled with exon numbers. Alternative spliced exons are indicated by capital characters following exon numbers. Peaked lines indicate the linkage between exons in splicing positions.

The Ca_Vß subunit carries several potential PKA and PKC phosphorylation sites. It has been demonstrated that PKC and PKA in vitro effectively phosphorylate S182 and T205, respectively, in the Ca_Vß1a subunit (102, 103). However, it is not clear whether phosphorylation of these two residues contributes to the regulation of channel activity (104). The Ca_Vß2a subunit also contains several potential PKA and PKC phosphorylation sites (105). The in vivo PKA phosphorylation of this subunit has been demonstrated in intact heart stimulated with ß-adrenoreceptor agonists (106, 107). Electrophysiological and mutation analysis revealed that S478 and S479, nonclassical PKA phosphorylation sites, in the Ca_Vß2a subunit are critical for the regulation of the activity of truncated Ca_V1.2 channels (108). Recently, the Ca_Vß subunit has also been demonstrated to mediate MAPK modulation of Ca_V2.2 channels (109). Palmitoylation of the rat Ca_Vß2a subunit has been extensively studied. The two N-terminal cysteines are palmitoylation sites (104, 110, 111). Functional effects of posttranslational modifications of Ca_Vß2a subunits will be discussed in Section II.B.3.b.

c. Ca_V subunits.
Originally, the Ca_V subunit was revealed exclusively in skeletal muscle from different species (28, 31, 44, 50, 51). In 1998, Letts et al. (112) found a spontaneous mutant mouse line, stargazer, resulting from a single defective gene. Interestingly, the corresponding healthy gene shows obvious similarities to the skeletal muscle Ca_V subunit gene. Therefore, this gene was named Ca_V2, and the first-identified skeletal muscle Ca_V subunit gene was termed Ca_V1 (112). Soon, six other Ca_V subunit isoforms (Ca_V3–8) were identified by searching the DNA database in terms of sequence homology to Ca_V1 and Ca_V2 genes (113, 114, 115, 116, 117). The chromosomal locations of human Ca_V genes have been identified. They span chromosomes 17q24, 22q12-q13, 16p12-p13.1, 17q24, 17q24, 19q13.4, 19q13.4, and 19q13.4 in the order from Ca_V1–8 (114, 116). The Ca_V5 and Ca_V 7 genes comprise five exons, but other isoforms consist of four exons (50, 117, 118). The Ca_V1 is tightly associated with the Ca_V1.1 subunit and copurified with the Ca_V1.1 subunit (31, 32, 44). Amino acid sequence analysis indicates that the Ca_V subunit is composed of a conserved four-transmembrane domain topology together with intracellular N and C termini (Fig. 1). All known Ca_V subunits carry a signature motif (GLWXXC), a conserved N-glycosylation site, and a pair of conserved cysteine residues, forming a disulfide bridge in the first extracellular loop (28, 50). In addition, Ca_V2–5 and Ca_V7–8 bear a PDZ binding or related motif at the C terminus (117). Ca_V subunits inhibit Ca_V channel activity and modulate its activation and inactivation kinetics (28). Ca_V subunits have little effect on Ca_V channel trafficking (see Section II.B.3.b). However, the Ca_V2 subunit facilitates trafficking and targeting of -amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors to postsynaptic membranes through interaction with PSD95 (50, 51).

d. Ca_V2 subunits.
Like other Ca_V channel subunits, the Ca_V2 subunit was first copurified with the Ca_V1.1 subunit from the skeletal muscle preparation (32, 45, 46, 47). Subsequently, the Ca_V2 subunit was found to associate with other Ca_V1 subunits from other tissues (119, 120). In 1988, molecular cloning of the Ca_V2 subunit was accomplished (121). Since then, no further Ca_V2 subunit genes were uncovered for about 10 yr. After the successful identification of multiple Ca_V subunit genes by searching the sequence database, the same strategy was applied to disclose novel Ca_V2 subunit genes (122). It turned out that in the database, two novel genes encoding proteins with significant similarities to the previously identified Ca_V2 subunit were identified (122). Thereafter, the previously identified Ca_V2 subunit was referred to as the Ca_V21, and the other two were known as the Ca_V22 and Ca_V23 (122). Recently, the human Ca_V24 gene has been isolated and characterized (123). Human Ca_V21, Ca_V22, Ca_V23, and Ca_V24 genes are mapped to chromosome 7q21-q22, 3p21.3, 3p21.1, and 12p13.3, respectively (28). It is known that there are three alternatively spliced regions in the Ca_V21 subunits. The Ca_V21 subunit exists in five splice variants within mouse tissues: Ca_V21a, Ca_V21b, Ca_V21c, Ca_V21d, and Ca_V21e. Only the single isoforms Ca_V21a and Ca_V21b express in skeletal muscle and brain, respectively. Heart and smooth muscle tissues contain multiple isoforms of Ca_V21 subunits (124). Two alternative spliced regions have been revealed in the human Ca_V22 subunit. Three isoforms of this subunit, Ca_V22a, Ca_V22b, and Ca_V22c subunit, have been found in human tissues. The human heart only expresses Ca_V22a, whereas a human medullary thyroid carcinoma cell line contains all three isoforms, Ca_V22a, Ca_V22b, and Ca_V22c (125). The human Ca_V23 gene produces a full-length and a truncated transcript. No additional splice variants of the human Ca_V23 gene are detected (126). The gene structure of the human Ca_V24 is best characterized in the Ca_V2 family. There are 39 exons in the human Ca_V24 gene. Exons 2–37 are invariant exons, whereas exon 1, 1B, 37L, and 38 are alternatively spliced. Rapid amplification of cDNA ends-PCR product sequencing identified two different N termini and two different C termini of the human Ca_V24 subunit. Therefore, four splice variants, Ca_V24a, Ca_V24b, Ca_V24c, and Ca_V24d have been expected. However, these splice variants remain to be identified (123).

The Ca_V2 subunit is a most highly glycosylated protein among Ca_V channel subunits (32, 127, 128). The premature Ca_V2 subunit is a single polypeptide encoded by a single Ca_V2 gene (121, 129). The posttranslational cleavage and disulfide linkage make up the mature form of the Ca_V2 subunit, a two-peptide dimer linked by disulfide bonds, from the single precursor Ca_V2 subunit (129, 130). Ca_V2 subunits contain numerous glycosylation sites, cysteine residues, and hydrophobic sequences (121, 122, 123). Topological analysis indicates that Ca_V2 is an entirely extracellular polypeptide and Ca_V possesses a single transmembrane domain with a very short intracellular C terminus and long extracellular portion (127). The transmembrane domain serves as an anchor of the Ca_V polypeptide in the plasma membrane. Interestingly, neither the transmembrane domain nor the intracellular part of the Ca_V polypeptide is involved in the interaction of the Ca_V2 subunit with the Ca_V1 subunit. By contrast, both the entire Ca_V2 and Ca_V extracellular portion are responsible for their association with the Ca_V1 subunit (131). The Ca_V2 polypeptide functions as a crucial determinant required for stimulation of the current amplitude, whereas the Ca_V domain mainly modulates voltage-dependent activation and steady-state inactivation (132). Distinct Ca_V2 subunits have slightly different contributions to channel function (28, 132, 133). For example, coexpression of the Ca_V21 subunit promotes Ca_V1 subunit trafficking to the plasma membrane (also see Section II.B.3.b), increases current amplitude, and fastens activation and inactivation (132). The Ca_V22 subunit only appears to enhance the current amplitude but does not affect activation and inactivation (133). In addition, some of these effects occur in the presence of the Ca_Vß subunit, whereas others do not require the coexpression of the Ca_Vß subunit (122, 127).

2. Molecular architecture of Ca_V channels.
Lack of knowledge about atomic level structures of Ca_V channel subunits and in particular Ca_V subunit complex prevents us from understanding a large number of critical issues concerning Ca_V channel subunit assembly, biophysical properties, and modulation mechanisms. The large size and multiple transmembrane segments of pore-forming Ca_V1 subunits make them difficult to solubilize and purify from native tissues without severe interference with their proper folding. It is also very hard to obtain pure and properly folded Ca_V1 subunits from heterologous expression systems including yeasts, bacteria, and mammalian cells. Therefore, Ca_V1 subunits have not yet been crystallized and visualized at the atomic level. Although Ca_V2 and Ca_V subunits are smaller in size and they are either entirely extracellular (Ca_V2 subunits) or equipped with fewer transmembrane segments (Ca_V and Ca_V subunits) than Ca_V1 subunits, there is still no published information about crystal structures of these subunits.

a. Crystallographic structures of AID-Ca_Vß subunit complexes.
Recently, a breakthrough has been made in understanding the structure of the AID-Ca_Vß subunit complex at the atomic level. High-resolution x-ray crystallographic analysis not only allows us to understand the atomic disposition within the AID-Ca_Vß subunit complex but also provides an insight into the molecular mechanisms of Ca_V channel regulation. The studies done by three different groups present several novel findings (97, 98, 99). First, the Ca_Vß subunit core contains two well-conserved domains, a SH3 domain (also referred to as domain I) and a GK domain (also termed nucleotide kinase domain or domain II). The Ca_Vß subunit SH3 domain mainly consists of five ß-strands and two -helices. The Ca_Vß subunit GK domain primarily comprises five ß-strands and seven -helices. In the AID-Ca_Vß subunit complex, the AID, folded into an -helix, binds to a hydrophobic groove (also called ₁ subunit binding pocket) in the GK domain, but not to the previously proposed BID of Ca_Vß subunits. Instead, this domain preserves the structural integrity of the SH3 and GK domains and links these two domains together. Furthermore, they also show that three of the putative Gß-AID interacting residues in the AID are deeply buried by the hydrophobic groove of the Ca_Vß subunit GK domain (97, 98, 99). Second, the Ca_Vß subunit directly modulates the movement of the IS6 segment in the Ca_V1 subunit to influence channel pore gating, because the AID helix N terminus is very close to the IS6 segment, a pore-lining segment (97, 98, 99, 134). Finally, the Ca_Vß subunit can perform a diverse range of tasks in terms of its common protein interaction domains (97, 98, 99).

b. Electron-microscopic structures of Ca_V channel subunit complexes.
As mentioned before, so far it has not been possible to crystallize Ca_V1 subunits. Electron microscopy as an alternative approach has been used to visualize the molecular architecture of Ca_V channels. In the early 1980s, the two-dimensional electron microscopic structure of Ca_V1.1 channels was observed by Osame et al. (135) using freeze-fracture techniques in the plasmalemma of human muscles. This two-dimensional structure was confirmed by Franzini-Armstrong and Nunzi (136, 137) using the same techniques in different types and subcellular compartments of muscle fibers from different species, e.g., the transverse tubules of the fast twitch muscle fiber of the toadfish and the slow frog fiber. Later on, electron microscopy of the freeze-dried, rotary-shadowed sarcoplasmic reticulum-transverse tubule junctions clearly showed en face three-dimensional (3D) views of Ca_V1.1 channels (138). Typically, four Ca_V1.1 channels are disposed in the corners of a small square. Therefore, this profile of Ca_V1.1 channels was originally called a tetrad. The geometry of tetrads in the transverse tubular membrane matches well with the disposition of four large cytoplasmic domains protruding from a tetrameric complex of ryanodine receptors in the adjacent sarcoplasmic membrane (138). This encouraged one to propose the interaction between Ca_V1.1 channels and ryanodine receptors in excitation-contraction coupling (139). Successful purification of Ca_V1.1 channels from the transverse tubular membranes of skeletal muscle made more adequate visualization of these channels possible. The Campbell group (140) presented the electron microscopic structure of purified Ca_V1.1 channels by freeze-dried, rotary shadow electron microscopy. They revealed that the purified Ca_V 1.1 channels appeared as a homogeneous population of 16 x 22-nm ovoidal particles in the preparation. The electron microscopic data together with hydrodynamic characteristics of the purified Ca_V1.1 channels demonstrated that the ovoidal particle corresponds to a Ca_V channel complex composed of Ca_V1, Ca_Vß, Ca_V, and Ca_V2 subunits (140, 141).

All of the above two-dimensional structures of a Ca_V channel subunit complex were obtained in the 1980s. There were few documents about electron microscopic structures of Ca_V channels published in the 1990s due to technical limitations. In the 2000s, the development of the powerful single particle analysis method coupled to cryoelectron microscopy raises a new tidal wave on the research of the Ca_V channel ultrastructure. The single particle analysis of cryoelectron microscopic images can reconstruct the refined 3D structure of a Ca_V1.1 channel subunit complex showing disposition and shape of different subunits within the complex (142, 143, 144, 145, 146). Murata et al. (145) showed that the Ca_V1.1 channel complex is built from a 9 x 20-nm cylinder and a 7-nm diameter ball, which is decorated on the side of the extracellular part of the cylinder. The cylinder consists of a Ca_V1.1 and a Ca_Vß subunit. However, the Ca_Vß subunit does not completely cover the intracellular edge of the Ca_V1.1 cylinder. This results in four asymmetric domains when viewed from the top of the cylinder. These four domains, representing the four repeated domains of the Ca_V1.1 subunit, form a 2-nm central ion-conducting pore. The ball was identified as the Ca_V2 subunit (145). Shortly afterward, the 3D structure of the Ca_V1.1 channel complex was reconstructed at 30-Å resolution (142). It was also demonstrated that this channel complex exhibited an asymmetrical structure comprising two major regions, a heart-shaped region whose widest end attaches to a handle-shaped region. The rough volume is 115 x 130 x 120 Å. A 30-Å diameter cavity was seen between the two regions. The heart-shaped region accounts for 65% of total volume, corresponding to about 280 kDa, and is proposed to be the pore-forming Ca_V1.1 subunit associated with the Ca_Vß and Ca_V subunits. However, they did not observe any ion-conducting pore feature within the heart-shaped region. The handle-shaped region was regarded as the Ca_V2 complex, representing approximately 35% of total volume equal to about 140 kDa (142). Recently, the 3D structure of the Ca_V1.1 channel complex has been visualized at a 23-Å resolution, the highest resolution of an electron microscopic structure of the Ca_V1.1 channel complex achieved so far (143). The superior elegance in this observation is the high-resolution details of the five different subunit profiles and dispositions within the channel complex. In agreement with the previous observations, the asymmetric contour is the most striking feature of the 3D Ca_V1.1 channel complex structure. The reconstructed 3D structure with an estimated total molecular mass of 550 kDa corresponds approximately to the assembly of the five Ca_V channel subunits, i.e., the Ca_V1.1, Ca_Vß, Ca_V, and Ca_V2 subunits. The contour of the channel complex obtained at a density level of 550 kDa displays a curling stone-like appearance, namely a large globular domain with a handle-shaped (called leg-shaped in the original paper) protrusion. The globular domain is ellipsoidal in shape with a length of 165 Å, a width of 145 Å, and a height of about 95 Å. At a higher density level, the contour reveals that the globular domain is separated by a gap into a smaller and a larger subvolume. The larger subvolume corresponds to the Ca_V1.1 subunit. Antibody labeling and the calculated molecular mass indicate that the smaller subvolume contains both the Ca_Vß and Ca_V subunits. The leg-shaped density with a length of about 95 Å protrudes from the edge of the top flat side (extracellular side) of the larger subvolume. This protrusion is recognized by an anti-Ca_V2 antibody. This indicates that the Ca_V subunit is adjacent to or partially embedded within the Ca_V1.1 subunit (the larger subvolume) because the Ca_V2 subunit is linked to the Ca_V subunit by a disulfide bond (143). Additionally, Wang et al. (144, 146) reported a ring-shaped dimer of both Ca_V1.1 and Ca_V1.2 channels. The lack of consistency in the findings from the different groups reflects weaknesses of the current methodology. We still face a big challenge in understanding the molecular architecture of Ca_V channels.

3. Expression and trafficking of Ca_V channels
a. Expression and posttranslational modification of Ca_V channels.
To date, molecular cloning has documented 26 distinct genes encoding different Ca_V channel subunits (3, 28). Ca_V channel gene expression is controlled by the interaction of the Ca_V channel gene promoters and other regulatory DNA sequences with transcription factors, activators, and repressors. This interaction determines in which cell type, at what developmental stage, and to what extent individual Ca_V subunit genes will be expressed (147). However, it should be noted that Ca_V channel gene expression is highly plastic and changes in response to numerous physiological and pathological stimuli. It has been demonstrated that cytokine incubation can turn on the gene expression of Ca_V3 channels in mouse ß-cells (for further details, see Section IV.B) (148). Before innervation, myotubes are characterized by the expression of Ca_V3 channels. The Ca_V channel expression switches from Ca_V3 to Ca_V1.1 resembling the adult pattern shortly after myotubes are innervated. On the contrary, denervated muscles display the embryonic profile of Ca_V channel expression. However, these Ca_V channel phenotype switches depend on electrical activity rather than the presence of nerves because electrical stimulation suffices to keep the adult pattern of Ca_V channel expression in the denervated muscles (64).

Another important process is splicing of Ca_V channel subunit pre-mRNAs, encompassing invariant and alternative splicing. This process not only removes introns but also generates a variety of different combinations of exons from the same gene to make different isoforms of a Ca_V channel subunit (149, 150). One has estimated that alternative splicing could give rise to over 1000 distinct mRNAs from each Ca_V1 gene in terms of possible number of alternative exons (see Section II.B.1.a) (53).

The Ca_Vß subunit as an intracellular protein is synthesized by free ribosomes. In contrast, Ca_V1 and Ca_V subunits as well as the precursor polypeptide of Ca_V2 subunits are transmembrane proteins. The translation of these subunit transcripts takes place on ribosomes situated on the rough endoplasmic reticulum (ER). Like other voltage-gated ion channels, these Ca_V channel subunits do not possess an N-terminal ER signal sequence (64). They use internal ER signal sequences to direct their insertion in the ER membrane. Charged residues flanking the hydrophobic core of ER signal sequences primarily determine the orientation of these sequences, namely the more positive end is predominantly orientated toward the cytoplasmic side of the ER membrane. This phenomenon is generally accepted as the "positive-inside rule" (151). According to this positive-inside rule, the region immediately upstream of the hydrophobic core of the first start transfer sequence in these Ca_V channel subunits is more positively charged in comparison with that adjacent to the end of this hydrophobic core. Therefore, their N termini are orientated to the cytoplasmic side of the ER membrane. The polypeptide chain of individual Ca_V channel subunits remains and passes once or repeatedly across the ER membrane depending on the number of transmembrane segments (152). Normally, a peptide chain grows at 3–5 amino acids per second at 37 C. It probably takes about 10 min for a ribosome to synthesize a 2000-residue Ca_V1 subunit (64).

Before a newly synthesized Ca_V channel subunit targets to the right location to function, it has to be subjected to several forms of posttranslational alterations, such as glycosylation, proteolytic cleavage, disulfide bridge cross-linkage and phosphorylation. Glycosylation does not occur at intracellular Ca_Vß subunits (32). However, these subunits can be reversibly phosphorylated (48). The rat Ca_Vß2a subunit, a particular isoform of Ca_Vß2 subunits, is also reversibly palmitoylated at its N-terminal dicysteine motif (104, 110, 111). The transmembrane subunits Ca_V and Ca_V2 are permanently glycosylated (28, 32, 50, 127). Both in vitro and in vivo phosphorylation of Ca_V1 have been experimentally demonstrated (3, 81). Several potential sites for serine or threonine phosphorylation have been revealed in most of the Ca_V subunits (51, 115). Disulfide bridges between Ca_V2 and Ca_V are built in the ER lumen by protein disulfide isomerase (153, 154). Two other important processes in the maturation of Ca_V subunits are proper folding and assembly. Like other proteins, Ca_V subunits begin to fold during their synthesis. Eventually, molecular chaperones and posttranslational alterations help the Ca_V subunit molecule fold up into its unique 3D conformation. The proper folding and assembly of the transmembrane Ca_V1, Ca_V, and Ca_V subunits, the intracellular Ca_Vß subunit, and the extracellular Ca_V2 subunit in the ER are prerequisites for Ca_V channel trafficking to the plasma membrane. The assembly of the Ca_V1/ß complex in ER has been indicated by a heterologous expression system (155). The Ca_V1.1 subunit expressed on its own in HEK-tsA201 cells distributes in a tubular/reticular network throughout the entire cytoplasm, being much denser in the perinuclear region, a typical ER structure. Furthermore, these cells do not give rise to any detectable Ca_V currents. In contrast, when Ca_Vß1a is expressed alone in these cells, its distribution pattern is quite different from that of the expressed Ca_V1.1. The expressed Ca_Vß1a is evenly distributed all over the cytoplasm without association with any cytoplasmic structure. However, the Ca_Vß1a becomes colocalized with the coexpressed Ca_V1.1 in ER. Most importantly, coexpression of these two subunits endows the transfected cell with genuine Ca_V1.1 channels. This demonstrates that assembly of the Ca_V1/ß complex in ER is a prerequisite for the maturation of Ca_V channels (155).

b. Trafficking and targeting of Ca_V channels.
Ca_V channel trafficking and targeting enable distinct areas of the cell to be equipped with particular types and densities of Ca_V channels for specific functions (156, 157, 158, 159, 160). Similar to other ion channels, newly synthesized Ca_V channel subunits are properly folded and assembled in the ER to build up Ca_V channel subunit complexes. Subsequently, these Ca_V channel subunit complexes are carried by the transport vesicles and sequentially transferred to the cis-Golgi network, the Golgi apparatus, and the trans-Golgi network. The constitutive secretory vesicles carrying the Ca_V channel subunit complex separately bud off from the trans-Golgi network using sorting mechanisms. Subsequently, these vesicles carry Ca_V channel subunits to the targets, the different areas of the plasma membrane. Finally, the channels are anchored by as yet unknown mechanisms in the right place to function (Fig. 4) (161). Actually, these processes are very sophisticated and highly regulated. The posttranslational modification plays an important role not only in Ca_V channel subunit folding and assembly but also in Ca_V channel subunit complex trafficking (127). For example, the N-glycosylation in the ER, as a marker of the state of protein folding, is an important control point in Ca_V channel trafficking (127, 153). PKA phosphorylation significantly facilitates budding off constitutive secretory vesicles from the trans-Golgi network (162). Interactions among Ca_V channel subunits themselves and between Ca_V channel subunits and other nonchannel proteins take center stage in Ca_V channel trafficking, targeting, and anchoring (161, 163, 164). The section below discusses this aspect in detail.

View larger version (49K): [in this window] [in a new window]   FIG. 4. A model depicting the expression and trafficking of CaV channels. CaVß subunits are synthesized by free ribosomes, whereas CaV1 and CaV subunits as well as the precursor polypeptide of CaV2 subunits are manufactured by ribosomes situated on the ER. The CaV1 subunit harbors multiple ER retention signals. In the correctly folded CaV1 subunit, the ER retention signals can interact to mask each other. However, the correctly folded CaV1 subunit is still locked by the ER retention protein in the ER. The CaVß subunit serves as a key to unlock the CaV1 subunit in the ER and then chaperones it to the plasma membrane. The incorrectly folded CaV1 subunit cannot traffic to the plasma membrane because its ER retention signals cannot be properly hidden even in the presence of the CaVß subunit. The CaV channel traffics to the plasma membrane not only in a constitutive manner but also via regulated secretory vesicles with and without transmitters.

i. The interaction between Ca_V1 and Ca_Vß subunits is critical for Ca_V channel trafficking.

Nevertheless, strong experimental evidence of a chaperone-like effect of Ca_Vß subunits in Ca_V1 subunit trafficking in the Xenopus oocyte has emerged since the late 1990s. Injection of purified Ca_Vß3 subunit proteins into oocytes expressing human Ca_V1.2 subunits produced not only a rapid allosteric modulation on Ca_V1.2 subunit function, but also a chaperone-like effect on Ca_V1.2 subunit trafficking gradually occurring over 4 h (169). The latter conferred a massive augmentation of Ba²⁺ current density and a drastic elevation in the amount of Ca_V1.2 subunit protein, which were abolished by bafilomycin A1, an inhibitor of intracellular glycoprotein transport (169). Similarly, the Ca_Vß2a subunit dramatically increased both the Ba²⁺ current density and the surface expression of human Ca_V1.2 subunit when these two subunits were coexpressed in the oocyte (168). Moreover, facilitation of Ca_V2.2 subunit trafficking by the Ca_Vß3 has been visualized in the Xenopus oocyte (174).

Identification of the endogenous Ca_V channel subunits in oocytes helped understand what caused the discrepancies about the role of Ca_Vß subunits in Ca_V channel trafficking. Lacerda et al. (175) found that endogenous oocyte Ca_V channels appeared approximately once every 2 or 3 months in their experiments. More interestingly, molecular cloning has identified an endogenous oocyte Ca_Vß subunit gene (ß3xo) corresponding to mammalian Ca_Vß3 subunits. The injection of antisense oligonucleotides to ß3xo can significantly blunt currents through endogenous Ca_V channels and completely suppress the functional expression of injected Ca_V1.2 and Ca_V2.3. Therefore, it was believed that ß3xo genes do express active proteins functioning as chaperones aiding in the folding and trafficking of Ca_V1 to the oocyte surface. This also led to the hypothesis that the endogenous concentration of ß3xo is capable of transporting the newly synthesized channel to the oocyte surface via its high affinity interaction with Ca_V1 subunits (176). In contrast, the higher concentrations of exogenous Ca_Vß subunits mainly produce the allosteric modulation on the targeted Ca_V1 subunits through their lower affinity interaction. Indeed, coexpression of low concentrations of Ca_Vß3 subunit with a fixed concentration of Ca_V2.2 subunits only increases the maximum conductance at a fixed depolarizing voltage without changing current-voltage relationship, suggesting an increase in the surface expression rather than alteration in channel function (174). This hypothesis reconciles disputes among different groups on the role of Ca_Vß in Ca_V channel trafficking and targeting. It is plausible that the oocytes used in the different experiments might contain different concentrations of endogenous Ca_Vß exhibiting marked variation in the regulation of Ca_V1 subunit trafficking. Furthermore, it should be noted that other experimental conditions, e.g., the holding potential, concentration of extracellular Ba²⁺, and abundance of expressed Ca_V subunits, may also contribute to the observed discrepancies (177).

It has been shown that some mammalian proteins traffic differently when heterologously expressed in Xenopus oocytes (178). To better understand Ca_V channel trafficking and targeting in mammalian cells, CHO, COS-7, HEK 293, HEK-tsA201, and Ltk^– cell lines have been widely employed for heterologous expression of these channel subunits in different combinations (105, 110, 179, 180, 181, 182). The early literature reported controversial results pertaining to the role of Ca_Vß subunits in trafficking and targeting of functional Ca_V channels in mammalian heterologous expression systems (105, 180, 181, 182, 183). In tackling the problem, more critical analyses have been performed in mammalian heterologous expression systems since the middle of the 1990s. Numerous combinations of Ca_V1 and Ca_Vß subunits expressed in a series of cell lines have been examined by combining the techniques of electrophysiology, molecular biology, biochemistry, immunocytochemistry, and confocal microscopy (179, 184, 185, 186, 187). In general, all tested Ca_Vß subunits augment functional Ca_V1 subunit density in the plasma membrane. This is demonstrated by massive increases in ionic currents conducted by the Ca_V1 subunit, gating currents derived from the movement of charged amino acid residues in this subunit, the abundance of this subunit and DHP binding sites (48, 161). Furthermore, it is noteworthy that the same Ca_Vß subunit displays different possibilities to chaperone different Ca_V1 subunits and the same Ca_V1 subunit traffics differently in the presence of the various Ca_Vß subunits (188, 189). Fairly consistent results from these careful studies led to the consensus in the literature that Ca_Vß subunits play an important role in Ca_V channel trafficking and targeting (48, 161).

The question that immediately arises is how Ca_Vß subunits function in Ca_V channel trafficking and targeting. To answer this question, one would inevitably need to ask whether the Ca_Vß subunit carries the specific plasma membrane-targeting signal. Chien et al. (110, 111, 187, 190) has carried out a series of experiments to examine whether palmitoylation, a posttranslational modification, can function as such a signal. Originally, they observed that the rat Ca_Vß2a subunit, when expressed alone in mammalian cell lines, is targeted to the plasma membrane although it is hydrophilic and has no membrane-spanning domains (187). In contrast, the Ca_V1.2 subunit alone distributes predominantly in the cytoplasm and in the perinuclear region but to a limited extent in the plasma membrane. However, when the rat Ca_Vß2a subunit is coexpressed, a greater proportion of the Ca_V1.2 subunit is distributed in the plasma membrane, resulting in significantly enhanced both DHP binding sites in intact cells and peak Ca_V current density (187). Further careful characterization denies any increases in either the expression or the half-life of the Ca_V1.2 subunit by the coexpressed rat Ca_Vß2a subunit (187). Later, it was found that the rat Ca_Vß2a subunit is the only palmitoylated form among the tested Ca_Vß subunits including rat Ca_Vß1b, Ca_Vß2a, Ca_Vß3, and Ca_Vß4 as well as rabbit Ca_Vß2a and Ca_Vß2b (110, 111). The palmitoylation makes the Ca_Vß2a subunit itself capable of associating with the plasma membrane. Replacement of the N-terminal region of the Ca_Vß1b or the Ca_Vß3 with that of the Ca_Vß2a efficiently confers palmitoylation to these chimeras, Ca_Vß2a/1b and Ca_Vß2a/3, but is insufficient to target them to the plasma membrane (111). This suggests that there are additional sequences in the Ca_Vß2a subunit contributing to the autonomous trafficking of this subunit to the plasma membrane (111). Furthermore, the palmitoylation was not a necessary condition for Ca_Vß subunits to bring Ca_V1 subunits to the plasma membrane. A double mutation (C3S-C4S) of the Ca_Vß2a subunit abolishes palmitoylation of this subunit (110). Consequently, this mutant lost the capacity to traffic to the plasma membrane by itself and was distributed throughout the cytoplasm when expressed alone (111). Interestingly, the mutation drastically decreased whole-cell Ca²⁺ currents while coexpressed with the Ca_V1.2 subunit. However, the reduction in whole-cell Ca²⁺ currents is not due to less Ca_V1.2 subunits in the plasma membrane because there is no decrease in the size of intramembrane charge movement, a readout of the number of functional channels (110). This suggests that the palmitoylation-deficient Ca_Vß2a subunit is still able to chaperone the Ca_V1.2 subunit to the plasma membrane (110). These data demonstrate that the palmitoylation-deficient Ca_Vß2a mutant exhibits the same potency to traffic and targets the Ca_V1.2 subunit to the plasma membrane, but that it is less efficient to increase the ion conductivity of the Ca_V1.2 subunit in comparison with the wild-type Ca_Vß2a (110). Overall, palmitoylation indeed is pivotal for the plasma membrane trafficking of the rat Ca_Vß2a subunit itself. Although the palmitoylation of the rat Ca_Vß2a subunit can facilitate Ca_V1 subunit trafficking to the plasma membrane, the polypeptide part of the Ca_Vß2a subunit seems to play a more important role in this respect.

Recently, it has been revealed that two other Ca_Vß subunits, Ca_Vß1b and Ca_Vß2e, can themselves traffic to the plasma membrane (191, 192). Like the rat Ca_Vß2a subunit, the Ca_Vß1b subunit exhibits clear plasma membrane association when expressed in COS-7 cells, but not in other cell types. The chimera containing the major region of Ca_Vß3 and C terminus of Ca_Vß1b also displays the same distribution pattern. On the contrary, the chimera carrying the major region of Ca_Vß1b and C terminus of Ca_Vß3 distributes in the cytoplasm, similar to the Ca_Vß3 parent. This indicates that the plasma membrane association of Ca_Vß1b subunits depends on its C terminus (191). Further mutation analysis has demonstrated that the deletion of an acidic motif, comprising 11-amino acid residues (WEEEEDYEEE), in the C terminus of Ca_Vß1b subunits can significantly diminish the plasma membrane association of this subunit. Therefore, it is believed that this acidic motif is in charge of plasma membrane association of Ca_Vß1b subunits (191). Indeed the Ca_Vß1b subunit effectively chaperones the Ca_V2.1 subunit to the plasma membrane. The acidic motif situated in the Ca_Vß1b subunit should contribute to this subunit-mediated Ca_V channel trafficking to the plasma membrane. However, the acidic motif is not an absolute necessity shared by all Ca_Vß subunits to chaperone the Ca_V1 subunit (191). Additionally, the autonomous association of the Ca_Vß2e subunit with the plasma membrane has been visualized in HEK 293 cells. The motif responsible for plasma membrane targeting has not yet been identified, although the unique D1 domain is believed to be the area where the motif should localize (192).

The interaction between Ca_V1 and Ca_Vß subunits plays an important role in Ca_V channel trafficking. Experimental evidence manifested that this interaction not only brings about regulation of biophysical and pharmacological properties of Ca_V1 subunits by Ca_Vß subunits but also underlies plasma membrane expression of Ca_V channels (193). To simplify the evaluation of the contribution of the association of Ca_Vß and Ca_V1 subunits to Ca_V channel trafficking, Shaker channel- and CD8-Ca_V1 L_I-II chimeras were created. Fusion of the Ca_V2.1 L_I-II with the C terminus of Shaker channels or the chain of CD8 receptors made these plasma membrane proteins hardly traffic to the plasma membrane (193). The same was true for the L_I-II of Ca_V1.2, Ca_V2.2, and Ca_V2.3 subunits when they were fused with CD8 (193). Further analysis demonstrated that Ca_V2.1 L_I-II effectively retained its chimeric partner CD8 in the ER. The Ca_V2.1 L_I-II in the chimeras can bind to the Ca_Vß3 subunit very well. Upon coexpression with the Ca_Vß3 subunit, the Shaker channel-Ca_V2.1 L_I-II or CD8-Ca_V2.1 L_I-II chimeras were clearly visualized in the plasma membrane. Pulse-chase experiments demonstrated that the association of the Ca_Vß3 subunit with the CD8-Ca_V2.1 L_I-II chimera took place early in the biosynthesis pathway, likely at the ER level. Furthermore, the Ca_V2.1 subunit lacking part of the L_I-II (389–423) is capable of reaching the plasma membrane in the absence of the Ca_Vß3 subunit (193). These data suggest that the AID-containing L_I-II harbors an ER retention signal, which inhibits trafficking of Ca_V1 subunits to the plasma membrane. Binding of the Ca_Vß subunit to the L_I-II releases the brake and triggers a rapid departure of Ca_V1/ß complex from the ER to the plasma membrane (Fig. 4) (193).

Actually, the ER retention of the Ca_V1 subunit is much more complicated than that in the above simplified case. In addition to the ER retention signal masked by the interaction with the Ca_Vß3 subunit, the L_I-II of Ca_V2.1 subunit contains multiple ER retention determinants that interact with other ER retention sequences in L_III-IV and N and C termini of the Ca_V2.1 subunit (194). It has been proposed that the L_I-II forms a ternary interaction complex with these internal ER retention determinants to mask each other in the correctly folded Ca_V1 subunit. However, this correctly folded Ca_V1 subunit is still locked in the ER at this stage. It must be unlocked by the key Ca_Vß subunit and then leave from the ER for the plasma membrane. On the contrary, the incorrectly folded Ca_V1 subunit cannot traffic to the plasma membrane because its internal ER retention determinants cannot be properly masked (194).

Interestingly, it has been demonstrated that two ER export signals are present in the inwardly rectifying potassium (Kir) channels Kir1.1 and Kir2.1, respectively, and essential for export of the channels from the ER (195). Likewise, the Ca_V1 subunit not only harbors ER retention signals to immobilize itself in the ER but also carries such ER export signals to propel itself to the plasma membrane (196). It seems likely that a 43-amino acid stretch from 1623 to 1666 in the C-terminal region of the Ca_V1.2 subunit is critical for Ca_V1.2 subunit folding. Deletion of this stretch ablated plasma membrane expression of Ca_V1.21623–1666/ß2a channels in tsA201 cells as evidenced by complete loss of cell surface staining of Ca_V1.21623–1666, cell surface binding of the DHP antagonist [³H]PN200-110, and L-type Ba²⁺ currents (196). This indicates that this misfolded Ca_V1.2 subunit can no longer traffic to the plasma membrane. These results add more complexity to Ca_V channel trafficking. It seems likely that masking ER retention signals and keeping export signals intact are of equal importance in Ca_V channel trafficking.

ii. The role of Ca_V2 subunits in Ca_V channel trafficking.
It has been ascertained that Ca_V2 subunits regulate Ca_V channel trafficking. Some isoforms enhance membrane trafficking of Ca_V1 subunits in either the absence or the presence of Ca_Vß subunits, whereas others strictly require the presence of Ca_Vß subunits to exert the effect (28). The coexpressed Ca_V21 subunits drastically increase the current amplitude and [³H]PN200-110 binding in tsA201 cells transfected with Ca_V1.2 subunits (132). Furthermore, Ca_V1.2/₂1/ß2a channels expressed in HEK cells display larger amplitude in both ionic conductance and intramembrane charge movement than Ca_V1.2/ß2a channels (197). Interestingly, coexpression of the peptide of Ca_V21 subunits failed to produce an effect on either current amplitude or [³H]PN200-110 binding in cells expressing Ca_V1.2 channels (132). This strongly suggests that the insertion of Ca_V1.2 subunits into the plasma membrane occurs in the absence or presence of Ca_Vß2 subunits and the effect depends on the presence of the ₂ domain of Ca_V21 subunits (132, 197). Actually, neither the ₂ nor domain of Ca_V21 subunits can independently promote Ca_V2.1/ß4 channel trafficking. The coexpressed Ca_V21 subunits result in a 9-fold increase in current amplitude without altering voltage-dependence of activation. This effect cannot be mimicked by coexpression of either the ₂ or peptide. Furthermore, the peptide massively counteracted the enhanced current through the Ca_V2.1/ß4 channel induced by Ca_V21 subunits (127). Therefore, the structural integrity of Ca_V21 subunits is fundamental for Ca_V2.1/ß4 channel trafficking (127). The truncated version of Ca_V21 subunits at the N-terminal region (N28–184) results in a loss of the stimulatory effect on the current amplitude. Although both glycosylated and deglycosylated Ca_V21 subunits remain associated with Ca_V1 subunits, the degree of association is reduced by 60% when Ca_V21 subunits are deglycosylated. Deglycosylation of intact oocytes expressing Ca_V2.1/₂1/ß4 channels causes a 67% reduction in the current amplitude (127). All these data strongly support the notion that the glycosylation may stabilize the channels in the plasma membrane (127).

The possible effect of Ca_V22 subunits on channel trafficking has been suggested from functional analysis in heterologous expression systems (125, 133, 198). The current amplitude measured in oocytes expressing either Ca_V1.2/₂2 or Ca_V3.1/₂2 is 2-fold enhanced in comparison with those expressing either Ca_V1.2 or Ca_V3.1 alone. However, Ca_V22 subunits have no effect on current-voltage relationship. Interestingly, Ca_V22 subunits more prominently increase the current amplitude without significantly affecting gating kinetics when they are coexpressed with Ca_V2.2/ß3 in oocytes (133). Further investigations demonstrated that the coexpressed Ca_V22 increases the current density when coexpressed with Ca_V1.2/ß2a, Ca_V2.1/ß2a, and Ca_V2.3/ß3, respectively, in HEK 293 cells, suggesting enhanced surface expression of the channels. However, Ca_V22 also changes gating kinetics indicating functional regulation of the expressed channels (125). Recently, the effect of Ca_V22 subunits on the expression of Ca_V2.1/ß4 channels in the plasma membrane has been carefully characterized (198). The coexpressed Ca_V22 subunits significantly increased current density without altering biophysical properties at both the single channel and the whole-cell level. Taken together, these results may indicate that Ca_V22 subunits promote both channel insertion into the plasma membrane and stabilization of these inserted channels (198).

The involvement of Ca_V23 subunits in the surface expression of Ca_V1.2 and Ca_V2.3 channels has been characterized (122). It seems that Ca_V23 subunits require the presence of Ca_Vß2a to regulate trafficking of the coexpressed Ca_V1.2 to the plasma membrane. Ca_V23 subunits do not produce an effect on the density of currents through Ca_V1.2 channels expressed in HEK 293 cells in the absence of Ca_Vß2a. However, in the presence of Ca_Vß2a, Ca_V23 subunits significantly increase current density reflecting more functional Ca_V1.2 channels in the plasma membrane. Furthermore, Ca_V23 also dramatically increases the current amplitude measured in cells expressing Ca_V2.3/ß3 (122).

Ca_V24 also participates in the regulation of membrane expression of Ca_V channels (123). Functional analysis showed that the high K⁺-induced increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in HEK 293 cells transfected with Ca_V1.2/ß3/₂4 was significantly greater than that in HEK 293 cells transfected with Ca_V1.2/ß3. This suggests that the coexpressed Ca_V24 facilitates Ca_V1.2 channel targeting to the plasma membrane (123).

iii. Ca_V subunits seem to play a negative role in Ca_V channel trafficking.
Several isoforms of Ca_V subunits, 2–4, can traffic to the plasma membrane in the absence of other Ca_V channel subunits (199). It is not clear whether Ca_V subunits are involved in Ca_V channel trafficking. Some experimental data showed that Ca_V subunits had no detectable effect on Ca_V channel trafficking (199). Others demonstrate that Ca_V subunits play a negative role in surface expression of Ca_V channels (118, 200).

iv. The A-kinase anchoring protein (AKAP79) participates in Ca_V channel trafficking.
In addition to Ca_V channel subunits, the nonchannel protein AKAP79 also plays a role in the surface expression of Ca_V channels (164). Expression of AKAP79 significantly increases the current amplitude measured in oocytes expressing Ca_V1.2 channels but has no effect on the activity of the coexpressed Ca_V2.1, Ca_V2.3, Ca_V3.1, and Ca_V3.2 channels. The effect of AKAP79 on Ca_V1.2 channels is independent of Ca_Vß subunits. AKAP79 exerts no action on the biophysical property of Ca_V1.2 channels at either the single channel or the whole-cell level (164). However, cell-attached recordings showed a significant increase in the number of functional Ca_V1.2 channels per patch in the oocytes expressing Ca_V1.2/ß1b/₂ and AKAP79. Interestingly, deletion of the PKA interacting domain in AKAP79 does not influence the effect of AKAP79 on the current density in oocytes expressing Ca_V1.2/ß1b/₂. The same is true for either activation or inhibition of PKA. These data indicate that AKAP79 enhances surface expression of Ca_V1.2 channels through interaction with Ca_V1.2 subunits independent of PKA signaling (164). Furthermore, immunoassay of the hemagglutinin-tagged Ca_V1.2 subunit revealed that AKAP79 dramatically increases the surface expression of Ca_V1.2 subunits with a resulting enhanced current density. Chimeric engineering manifested that the structural determinants in Ca_V1.2 subunits responsible for the stimulatory effect of AKAP79 on the current density are localized at the carboxyl half of Ca_V1.2 L_II-III. In this region, an 11-amino acid sequence containing five prolines was further identified to interact with AKAP79, mediating its effect on the surface expression of Ca_V1.2 channels (164). Overall, AKAP79 chaperones Ca_V1.2 channels to the plasma membrane through the direct interaction of AKAP79 with the proline-rich motif in the carboxyl half of Ca_V1.2 L_II-III (164).

v. Ras-related G proteins compete for Ca_Vß subunits with Ca_V1 subunits, thereby leaving Ca_V1 subunits in the ER.
Nonchannel proteins interact not only with pore-forming Ca_V1 subunits but also with auxiliary Ca_Vß subunits to modulate Ca_V channel trafficking. The Ras-related G protein kir/Gem directly interact with Ca_Vß subunits to down-regulate Ca_V1.2 channel trafficking to the plasma membrane (for details, see Section V.I) (163).

vi. Phosphatidylinositol 3-kinase (PI3K) plays an important role in Ca_V channel trafficking.
The molecular mechanisms underlying Ca_V channel trafficking are very sophisticated. In addition to the physical association of pore-forming Ca_V1 subunits with auxiliary Ca_Vß subunits and other nonchannel proteins, PI3K is also involved in Ca_V channel trafficking (201). Expression of PI3K massively enhances current density without altering activation and inactivation kinetics of Ca_V1.2/ß2a channels expressed in COS-7 cells. The effect is mediated by phosphatidylinositol 3,4,5-trisphosphate generated by the expressed PI3K. This phosphatidylinositol 3,4,5-trisphosphate-mediated action selectively depends on Ca_Vß2a subunits because PI3K functions only when coexpressed with Ca_Vß2a, but not with Ca_Vß1b, Ca_Vß3, or Ca_Vß4 subunits (201). Moreover, the PI3K-induced modulation on the Ca_V current density is due to increased expression of Ca_V channels at the cell surface. Overexpression of PI3K makes the green fluorescent protein (GFP)-tagged Ca_V2.2 subunit effectively traffic to the plasma membrane in the presence of Ca_Vß2a subunits. As mentioned before, palmitoylation of the Ca_Vß2a subunit makes this subunit itself capable of targeting to the plasma membrane. However, this posttranslational modification of the Ca_Vß2a subunit is not involved in PI3K-induced modification of Ca_V channels. Instead, the serine/threonine kinase Akt/protein kinase B (PKB) functions downstream of PI3K and associates with the Ca_Vß2a subunit. Akt/PKB is effectively coimmunoprecipitated with the Ca_Vß2a subunit. Overexpression of PI3K causes phosphorylation of Akt/PKB on T308 and S473. Coexpression of the dominant-negative mutant AAA-Akt/PKB with PI3K to prevent activation of endogenous Akt/PKB significantly decreases accumulation of GFP-Ca_V2.2/ß2a at the plasma membrane. This also abolishes the PI3K-induced increase in Ca_V currents through Ca_V2.2/ß2a channels (201). Additionally, coexpression of a myristoylated Akt/PKB with GFP-Ca_V2.2/ß2a significantly increases both GFP fluorescence at the plasma membrane and Ca_V currents through GFP-Ca_V2.2/ß2a channels. Further analysis identified a unique putative consensus site (RXRXXS) for Akt/PKB phosphorylation in the C-terminal region of Ca_Vß2a. The replacement of serine 574 by alanine (S547A) at this site of Ca_Vß2a significantly reduces the phosphorylation of the Ca_Vß2a subunit by PI3K overexpression. It also makes the expressed PI3K or myr-Akt/PKB incapable of promoting the GFP-Ca_V2.2/ß2aS574A channel trafficking to the plasma membrane (201). Furthermore, the substitution of serine 574 with glutamate (S547E) in the Ca_Vß2a subunit can mimic the effect resulting from the phosphorylation of the Ca_Vß2a subunit at serine 574. This mutant of the Ca_Vß2a subunit not only increases the targeting of GFP-Ca_V2.2 to the plasma membrane but also augments Ba²⁺ currents through the coexpressed Ca_V2.2 subunit. The effect of Ca_Vß2a S547E can neither be prevented by AAA-Akt/PKB nor further increased by PI3K and myr-Akt/PKB (201). Additionally, PI3K produces an effect on the Ca_V channel trafficking similar to PI3K, indicating that the effect is independent of the PI3K isoform. Taken together, these results demonstrate that the PI3K-Akt/PKB cascade-mediated phosphorylation at S547 of Ca_Vß2a plays an important role in Ca_V channel trafficking (201).

vii. Does regulated exocytosis mediate Ca_V channel trafficking?
It is believed that Ca_V channels traffic to the plasma membrane via the constitutive exocytotic pathway, which is responsible for the renewal of integral membrane proteins and lipids. However, it has been demonstrated that neurons and endocrine cells use regulated exocytosis not only to secrete neurotransmitters and hormones but also to transport Ca_V channels to the plasma membrane (Fig. 4). For example, N-type Ca_V channels have been demonstrated to localize in PC12 cell secretory granules and to be translocated to the plasma membrane during regulated exocytosis (202). Recently, we have found that abundant granular structures in the mouse ß-cell do not contain insulin but are richly equipped with ATP-sensitive potassium (K_ATP) channel subunits and undergo exocytosis in response to glucose. Consequently, K_ATP channel subunits are acutely recruited to the ß-cell plasma membrane (203). It has been demonstrated that glucose stimulation can induce Ca_V2.2 channel translocation to the plasma membrane of the insulin-secreting RINm5F cells (204). It is intriguing to investigate the pathways responsible for trafficking of ß-cell Ca_V channels to the plasma membrane.

viii. How do Ca_V channels target to specific zones in the plasma membrane?
Microscopically, different types of Ca_V channels distribute in distinct areas of the plasma membrane to perform specific tasks. For example, nerve terminals use Ca_V2.1, Ca_V2.2, and Ca_V2.3 channels to trigger neurotransmitter release (157). Skeletal muscle triads employ Ca_V1.1 channels to initiate excitation-contraction coupling (158). Even in the pancreatic ß-cell, Ca_V1 channels predominantly localize at exocytotic sites, which are not evenly distributed (159, 160). It is poorly understood how different types of Ca_V channels target to distinct functional zones. However, there are experimental hints that Ca_V1 and Ca_Vß subunits carry specific targeting signals. The polarized Madin Darby Canine Kidney epithelial cell line has been used to evaluate targeting of Ca_V channels in different regions of this cell (186). Ca_V1.2/ß1b/₂, Ca_V1.2/ß2a/₂, Ca_V1.2/ß3/₂, and Ca_V1.2/ß4/₂ channels mainly target to the basolateral membrane. Interestingly, Ca_V2.1 subunits coexpressed with different Ca_Vß subunits display distinct targeting. Ca_V2.1/ß1b/₂ and Ca_V2.1/ß4/₂ channels predominantly reach the apical membrane. Ca_V2.1/ß2a/₂ channels mainly localize in the basolateral membrane. Ca_V2.1/ß3/₂ channels can target to the plasma membrane without selective distribution. However, this combination of Ca_V channel subunits also leaves a substantial amount of Ca_V2.1 subunits in intracellular compartments. Ca_V2.2/ß1b/₂, Ca_V2.2/ß2a/₂, Ca_V2.2/ß3/₂, and Ca_V2.2/ß4/₂ channels appear in the apical membrane (186).

In skeletal muscle cells, both Ca_V1.1 and Ca_Vß1a subunits have been demonstrated to harbor important sequences to target Ca_V1.1 channels to triads, i.e., the functional sites of excitation-contraction coupling (205, 206, 207, 208). Expression of Ca_V1.1, Ca_V1.2, Ca_V2.1, and Ca_V2.2 subunits effectively reconstitutes corresponding Ca_V channels in dysgenic myotubes, which lack Ca_V1.1 subunits. However, only the expressed Ca_V1.1 subunits can restore skeletal muscle type excitation-contraction coupling in these Ca_V1.1 null cells (208). A 55-amino acid sequence (aa 1607–1661) in the C-terminal region of Ca_V1.1 subunits has been identified to function as the triad-targeting signal of the skeletal muscle Ca_V channel. An exchange of the C terminus between Ca_V1.1 and Ca_V2.1 subunits makes the chimeric Ca_V1.1 subunit lose its ability to target to triads but brings the chimeric Ca_V2.1 subunit to triads for excitation-contraction coupling (207). Although Ca_V1.1 subunits carry the molecular determinant for triad targeting, the C terminus of Ca_Vß1a subunits also plays an important role in this process. Both Ca_Vß1a and Ca_Vß2a subunits can restore Ca_V currents in the myotubes lacking Ca_Vß1 subunits. However, Ca_Vß1a subunits rescue excitation-contraction coupling in these cells more efficiently than Ca_Vß2a subunits (206). Furthermore, the chimeric Ca_Vß2a subunit containing the C terminus of Ca_Vß1a subunits brings about excitation-contraction coupling in Ca_Vß1 null myotubes as efficiently as the wild-type Ca_Vß1a does (205).

Polarized distribution of distinct Ca_V channels in neurons, myocardiocytes, and endocrine cells, including the pancreatic ß-cells, is obvious (156, 157, 159, 209). However, targeting of Ca_V channels is investigated much less in these cells than in skeletal muscle cells. Overexpression of GFP-tagged Ca_Vß4 subunits in hippocampal neurons indicates that Ca_Vß4 subunits are targeted to synaptic sites (210). GFP-tagged Ca_Vß4 fragments reveal that the N terminus (aa 1–49) and C terminus (aa 408–519) are minimal segments required for targeting of Ca_Vß4 subunits to synaptic sites. Furthermore, either the N terminus (aa 1–49) or the C terminus (aa 408–519) alone is sufficient for synaptic targeting (210). Interestingly, the cardiac Ca_V1.2/ß2 channel is confined to the transverse tubules by as yet unknown mechanisms (156). Moreover, it has been demonstrated that Ca_V1.2 and Ca_V1.3 channels directly interact with exocytotic proteins (see Section III.E.1) and distribute in polarized exocytotic sites of the pancreatic ß-cell (211, 212). The targeting mechanisms of ß-cell Ca_V channels are unknown.

c. Turnover and recycling of Ca_V channels.
Ca_V channels turn over and recycle constitutively and also in a regulated manner in the plasma membrane to keep the proper number of these channels at the cell surface. Different Ca_V subunits seem to have distinct turnover rates. Pulse-chase studies reveal that the half-life of Ca_V1.2 subunits is approximately 3 h, regardless of the presence or absence of Ca_Vß subunits (187). Measurements with ¹²⁵I--CTX GVIA in several neuronal cell lines reveal that the half-life of Ca_V2.2 channels is about 15–18 h (213, 214). Immunocytochemical quantification with an antibody recognizing all Ca_Vß subunits shows that the Ca_Vß subunit level in cultured rat dorsal root ganglion neurons is maximally decreased after 108 h of antisense knockdown. The half-life of the endogenous Ca_Vß subunits in this preparation is estimated to be 50 h (215). The Ca_Vß3 subunit is rapidly removed from the plasma membrane when this subunit is expressed alone in COS-7 cells. The Ca_Vß3 subunit immunoreactivity completely disappears after 2–6 h of treatment with the protein synthesis inhibitor cycloheximide (191). The Ca_Vß1b subunit has a slow turnover and is not affected by inhibition of protein synthesis for up to 6 h (191). The half-life of Ca_V2.2 subunits can change dramatically. For example, differentiating agents significantly slow down the Ca_V2.2 channel turnover in the aforementioned neuronal cell lines (216). Conversely, the autoantibody found in the serum of Lamber-Eaton myasthenic syndrome patients drastically speeds up the Ca_V2.2 channel turnover (213). In this context, it is not known whether different subunits in the same Ca_V channel complex have a simultaneous or separate turnover.

Interestingly, it has been demonstrated that secretory vesicles of neuroendocrine cells, including insulin-secreting RINm5F cells, are equipped with Ca_V2.2 channels. More interestingly, these vesicular Ca_V channels can transiently shuttle between the intracellular pool and the plasma membrane during regulated exocytosis/endocytosis (204, 217). Recently, it has been reported that high [Ca²⁺]_i induces a significant internalization of Ca_V1.3 channels from the plasma membrane to intracellular compartments in insulin-secreting INS-1 cells (218). Therefore, it is attractive to envisage that the functional Ca_V channel complex in the plasma membrane, like other membrane proteins, may recycle to economically adapt to different physiological and pathophysiological situations.

	III. Role of CaV Channels in ß-Cell Physiology

Top Abstract I. Introduction II. General Aspects of... III. Role of CaV... IV. Role of CaV... V. ß-Cell CaV Channel... VI. Future Perspectives References

 
A. Types of ß-cell Ca_V channels
Patch-clamp studies have been extensively performed in the characterization of ß-cell Ca_V channels (2, 219, 220). The identity of islet ß-cells used in electrophysiological studies can be defined by a number of accepted criteria, such as glucose responsiveness, complete inactivation of Na⁺ currents at physiological voltages, cell size reflected by cell capacitance and cell morphology (13, 221, 222, 223, 224, 225). Whole-cell L-type Ca_V currents were first reported in the cultured neonatal rat pancreatic ß-cell (226). Soon, whole-cell Ca_V currents were visualized in a variety of ß-cells, including insulin-secreting cell lines and islet ß-cells from different species (Table 2) (2, 227). There is now considerable consensus that the L-type Ca_V current is the major Ca_V current subtype in the ß-cell from all the tested species (2, 219, 220). For other subtypes of Ca_V channels, there are obvious interspecies differences (2, 219, 220). The different subtypes of ß-cell Ca_V channels are further discussed below.

Whole-cell patch-clamp studies have revealed that a major proportion of Ca_V currents in the rat pancreatic ß-cell is L-type, although this cell carries more subtypes of Ca_V channels than the mouse pancreatic ß-cell (239). The rat ß-cell Ca_V1 channel displays the activation threshold, inactivation rate, current-voltage relationship, and sensitivity to DHP similar to those observed in the mouse ß-cell (228, 229, 230, 231, 239, 240). The Ca_V1 channel in the rat ß-cell has been well characterized at the single-channel level (223, 241). It shows a large unitary Ba²⁺ conductance (20 pS in 100 mM Ba²⁺), is activated at potentials greater than –30 mV, and displays little inactivation. Its openings are selectively prolonged by the DHP agonist BAY K8644 (223). The whole-cell Ca_V current recording from the RINm5F cell was reported just 2 months after the publication of the first whole-cell Ca_V current recording in the neonatal rat pancreatic ß-cell (226, 242). Thereafter, HVA Ba²⁺ currents have been well characterized in RINm5F cells. Whole-cell Ba²⁺ currents recorded in these cells are activated by voltage pulses to potentials more positive than –40 mV, reach maximal amplitude at around –10 mV, and reverse at about +50 mV. They show little inactivation (243, 244). Maximally 80% of the whole-cell Ca_V currents in RINm5F cells can be blocked by a saturating dose of DHPs (244). The characterization of single L-type Ca_V currents has been carried out in cell-attached and outside-out patches of RINm5F cells (245). Using Ba²⁺ (100 mM) as a charge carrier, single L-type Ca_V currents with little inactivation can be visualized in most patches at potentials between –30 and +30 mV. The Ca_V1 channel blocker nifedipine completely abolishes these single channel currents. In striking contrast, BAY K8644, a Ca_V1 channel opener, drastically prolongs channel open time. Unitary Ba²⁺ conductance of the channel is 21 pS in the absence of BAY K8644 and 24 pS in the presence of BAY K8644 (245). Electrophysiological and pharmacological studies have also revealed L-type Ca_V currents in rat INS-1 cells. The proportion of L-type Ca_V currents in these cells appears to be less than that in RINm5F cells (246, 247, 248, 249).

The L-type Ca_V current has also been well examined in HIT-15T cells, a hamster insulin-secreting cell line. Although there is controversy regarding the subtype of Ca_V channels in HIT-15T cells, most researchers would agree that the Ca_V1 channel is predominant (220). Single Ca_V channel analysis shows that single Ca_V channels in cell-attached patches of HIT-15T cells exhibit clear Ca_V1 channel properties. These channels display a mean conductance of 26 pS when conducting Ba²⁺ (100 mM), activate at potentials positive to –40 mV, and exhibit little inactivation. The DHP agonist BAY K8644 significantly prolongs their openings, and the DHP antagonist nifedipine dramatically decreases their open probability (250). Likewise, the majority of the whole-cell Ca_V currents in HIT-15T cells are sensitive to DHPs. More than 80% of these currents can be blocked by 5 µM nifidipine (250). The whole-cell Ca_V currents in HIT-15T become detectable at about –40 mV and reach their maximal magnitude at about 0 mV. These currents are also characterized by little inactivation (251, 252, 253, 254, 255).

Although a smaller number of Ca_V channel studies have been performed in human islet ß-cells, the data available clearly demonstrate that Ca_V1 channels underlie the principal HVA Ca²⁺ currents (2, 219). Three types of single-channel Ba²⁺ currents have been recorded in cell-attached patches of the human islet ß-cell. One of them resembles L-type Ca_V currents. The channels conducting this type of Ba²⁺ currents show a unitary conductance of 20–22 pS (90 mM Ba²⁺), are activated by depolarizations positive to –30 mV, and exhibit slow inactivation. They are opened much longer when exposed to the DHP agonist BAY K8644 (256). In contrast to rodent islet ß-cells, human islet ß-cells show a greater complexity and remarkable heterogeneity in terms of subtypes of Ca_V currents (244, 257, 258, 259). Whole-cell patch-clamp analysis of Ca_V currents has revealed at least three groups of human ß-cells with different profiles of Ca_V currents. The first and second group are characterized by predominately HVA and LVA Ca²⁺ currents, respectively. The third group displays a mixture of HVA and LVA Ca²⁺ currents. The whole-cell patch-clamp technique in combination with pharmacological manipulation demonstrates that a major proportion of the HVA Ca²⁺ current is L-type. Therefore, the maximal blockade of HVA Ca²⁺ currents in the human preparation by DHP antagonists (10 µM nifedipine, nimodipine, or nitredipine) is more than 80%. The DHP agonist BAY K8644 significantly enhances macroscopic L-type Ca_V currents, measured using the whole-cell patch-clamp technique (244, 257, 258, 259). The Ca_V current recorded from the first group exhibits characteristic features of L-type. It is activated by depolarizations to potentials positive to –40 mV, peaks at between 0 and +20 mV, and reverses at around +60 mV. The inactivation of this current is slow (244, 257, 258, 259).

Interestingly, the genetic ablation of the Ca_V1.3 subunit gene and heterologous expression of Ca_V1.3 channels have clearly demonstrated that Ca_V1.3 channels display distinct features distinguishing them from the classical DHP-sensitive Ca_V channels (260, 261, 262, 263, 264). Ca_V1.3 channels become activated at about –55 mV, which is 20–25 mV more hyperpolarized than the activation threshold of Ca_V1.2 channels. They are significantly less sensitive to DHP (262, 263, 264). This suggests that the lower activation threshold of ß-cell Ca_V1 channels may be attributed to the contribution of Ca_V1.3 channels.

2. ß-Cell Ca_V2 channels.
Although the primary role of Ca_V1 channels in mouse ß-cells has been heavily stressed, the Ca_V1 channel blockers DHP or D-600 cannot fully block Ca_V currents recorded from these cells (228, 229, 236, 265). Therefore, it is not possible to exclude the presence of other types of HVA Ca²⁺ channels such as the Ca_V2.1 channel in mouse ß-cells. Recently, the presence of Ca_V2.1 channels in the mouse islet ß-cell has been verified by pharmacological dissection (236). A mixture of the Ca_V1 channel blocker isradipine and Ca_V2.3 channel blocker SNX482 reduced Ca_V currents recorded from the mouse islet ß-cell by about 80%. A cocktail of isradipine, SNX482, and the Ca_V2.1 channel blocker -Aga IVA almost fully blocked mouse ß-cell Ca_V currents (236).

Rat ß-cells are also equipped with the Ca_V2.1 channels (224). When the rat islet ß-cell is preincubated with the Ca_V1 channel blocker nimodipine, it still exhibits HVA Ca²⁺ currents. These DHP-resistant Ca_V currents rapidly activate at a threshold near –30 mV, peak at +10 mV, and inactivate slowly. These properties are carried by the P/Q-type Ca_V currents in other types of cells. Most importantly, the Ca_V2.1 channel blocker -Aga IVA significantly blocks these DHP-resistant Ca_V currents. This demonstrates that the Ca_V2.1 channel is present in the rat islet ß-cell (224). The P/Q-type Ca_V currents have also been detected in various types of rat insulin-secreting cell lines (243, 245, 246, 247, 266, 267). In RINm5F cells, -Aga IVA dose-dependently inhibits whole-cell Ca_V currents. The P/Q-type Ca_V current contributes to 30% of the total whole-cell Ca_V current in this insulin-secreting cell line according to the inhibition by the maximal dose of -Aga IVA (243). Single channel patch-clamp analysis revealed a unitary Ba²⁺ current in the RINm5F cell pretreated with the Ca_V1 channel blocker nifedipine and the Ca_V2.2 channel blocker -CTX GVIA. This unitary current is characterized by an activation threshold at –10 mV, a slope conductance of 21 pS, little inactivation, and persistent flickering kinetics above 0 mV, i.e., most likely to be of P/Q-type (245). INS-1 cells also display the -Aga IVA-sensitive Ca_V current (246, 247). Treatment with -Aga IVA significantly reduces whole-cell Ca_V currents in these cells (246).

The presence of the Ca_V2.1 channel in human ß-cells was indicated by the fact that a portion of Ca_V currents in human islet ß-cells remained in the presence of both the Ca_V1 channel blocker nifedipine and the Ca_V2.2 channel blocker -CTX GVIA (258). Indeed, about 25% of human ß-cell Ca_V currents have been verified as P/Q-type Ca_V currents by application of -Aga IVA (219).

In earlier patch-clamp studies, the ß-cell Ca_V current kinetics was overemphasized when classifying HVA Ca²⁺ currents. All long-lasting HVA Ca²⁺ currents visualized in the ß-cell were unconvincingly classified as L-type. The selectivity of DHP was also largely overstressed in the classification of HVA Ca²⁺ currents in the ß-cell. DHP blockade was accepted as definitive evidence for the presence of the Ca_V1 channel. The usage of high concentrations of DHPs and the lack of selective antagonists for other types of Ca_V channels delayed the discovery of other types of ß-cell Ca_V currents. It was claimed that the mouse ß-cell possesses only Ca_V1 channels and that the rat and human ß-cell have both Ca_V1 and Ca_V3 channels, before application of peptide toxins from marine snails and spiders in ß-cell Ca_V channel studies (227). -CTX GVIA was first isolated, purified, and sequenced by Olivera et al. (268) in 1984. This 27-amino acid peptide irreversibly blocked stimulus-evoked neurotransmitter release from the frog skeletal neuromuscular junction by preventing Ca²⁺ influx through Ca_V2.2 channels (269). -CTX GVIA was applied in ß-cell Ca_V channel research in the early 1990s (270, 271). This has changed the aforementioned view on ß-cell Ca_V channel subtypes. Emerging evidence suggests that N-type Ca_V currents are present in some insulin-secreting cells (270, 271, 272, 273). However, the identity and function of N-type Ca_V currents are most controversial in insulin-secreting cells (219, 220). The mouse pancreatic ß-cell does not appear to possess Ca_V2.2 channels because application of -CTX GVIA does not affect the mouse ß-cell Ca_V currents (274). Evidence for the presence of Ca_V2.2 channels in the rat pancreatic ß-cell has been obtained using [Ca²⁺]_i measurements. This study revealed that arachidonic acid induced Ca²⁺ influx into purified rat pancreatic ß-cells. The Ca_V1 channel blocker nifedipine only partially blocked this effect. Interestingly, -CTX GVIA decreased arachidonic acid-induced Ca²⁺ influx to an extent similar to that observed by using nifedipine. This indicates that the rat ß-cell Ca_V2.2 channel mediates Ca²⁺ influx induced by arachidonic acid (273). Some groups have detected N-type Ca_V currents in RINm5F, INS-1, and HIT-15T cells (219, 245, 251, 270, 271). Inconsistently, other groups reported that whole-cell Ca_V currents in these insulin-secreting cell lines were insensitive to -CTX GVIA (246, 275, 276). Electrophysiological and pharmacological evidence does not indicate that Ca_V2.2 channels are situated in human ß-cells (244, 258).

The evaluation of ß-cell Ca_V2.3 channels was hindered by the lack of effective experimental approaches. It was not clear whether the Ca_V2.3 channel is present in ß-cells before the development of the Ca_V2.3 channel-selective blocker SNX-482. The presence of the Ca_V2.3 channel in the mouse ß-cell was reported after SNX-482 was made available (40). Whole-cell patch-clamp analysis showed that 60% of the isradipine-resistant Ca_V current in the mouse islet ß-cell is inhibited by SNX-482 (236). The SNX-482-sensitive Ca_V current is also detected in INS-1 cells. The residual currents in INS-1 cells after incubation with isradipine and -conotoxin-MIIC are significantly reduced by addition of SNX-482 (277). However, SNX-482 is capable of blocking other types of Ca_V channels as well (11, 41). Therefore, the blockade by this Ca_V channel blocker only suggests, but cannot ascertain, the identity of the Ca_V2.3 channel in cells containing multiple types of Ca_V channels. Genetic deletion of Ca_V channel genes is one of the most powerful tools in characterization of Ca_V channels. The Ca_V2.3 subunit knockout (Ca_V2.3^–/–) mice have been generated (13, 233, 234). The Ca_V2.3^–/– ß-cell showed a selective reduction in the HVA Ca²⁺ current component during depolarizations in the range from –10 mV to +20 mV in comparison with the wild-type ß-cell. When cells were depolarized to –10 mV, the reduction of Ca_V currents reached approximately 23%. Ca_V currents induced by depolarizations more negative than –10 mV were kept intact in the Ca_V2.3^–/– ß-cell. Furthermore, SNX-482 can no longer exert its action on Ca_V currents in the Ca_V2.3^–/– ß-cell. However, the Ca_V1 channel blocker isradipine still effectively blocks Ca_V currents by approximately 60%. The genetic ablation of the Ca_V2.3 subunit gene in combination with pharmacological manipulations firmly confirms that the Ca_V2.3 channel is present in the mouse islet ß-cell and contributes to the generation of Ca_V currents (13).

3. ß-Cell Ca_V3 channels.
As mentioned before, Ca_V3 channels have been detected in a wide range of cell types, even in nonexcitable cells (Table 1) (2, 3, 30, 42). However, the normal mouse islet ß-cell does not express Ca_V3 channels (2, 219, 220, 227, 236). Interestingly, the occurrence of Ca_V3 channels has been observed in the diabetes-prone mouse islet ß-cells from nonobese diabetic (NOD) mice (16). This indicates that the Ca_V3 channel genes are silenced in the normal mouse islet ß-cells, whereas the diabetes-prone milieu in the NOD mouse islet ß-cell likely switches on the expression of the Ca_V3 channel genes (see Section IV.B).

Rat ß-cells express Ca_V3 channels (2, 223, 240, 241). In rat islet ß-cells, the T-type Ca_V current is not obvious. The activation threshold cannot easily discriminate between T-type Ca_V currents and HVA Ca²⁺ currents. However, the whole-cell T-type Ca_V current in rat islet ß-cells inactivates rapidly and displays characteristic tail currents (see above). These features differentiate the T-type Ca_V current from HVA Ca²⁺ currents in rat islet ß-cells (240). The presence of the Ca_V3 channel in rat islet ß-cells was confirmed at the single channel level (223, 241). The single T-type Ca_V current in rat islet ß-cells is characterized by a unitary Ba²⁺ conductance of 8 pS (in 100 mM Ba²⁺), an activation threshold above –50 mV, openings gathering at the beginning and disappearing rapidly during depolarization, and insensitivity to BAY K8644 (223). RINm5F cells express Ca_V3 channels. Unlike rat islet ß-cells, this rat insulin-secreting cell line displays clear whole-cell T-type Ca_V currents activated by potentials more positive than –60 mV. The whole-cell Ca_V current inactivates rapidly during a depolarizing step from a holding potential of –80 mV to –30 mV following masking HVA Ca²⁺ currents with 20 µM Cd²⁺. An appreciable shoulder appears in the voltage range of the current-voltage curve, reflecting the presence of a population of Ca_V channels activated by low-voltage depolarizing pulses (24). The property of unitary T-type Ca_V currents in RINm5F cells is similar to that in rat islet ß-cells (24, 223). Like RINm5F cells, INS-1 cells show typical whole-cell T-type Ca_V currents evoked by either ramp or step depolarization (218, 246, 278, 279, 280).

Human islet ß-cells have been demonstrated to possess Ca_V3 channels (2, 256, 257, 259). In comparison with rat islet ß-cells, human islet ß-cells exhibit more clear T-type Ca_V currents. The whole-cell T-type Ca_V currents in some human islet ß-cells can be activated at a potential of –60 mV and peak at about –30 mV. They decay very rapidly during depolarization, are blocked by the Ca_V3 channel blocker amiloride, and are sensitive to holding potentials. The T-type component of whole-cell Ca_V currents evoked by depolarizing steps from a holding potential of –100 mV to either –30 or 0 mV completely disappears when the holding potential is changed to –50 mV (256, 257, 259). Unitary T-type Ca_V current analysis has also been performed in cell-attached patches of human islet ß-cells. The single Ca_V3 channel in these cells has a unitary Ba²⁺ conductance ranging from 8–10 pS in 90 mM Ba²⁺ and opens less frequently when the holding potential is increased (256).

B. Molecular components of ß-cell Ca_V channels
Although all known physiological types of Ca_V currents have been observed in ß-cells by combining electrophysiological techniques and pharmacological tools, the corresponding molecular identities mediating these currents are not completely understood, especially in islet ß-cells (2). The islet as a microorgan is embedded in the pancreas and contains four types of endocrine cells as well as nerve endings, blood cells, and capillaries. It is more difficult to isolate islets than just dissect homogeneous tissues such as muscle, fat, and neuronal tissue. These features hamper application of conventional molecular and biochemical approaches to identify ß-cell Ca_V channel genes, transcripts, and proteins. Although some Ca_V channel mRNAs and proteins have been revealed in islet tissues, they do not necessarily localize only in ß-cells. Therefore, caution is needed when interpreting the results from islet tissues. However, information obtained from islet tissues is still valuable when properly combined with results from studies at the resolution of single cells, for example, immunostaining, in situ hybridization, and electrophysiology. Furthermore, some Ca_V channel mRNA and protein isoforms have been identified in relative pure islet ß-cells obtained by fluorescence-activated cell sorting. In this context, well-refined approaches, such as optical tweezers and single-cell PCR, are encouraged for reevaluation and identification of Ca_V channel mRNA and protein isoforms in islet ß-cells. The following discusses available data, including those obtained from islet tissues.

The presence of multiple types of Ca_V currents in ß-cells indicates that ß-cell Ca_V channels are heterogeneous (2, 219, 220). Some ß-cell Ca_V channel subunit genes have been cloned from either pancreatic islets or insulin-secreting cell lines (84, 281, 282, 283). The ß-cell type Ca_V1.3 subunit cDNA CACNA1D (also known as CACH3, CACN4, CACNL1A2, or CCHL1A2) has been isolated from human pancreatic islets (84). Northern blot analysis demonstrates that this gene selectively expresses in pancreatic islets, brain, and RINm5F cells, among the numerous tissues tested. Furthermore, the CACNA1D transcript is clearly visualized in rat islet ß-cells by in situ hybridization analysis (84). The CACNA1D structure has been characterized (284). This gene spans more than 155 kb in chromosome 3p14.3 and comprises 49 exons whose sizes range from 27 bp (exon 44) to more than 519 bp (exon 49). Its exon-intron organization suggests that each transmembrane homologous repeat tends to be encoded by a single exon. The L_I-II, L_II-III, and C-terminal regions are encoded by 5, 5, and 13 exons, respectively (284). The predicted amino acid sequence shows that CACNA1D encodes a 2181-amino acid protein where four transmembrane homologous repeats are well conserved. Positively charged amino acids (arginine or lysine), believed to act as a voltage sensor, are distributed at every third position in the S4 segment of each transmembrane homologous repeat. N and C termini as well as L_I-II and L_II-III are unique regions. Further analysis revealed that this subunit contains three potential N-glycosylation sites, 11 potential PKA phosphorylation sites, nine potential PKG phosphorylation sites, and a potential PKC phosphorylation site (84).

In addition to human ß-cell CACNA1D, two isoforms, cacna1d1 and cacna1d2, of rat ß-cell cacna1d have been isolated from the insulin-secreting RINm5F cell. cacna1d1 encodes a 2203-amino acid protein. Ninety-five percent of cacna1d1 is identical to human CACNA1D. In cacna1d1, four transmembrane homologous repeats, including the putative voltage sensor, are highly conserved. cacna1d2 lacks 535 amino acids in the C-terminal region, likely due to alternative splicing (283, 285). The Xenopus oocyte injected with cRNA for cacna1d1 and cRNA for cacnb1 expresses functional Ca_V1.3 channels (285). CHO cells stably expressing cacna1d1 or cacna1d2 together with cacnb2 have been established. They display appreciable L-type Ca_V currents sensitive to BAY K8644 (283).

The HIT cell Ca_V1.3 subunit cDNA cacna1d (HCa3A) has also been cloned. The deduced amino acid sequence showed that the HIT cell Ca_V1.3 subunit consists of 1610 amino acids and shares 96% homology with the human ß-cell Ca_V1.3 subunit. Its C-terminal 15 amino acids are unique. The HIT cell Ca_V1.3 subunit lacks 20 amino acids in its L_I-II and has a considerably longer C terminus, compared with the human ß-cell Ca_V1.3 subunit. This indicates that they are splice variants of a common gene in these two species (84, 282). The HIT cell Ca_V1.3 subunit contains multiple potential phosphorylation sites and three N-glycosylation sites. The HIT cell Ca_V1.3 subunit gene expresses in hamster tissues (pancreas, brain, heart, and skeletal muscle), rat tissues (pancreas and brain), and islet cell lines (HIT, INR1-G9, STC-1, BTC-3, RIN 1056A, and RIN 1056C) (282). Functional expression of the HIT cell Ca_V1.3 subunit has been performed in Xenopus oocytes. The HIT cell Ca_V1.3 together with Ca_V2 and Ca_Vß3 expresses well and mediates typical L-type Ba²⁺ currents (282, 286).

The cDNA and amino acid sequences of Ca_V3.1 subunits from an insulin-secreting cell line INS-1 have been determined. The INS-1 Ca_V3.1 subunit cDNA cacna1g predicts a 2288-amino acid protein, which shares 96.3% identity to the neuronal isoform of Ca_V3.1 subunit. Only a single amino acid (G1667) substitution was found in the four transmembrane homologous repeats of the INS-1 Ca_V 3.1 subunit. The L_I-II is another highly conserved region. Three unique regions of the INS-1 Ca_V3.1 subunit are located at the N terminus (aa 1–34), L_II-III (aa 971–994), and L_III-IV (aa 1570–1588). Analysis of the genomic DNA sequence of the rat cacna1g indicates that the INS-1 Ca_V3.1 subunit is an alternative splice variant of the rat Ca_V3.1 subunit. The INS-1 Ca_V3.1 gene transcript was found in INS-1 cells and rat tissues, including pancreatic islets, heart, kidney, and brain. Typical T-type Ca_V currents were visualized in Xenopus oocytes injected with cRNA for the INS-1 cacna1g (281).

As listed in Table 2, multiple Ca_V subunit mRNAs and proteins have been identified in insulin-secreting cells or islets (12, 84, 218, 224, 235, 236, 246, 277, 280, 281, 287, 288, 289, 290, 291, 292, 293, 294). The Ca_V1.2 and in particular Ca_V1.3 subunit mRNAs and proteins are predominant in all tested insulin-secreting cells and islets (2). The Ca_V1.2 and Ca_V1.3 subunit mRNAs are detected in ß-cells from any tested species (Table 2) (12, 22, 84, 236, 246, 287, 288, 290, 292, 294). The Ca_V1.2 subunit protein has been identified in mouse islet ß-cells as well as HIT-15T, RINm5F, MIN6, and ßTC-3 cells (Table 2) (288, 289, 291, 293, 294). The Ca_V1.3 subunit protein is present in mouse islet ß-cells, INS-1, and RINm5F cells (Table 2) (12, 211, 218, 292). For the rest of the Ca_V1 subunits, there are obvious interspecies differences (Table 2). Table 2 lists Ca_V1 subunit mRNAs and proteins in islet ß-cells and insulin-secreting cell lines from different species. The Ca_Vß2 and Ca_Vß3 subunit mRNAs have been revealed in rat pancreatic islets. Competitive reverse transcription-PCR demonstrated that the Ca_Vß2 subunit mRNA level is much greater than the Ca_Vß3 subunit mRNA level, suggesting that the Ca_Vß2 subunit is predominant in rat pancreatic islets (17). We have visualized both Ca_Vß2 and Ca_Vß3 subunits at the protein level in mouse islets (295). It is not known whether the Ca_V subunit is expressed in the ß-cell. The Ca_V22 subunit mRNA has been identified in the human pancreas (133).

C. Ca_V channel regulation in insulin secretion
The ß-cell Ca_V channel exerts insulinotropic effects in many critical ways. The ß-cell Ca_V channel-mediated Ca²⁺ entry directly stimulates secretory granule trafficking and triggers insulin exocytosis. This is the paramount important way whereby ß-cell Ca_V channels regulate insulin secretion (2, 160). In addition to the direct effects on insulin secretory granules, the ß-cell Ca_V channel-mediated Ca²⁺ influx significantly contributes to the maintenance of ß-cell mass and function. For example, Ca²⁺ entry through ß-cell Ca_V channels plays a prominent role in insulin gene transcription, protein phosphorylation, mitosis, proliferation, and differentiation (for details, see Section III.D) (4, 12, 13, 14, 15, 296, 297). Furthermore, ß-cell Ca_V channel subunits can also act as nonchannel proteins to indirectly regulate the insulin secretory process. This can be exemplified by the enhanced insulin secretion from ß-cells lacking Ca_Vß3 subunits. The Ca_V ß3 subunit functionally crosstalks with the Ca²⁺ mobilization machinery. By this way, the Ca_Vß3 subunit functions as a brake for intracellular Ca²⁺ release, thus dampening insulin secretion (see Section III.E.2) (295). In this section, we focus on direct regulation by ß-cell Ca_V channels of insulin secretory granule trafficking and exocytosis with emphasis on glucose-stimulated insulin secretion.

1. Dynamics of insulin secretion.
Insulin secretion from the ß-cell is a complex process, which is precisely controlled by a molecular network with Ca_V channels as pivotal elements (11). Glucose-stimulated insulin secretion relies on not only common exocytotic machinery but also ß-cell-characteristic mechanisms. The ß-cell is exquisitely sensitive to glucose. Upon elevation of the plasma glucose level, the ß-cell efficiently takes up glucose through glucose transporters. Thereafter, subsequent glucose metabolism brings about the activation of a series of signal transduction events. A well-known paradigm is that an increase in the ATP/ADP ratio derived from glucose metabolism closes K_ATP channels, resulting in depolarization of the plasma membrane. The membrane depolarization in turn opens Ca_V channels, mediating Ca²⁺ influx. The resultant increase in [Ca²⁺]_i triggers direct interactions between exocytotic proteins situated in the insulin-containing granule membrane and those localized in the plasma membrane. Eventually, the interaction between exocytotic proteins initiates the fusion of insulin-containing granules with the plasma membrane, i.e., insulin exocytosis (see Figs. 5 and 7) (2, 11). There is no doubt that this K_ATP channel-dependent pathway plays a central role in the ß-cell stimulus-secretion coupling. However, elimination of this pathway does not entirely block glucose-stimulated insulin secretion. This observation has led to several significant discoveries of novel mechanisms of glucose-stimulated insulin secretion, which constitute a K_ATP channel-independent pathway (298). For example, high glucose together with both PKA and PKC activators appreciably stimulates insulin secretion from the ß-cell even under conditions where there is neither Ca²⁺ influx through the plasma membrane nor Ca²⁺ mobilization from intracellular stores (299). These K_ATP channel-dependent and K_ATP channel-independent mechanisms operate in a concerted manner guaranteeing dynamically adequate release of insulin to maintain normoglycemia (300).

View larger version (46K): [in this window] [in a new window]   FIG. 5. A scheme illustrating the regulation of dynamic insulin secretion by CaV channels. Glucose-stimulated insulin secretion is characterized by a rapid first phase of insulin release for about 10 min, followed by a nadir, and subsequently a gradually increasing second phase reaching a plateau after 25 to 30 min (inset). Insulin-containing granules (IG) are functionally divided into the reserve pool (RP) and the RRP/IRP. The KATP channel-dependent mechanisms trigger first-phase insulin secretion from the RRP/IRP by opening CaV1.2 and CaV1.3 channels. The KATP channel-independent mechanisms underlie second-phase insulin secretion by recruiting insulin-containing granules from RP to RRP/IRP. The Ca2+ influx through ß-cell CaV1.2, CaV1.3, CaV2.2, and CaV2.3 channels are involved in second-phase insulin secretion. CaV1.2/1.3, CaV1.2 and CaV1.3 channels; CaV1.2/1.3/2.2/2.3, CaV1.2, CaV1.3, CaV2.2, and CaV2.3 channels; GLUT, glucose transporter; KATP, ATP-sensitive potassium channels; , depolarization.

View larger version (57K): [in this window] [in a new window]   FIG. 7. A scheme illustrating the role of CaV channels in pancreatic ß-cell physiology and pathophysiology, summarizing the mechanisms of ß-cell CaV channel regulation. ß-Cell CaV channels take center stage in insulin secretion and play an important role in ß-cell development, survival, and growth by mediating physiological Ca2+ entry. ß-Cell CaV channels are regulated by a wide range of mechanisms, either shared by other cell types or specific to ß-cells to satisfy the needs of this cell for Ca2+ under different conditions. The dysregulated CaV channels either overload or underload ß-cells with Ca2+, causing ß-cell dysfunction and even death manifested in both type 1 and type 2 diabetes. Ca2+/CaM, Ca2+/calmodulin; CAC, citric acid cycle; G, GTP-binding protein; GMS, glucose metabolism-derived signal; GPCR, GTP-binding protein coupled receptor; IG, insulin-containing granule; InsP3R, InsP3 receptor; P, phosphoryl group; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC: phospholipase C; PPase, protein phosphatase; RyR, ryanodine receptor; TKR, tyrosine kinase receptor; , depolarization.

Great efforts have been made to understand the cellular mechanisms responsible for biphasic insulin secretion. Electron microscopy revealed that approximately 10,000 insulin-containing granules are distributed in a mouse islet ß-cell. Among the approximately 10,000 granules, about 600 are docked just beneath the plasma membrane, and about 2,000 localize within an area approximately 0.2 µm from the plasma membrane (309). Further refined functional analysis divided these granules into distinct functional pools. Only 50 to 100 docked granules, depending on the experimental conditions, are immediately available for exocytosis. This group of granules is defined as the readily releasable pool/the immediately releasable pool (RRP/IRP). The majority of granules are not immediately available for release and belong to the reserve pool (RP). As a matter of fact, the RRP/IRP size correlates well with the amount of insulin released during the first phase. Biphasic insulin secretion is most likely attributed to the existence of these two functional pools of insulin-containing granules. It is believed that glucose at first quickly empties the RRP/IRP by opening Ca_V1 channels causing the first phase of insulin secretion. Simultaneously, it also activates a series of intracellular signals such as ATP and Ca²⁺ to prime insulin secretory granules from the RP to the RRP/IRP in a time- and temperature-dependent manner. Release of the subsequently primed granules proceeds at a much lower rate leading to the second phase of insulin secretion (Fig. 5) (160, 309).

2. Regulation of insulin secretion by Ca_V channels.
Early studies convincingly illustrated that glucose simultaneously stimulates ⁴⁵Ca²⁺ uptake by and insulin release from pancreatic ß-cells (310, 311). They also showed that glucose-stimulated insulin release requires the presence of extracellular Ca²⁺, is blocked and facilitated by Ca_V channel antagonists and agonists, respectively, and is partially mimicked by depolarization with sulfonylurea compounds and high K⁺. All these observations indicate that Ca_V channels play an important role in pancreatic ß-cell stimulus-secretion coupling (310, 311). Now it is clear that the frequency of Ca²⁺-dependent action potentials in the pancreatic ß-cell deciphers information on fuel metabolism and determines the degree of insulin secretion (160, 227, 312, 313). Pancreatic ß-cells as paraneurons are equipped with neuronal protein families such as a similar set of exocytotic proteins and a rich assortment of Ca_V channels including Ca_V1.2, Ca_V1.3, Ca_V2.1, Ca_V2.2, Ca_V2.3, and Ca_V3.1 channels. All types of ß-cell Ca_V channels are likely to be involved in insulin secretion (2, 314).

There is now consensus that the Ca_V1 channel is the major Ca_V channel type playing a predominant role over other types of Ca_V channels in Ca²⁺-triggered insulin exocytosis (2). Pharmacological experiments demonstrate that 60–80% of glucose-induced insulin secretion from mouse, rat, and human islets, where ß-cells are equipped with various types of Ca_V channels, is attributed to Ca²⁺ influx through the Ca_V1 channel (236, 258, 315). Like in pancreatic islet ß-cells, Ca²⁺ entry through Ca_V1 channels also plays a major role in triggering insulin exocytosis in insulin-secreting cell lines (2, 219, 220). The role of Ca_V1 channels in dynamic insulin secretion has been extensively investigated (236, 258, 310, 315). An early study showed that the Ca_V1 channel blocker verapamil selectively inhibited the second phase of glucose-stimulated insulin secretion (316). However, more and more experimental data demonstrate that Ca_V1 channels participate in the regulation of both phases of insulin secretion and even play a more prominent role in triggering insulin release during the first phase in the mouse islet (Fig. 5) (236, 258, 315). For example, three Ca_V1 channel antagonists, nifedipine, diltiazem, and verapamil, significantly decreased both the first and the second phase of glucose-induced insulin release from perifused rat islets (315). The Ca_V1 channel agonist BAY K8644 dramatically enhanced glucose-stimulated insulin secretion at both the first and second phases in human perifused islets (258). Furthermore, perifused islets from Ca_V1.2 subunit knockout (Ca_V1.2^–/–) mice display a drastic reduction in first phase insulin secretion. Capacitance analysis showed that the Ca_V1.2^–/– selectively impairs the initial rapid component of insulin exocytosis (236).

There are two subtypes of Ca_V1 channels, Ca_V1.2 and Ca_V1.3 channels, in the ß-cell (12, 211, 212, 236). The distinct contribution of Ca_V1.2 and Ca_V1.3 subtypes to insulin exocytosis has not been thoroughly studied in different species and remains controversial. In rat ß-cells, the level of Ca_V1.3 subunit mRNA is 2.5 times higher than that of Ca_V1.2 subunit mRNA (22). Mouse ß-cells lacking Ca_V1.2 subunit exhibit a decrease in Ca_V currents by about 45%, an inhibition of first phase insulin secretion by about 80%, and glucose intolerance. Ca_V1 channel blockers had no effect on Ca_V channel currents and insulin release from ß-cells lacking Ca_V1.2 subunits (236). Furthermore, previous studies showed negative Ca_V1.3 subunit-like immunoreactivity in mouse pancreatic ß-cells (288). Those results led to the conclusion that only Ca_V1.2 subunits conduct L-type Ca_V currents in mouse pancreatic ß-cells and play a crucial role in stimulus-secretion coupling. However, the presence of Ca_V1.3 subunit mRNAs and proteins in mouse pancreatic ß-cells has been clearly demonstrated by other groups (12, 211). Additionally, Ca_V1.3 subunit knockout (Ca_V1.3^–/–) mice displayed a compensatory overexpression of Ca_V1.2 subunit proteins in ß-cells (12). Electrophysiological analysis showed that there was no difference in either total voltage-gated Ba²⁺ current density or L-type current density between mutant and control cells. However, the biophysical properties of L-type Ca_V currents in Ca_V1.3 subunit-deficient ß-cells were significantly altered. The current-voltage relationship of the mutant ß-cells was shifted by about 10 mV toward more positive potentials at the lower voltage range (12). Furthermore, mutant islets secreted less insulin than control islets in the presence of 3 mM glucose. However, insulin secretion from mutant islets was similar to that from control islets when subjected to 6 mM or higher concentrations of glucose. These data indicate that overexpression of Ca_V1.2 subunits indeed compensates for the loss of Ca_V currents conducted by Ca_V1.3 subunits and thereby maintains insulin secretion capacity (12). Hence, ß-cell Ca_V1.3 subunits in wild-type mouse ß-cells are likely to play an important role in basal insulin secretion and also in stimulus-secretion coupling at the lower range of glucose concentrations (12). Apparently, the distinct contribution of Ca_V1.2 and Ca_V1.3 subtypes to insulin exocytosis remains to be elucidated.

The involvement of Ca_V2.1 channels in glucose-stimulated insulin secretion from rat ß-cells has been demonstrated by electrophysiological and pharmacological means (224). The Ca_V2.1 channel blocker -Aga IVA partially blocks HVA Ca²⁺ currents in the rat ß-cell and inhibits the DHP-resistant component of glucose-induced insulin secretion by about 30% (224). It is clear that the Ca_V2.1 channel in the human ß-cell exerts a prominent effect in stimulus-secretion coupling. When the human ß-cell is exposed to -Aga IVA, it displays about 65% reduction in the glucose-induced secretory response (219). The role of Ca_V2.1 channels in the regulation of insulin secretion from mouse ß-cells remains to be examined. It has been demonstrated that Ca_V2.1 channels play an important role in stimulus-secretion coupling of rat insulin-secreting RINm5F cells (219).

The role of Ca_V2.2 channels in insulin exocytosis is controversial. Some experiments showed that the Ca_V2.2 channel blocker -CTX GVIA had no effect on glucose-dependent insulin secretion in human islets (258). However, others demonstrated that an appreciable inhibition occurred by this compound in this type of islet preparation (219). Furthermore, -CTX GVIA indeed promoted a measurable inhibition of the second phase glucose-induced insulin secretion from rat islets. In contrast, first phase insulin secretion and high K⁺-evoked insulin exocytosis, critically depending on Ca²⁺ influx, were intact in the -CTX GVIA-treated islets. Therefore, it was speculated that the impairment in the second phase of glucose-induced insulin secretion by -CTX GVIA was due to toxic effects on the secretory machinery rather than blockade of Ca_V2.2 channels (272). However, this speculation neglected the possibility that Ca²⁺ entry through ß-cell Ca_V2.2 channels does not trigger insulin exocytosis per se, but rather facilitates generation of signal(s) critical for second phase insulin secretion. It has been proposed that an increase in glucose concentration leads to a limited initial increase in cytosolic ATP, which then results in sequential events, K_ATP channel closure, Ca_V channel opening, and first phase insulin secretion. In addition to triggering first phase insulin secretion, Ca²⁺ influx through ß-cell Ca_V channels also subsequently facilitates mitochondrial metabolism to produce more ATP, which specifically regulates second phase insulin secretion in a K_ATP channel-independent manner (160, 317). Indeed, further inspection of the effect of -CTX GVIA on second phase insulin secretion demonstrated that -CTX GVIA significantly reduced the ATP/ADP ratio (315). It is plausible to envisage that Ca²⁺ entry through Ca_V2.2 channels plays an important role in Ca²⁺-dependent glucose metabolism, thereby facilitating later phase production of ATP, which is critical for second phase insulin secretion (Fig. 5) (160, 317). However, it should be noted that Ca²⁺ regulation of mitochondrial metabolism is complex. For example, the oxidative phosphorylation inhibitors carbonyl cyanide m-chlorophenylhydrazone and sodium azide increase [Ca²⁺]_i but decrease mitochondrial membrane potential and ATP generation in pancreatic ß-cells (318). It is worthwhile to note that Ca²⁺ entry through Ca_V2.2 channels in insulin-secreting RINm5F and INS-1 cells is likely to directly trigger insulin exocytosis. It has been demonstrated that -CTX GVIA significantly inhibits Ca_V currents and insulin secretion in these insulin-secreting cell lines, stimulated not only by glucose but also by high K⁺ (219, 271).

It has been difficult to evaluate the role of Ca_V2.3 channels in the regulation of insulin secretion. The problem has been solved by the application of the Ca_V2.3^–/– mouse model and the Ca_V2.3 channel selective peptide blocker SNX-482 (11, 13, 40, 234). Experimental evidence has demonstrated that Ca²⁺ entry through Ca_V2.3 channels regulates insulin secretion from both the pancreatic ß-cell line INS-1 and primary mouse ß-cells (11, 13, 234, 247, 277). Initially, it was found that Ca_V2.3^–/– did not alter ß-cell mass and insulin content. However, Ca_V2.3-deficient mice exhibited disturbances in glucose tolerance and insulin secretion as well as hyperglycemia. Unfortunately, dynamic insulin granule exocytosis and phasic insulin secretion from Ca_V2.3^–/– ß-cells were not measured in this initial study (234). Later, capacitance analysis revealed that SNX-482 dramatically inhibited the late component of depolarization-induced exocytosis without significant effect on the exocytotic response to the first depolarization in mouse pancreatic ß-cells (236). Similar to the treatment with SNX-482, the deletion of the Ca_V2.3 subunit gene selectively suppressed the late component without influencing the early component of the depolarization-induced capacitance responses (13). These results indicate that Ca_V2.3 channels may selectively control second phase insulin secretion. Indeed, phasic insulin secretion analysis revealed that either genetic deletion of the Ca_V2.3 subunit gene or pharmacological ablation of the Ca_V2.3 channel with SNX-482 significantly impaired second phase glucose-stimulated insulin secretion. These manipulations did not affect first phase insulin secretion. The above results demonstrate that Ca²⁺ entry through Ca_V2.3 channels is selectively coupled to second phase insulin secretion (Fig. 5) (13, 236). It has been proposed that unlike the ß-cell Ca_V1 channels, which are tightly coupled to the exocytotic machinery, the Ca_V2.3 channel seems to be distant from exocytotic sites in the ß-cell. The Ca_V2.3 channel-mediated Ca²⁺ influx may mainly recruit insulin-containing granules from the RP to the RRP/IRP to regulate second phase insulin secretion (11, 13). Interestingly, a 20% decrease in integral [Ca²⁺]_i, a 30% decrease in [Ca²⁺]_i oscillation frequency, and a 50% decrease in insulin secretion occurred in Ca_V2.3^–/– islets. This indicates that both the amount of [Ca²⁺]_i and the [Ca²⁺]_i oscillation frequency affect insulin secretion, especially at the second phase. This is strongly supported by the presence of the Ca²⁺-dependent adenylyl cyclase (AC) and phospholipase C in pancreatic ß-cells and the important roles of cAMP and diacylglycerol (DAG) produced by these enzymes in second phase insulin secretion (11, 13).

Experimental evidence suggests that the ß-cell Ca_V3 channel is likely to be a player in stimulus-secretion coupling. The Ca_V3 channel blocker NiCl₂ dose-dependently inhibits insulin secretion from INS-1 cells (278). Flunarizine, a nonselective antagonist of Ca_V1 and Ca_V3 channels, significantly reduced both glucose- and K⁺-induced insulin secretion in perifused rat islets (315). It is intriguing to investigate whether Ca_V3 channels are involved in insulin secretion induced by glucose stimulation. The role of the Ca_V3 channel in insulin secretion from human islet ß-cells is not known. It would be attractive to evaluate the possible contribution of the Ca_V3 channel to stimulus-secretion coupling in human islet ß-cells.

D. Effect of Ca_V channels on ß-cell development, survival, and growth
Ca²⁺ influx through ß-cell Ca_V channels could play an important role in ß-cell development, survival, and growth where a number of elementary cellular events, such as gene transcription, protein phosphorylation, mitosis, cell proliferation, and differentiation, are Ca²⁺-dependent (see Fig. 7) (4). Ca_V1 subunit knockout mouse models display overt defects in ß-cell development (12, 13). The Ca_V1.3^–/– mouse illustrates that the Ca_V1.3 channel is needed in postnatal pancreatic ß-cell generation. At birth, Ca_V1.3^–/– mice are smaller than wild-type mice, but both mice have an equivalent number of pancreatic islets when normalized by body weight. However, both the number and the size of islets in adult Ca_V1.3^–/– mice drastically decrease due to the reduced postnatal ß-cell generation. This is supported by the fact that there is no difference in ß-cell death between the knockout and control mice, whereas the proliferation rate of Ca_V1.3^–/– ß-cells drastically slows down (12). It is of particular interest that Ca_V2.3^–/– markedly retards islet cell differentiation. This phenotype may reflect the involvement of Ca_V2.3 channel-mediated Ca²⁺ influx in ß-cell differentiation (13). It is reasonable to assume that the Ca_V2.3 channel-mediated Ca²⁺ influx is likely to drive the expression of some genes critical for ß-cell differentiation. However, possible Ca_V1.3^–/– or Ca_V2.3^–/–-caused alteration in neuronal function and/or innervation of the islet may also contribute to the failure of ß-cell development. In addition, pharmacological manipulation of Ca_V channel opening and closure significantly affects ß-cell survival and proliferation. For example, the Ca_V1 channel blockers D-600 and diltiazem evidently inhibit ß-cell proliferation (14, 15). Depolarization and hyperpolarization of ß-cells with the selective K_ATP channel blocker glibenclamide and opener diazoxide significantly facilitate DNA synthesis and impede ß-cell growth, respectively (14).

Ca_V channel-dependent regulation of ß-cell development, survival, and growth is based on maintenance of expression of individual genes in the ß-cell. Expression of numerous genes in the ß-cell is critically dependent on Ca²⁺ influx through Ca_V channels (297, 319, 320, 321). It has been demonstrated that the glucose-stimulated expression of the insulin gene, the most specific ß-cell gene, relies on Ca²⁺ influx through ß-cell Ca_V channels. The Ca_V channel blockers, such as D-600 and verapamil, effectively prevent glucose-induced stimulation of insulin gene transcription (297, 319). The islet amyloid polypeptide amylin cosecretes with insulin and participates in normal regulation of glucose metabolism, although it was originally isolated from type 2 diabetic pancreas and is also involved in insulin resistance in skeletal muscle. Glucose stimulates amylin gene transcription in the ß-cell. This glucose-induced transcription is abolished by the Ca_V1 channel blocker verapamil (320). Ca²⁺ influx through Ca_V channels is also involved in the regulation of inositol 1,4,5-trisphosphate (InsP₃) receptor gene expression. The Ca_V1 channel blocker nimodipine completely blocks the effect of PKA activation on the expression of InsP₃ receptor type II and III genes (321).

Great efforts have been made to understand the molecular mechanisms underlying regulation of gene expression in the ß-cell by the Ca_V channel-mediated Ca²⁺ influx. Like in other types of cells, numerous protein kinases either require Ca²⁺ for their activation or work in concert with Ca²⁺ to regulate gene expression in the ß-cell. The MAPKs are very important serine/threonine protein kinases for controlling cell proliferation and differentiation and adapting the cell to its environment (322). It has been demonstrated that Ca²⁺-influx through Ca_V channels is critical to mediate activation of the MAPKs p42 and p44, also called extracellular-signal-regulated kinases 2 and 1 (ERK2 and ERK1), in insulin-secreting cells by physiological stimuli, e.g., glucose and glucagon-like peptide-1 (296, 323). Physiological concentrations of glucose rapidly activate these two MAPKs in the insulin-secreting MIN6 cells and subsequently translocate them to the nucleus of these cells. Depolarization of the MIN6 cells with either glibenclamide or high K⁺ mimics the effect of glucose on the MAPKs. Furthermore, glucose, glibenclamide, or high K⁺ can no longer activate these MAPKs when extracellular Ca²⁺ is depleted with the Ca²⁺ chelator EGTA. More importantly, activation of ERK1 and ERK2 can be prevented and mimicked by the Ca_V1 channel antagonist nifedipine and agonist BAY K8644, respectively. However, a global increase in [Ca²⁺]_i, induced by inomycin treatment, has no effect on ERK activity. This indicates that Ca²⁺ entry through Ca_V1 channels plays a specific role in ERK activation in these cells (296, 323). Given the involvement of Ca_V1 channels, the question that immediately arises is how Ca²⁺-influx through Ca_V1 channels mediates the glucose-induced activation of the MAPKs in the MIN6 cells. It is well-known that PKC is a Ca²⁺-dependent protein kinase, is activated by glucose in the ß-cells and stimulates ß-cell replication (11, 324). Either acute inhibition or chronic down-regulation of PKC ablates the activation of the MAPKs in response to glucose, whereas these pharmacological manipulations do not affect the depolarization-induced activation of the MAPKs. This indicates that PKC is involved in glucose-induced activation of the MAPKs, but does not mediate Ca²⁺-dependent activation of these protein kinases in the MIN6 cells. Interestingly, the protein tyrosine kinase inhibitor genistein suppresses the activation of the MAPKs by glucose or high K⁺. Moreover, the protein tyrosine phosphatase inhibitor vanadate stimulates the MAPKs in a manner dependent on Ca²⁺ entry through Ca_V1 channels, because the stimulatory effect of vanadate disappears in the absence of extracellular Ca²⁺ or in the presence of nifedipine. These data show that the Ca_V1 channel-mediated Ca²⁺ entry activates the MAPKs in the MIN6 cells through activation of tyrosine kinases and/or inhibition of tyrosine phosphatases (325).

The glucagon-like peptide receptor in the ß-cell is a major target for glucagon-like peptide-1, a peptide hormone secreted from intestinal L cells in response to oral ingestion of nutrients such as carbohydrates and fats. Glucagon-like peptide-1 executes multiple actions on the ß-cell including proliferation, differentiation, insulin gene transcription, and the facilitation of stimulus-secretion coupling (326). The effects of glucagon-like peptide-1 on proliferation, differentiation, and insulin gene expression are likely to be mediated by MAPKs. It has been demonstrated that glucagon-like peptide-1 glucose-dependently activates the MAPK ERK1 and ERK2 in MIN6 cells. The activation depends strictly on Ca²⁺ entry through Ca_V1 channels but not a global increase in [Ca²⁺]_i. The data suggest that the mode of Ca²⁺ entry is important in ERK activation by glucagon-like peptide-1. It is well known that [Ca²⁺]_i signals through interaction with Ca²⁺ binding proteins, e.g., calmodulin. This Ca²⁺ binding protein is very rich in the ß-cell and plays an important role in DNA synthesis and gene transcription through activation of CaMKII. This Ca²⁺-dependent kinase likely bridges Ca²⁺ entry through Ca_V1 channels and the glucagon-like peptide-1-induced activation of ERK1 and ERK2 in MIN6 cells. Indeed, stimulation with glucagon-like peptide-1 simultaneously increases CaMKII and ERK activity (323).

Given the specific role of the Ca_V1 channel-mediated Ca²⁺ entry in the activation of ß-cell MAPKs, it is reasonable to envision that all factors able to open ß-cell Ca_V1 channels or increase their activity may affect ß-cell development, survival, and growth. Only a limited number of factors have been examined so far. A large amount of work on the regulation of ß-cell development, survival, and growth by Ca_V channels remains to be performed.

E. Novel molecular networks of ß-cell Ca_V channels
Cellular proteins perform their activities in concert in complex molecular networks on the basis of protein-protein interaction. This constitutes a plethora of cellular signaling pathways. Ca_V channel subunits not only form Ca²⁺ conducting pores in the plasma membrane, but also interact with many other proteins to form complex molecular networks (Fig. 6). In these complex molecular networks, Ca_V channels no longer respond only to voltage depolarization but also are modulated by their interacting partners. Surprisingly, Ca_V channel subunits can even function as nonchannel proteins by crosstalking with other signaling molecules. For example, a short splice variant of the Ca_Vß4 subunit enters the nucleus where it directly interacts with the nuclear protein chromobox protein 2 and regulates gene silencing (327). The following discusses two Ca_V channel networks in the pancreatic ß-cell, i.e., the Ca_V1 channel-exocytotic protein network and the Ca_Vß3 subunit-intracellular Ca²⁺ store network.

View larger version (43K): [in this window] [in a new window]   FIG. 6. A scheme illustrating the CaV channel-centering molecular networks in the ß-cell. The ß-cell CaV1 channel complexes with syntaxin 1A and SNAP-25 to form a functional molecular network. This network serves both as a fine-tuning mechanism of ß-cell CaV1 channel function and an anchoring machinery to optimally organize this channel at the site of insulin exocytosis. The CaVß3 subunit is not a required building block of ß-cell CaV channels. Instead, it crosstalks with the intracellular Ca2+ release machinery to form a CaVß3 subunit-intracellular Ca2+ store network. This network negatively regulates intracellular Ca2+ release from InsP3-sensitive stores. G, GTP-binding protein; GPCR, GTP-binding protein coupled receptor; IG, insulin-containing granules; InsP3R, InsP3 receptor; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C.

Fluorescence microscopy in conjunction with deconvolution analysis reveals that the expressed Ca_V1.3 subunit-enhanced GFP and enhanced blue fluorescent protein-syntaxin 1 targeted to and colocalized in the ß-cell plasma membrane. Furthermore, subcellular fractionation showed that the endogenous Ca_V1.3 subunit and syntaxin 1A also coresided in the ß-cell plasma membrane fractions. This opened up the possibility that syntaxin 1A might interact with the Ca_V1.3 subunit in the pancreatic ß-cell, and this was subsequently found to be the case. Indeed, the polyclonal antibody against the intracellular domain of syntaxin 1A efficiently coimmunoprecipitated the Ca_V1.3 subunit from the ß-cell plasma membrane fractions. These data strongly suggest that syntaxin 1A forms a complex with the Ca_V1.3 subunit (211). Furthermore, this physical association of the Ca_V1.3 subunit with syntaxin 1A gives rise to clear functional consequences. Anti-syntaxin 1A antibody interference with the formation of a syntaxin 1A/Ca_V1.3 subunit complex not only makes ß-cell Ca_V1 channel activity drastically run down, but also impairs insulin exocytosis independent of the rundown of Ca_V1 channel activity. This demonstrates that interaction between the ß-cell Ca_V1 channel and syntaxin 1A is necessary for a proper ß-cell function (211).

The ß-cell Ca_V1.2 subunit also complexes with exocytotic proteins (212). His₆-fused Ca_V1.2 subunit peptides corresponding to the II-III loop of the Ca_V1.2 subunit [L_C(753–893)] effectively pulled down syntaxin 1A, SNAP-25, and synatotagmin. This indicates that these exocytotic proteins physically associate with the Ca_V1.2 channel at the II-III loop of the Ca_V1.2 subunit. The functional consequence of this physical association has been characterized in both a heterologous expression system and the pancreatic ß-cell. The coexpressed syntaxin 1A slightly alters the inactivation and activation rate but massively reduces the amplitude of Ca_V1.2 currents recorded in Xenopus oocytes injected with Ca_V1.2/ß2a/₂. The expressed synaptotagmin partially reverses the effects of the coexpressed syntaxin 1A on Ca_V1.2 channels (212). In the pancreatic ß-cell, the intracellular application of Ca_V1.2_(753–893) peptide effectively interrupts the physical association of the Ca_V1.2 channel with exocytotic proteins and thus almost completely blocks depolarization-evoked exocytosis without significant influence on Ca²⁺ influx (212). Similar effects were confirmed in insulin-secreting cell lines where overexpression of syntaxin 1A and 3 significantly decreased Ca_V1 channel activity and Ca²⁺-dependent insulin secretion (289).

The functional interaction of Ca_V1 channels with distinct domains within SNAP-25 has been visualized in ß-cells (337). Intracellular application of SNAP-25_(1–206) significantly reduces L-type Ca_V currents in mouse ß-cells. The coapplication of Ca_V1.2_(753–893) peptide prevents the reduction in L-type Ca_V currents by SNAP-25_(1–206). Furthermore, HIT cells overexpressing or injected with wild-type SNAP-25 display smaller L-type Ca_V currents than control cells. This inhibition is also prevented by the Ca_V1.2_(753–893) peptide. Intriguingly, the long N terminus [SNAP-25_(1–197)] and the short C terminus of this exocytotic protein [SNAP-25_(198–206) ] exert opposite actions on L-type Ca_V currents. L-type Ca_V currents are significantly enhanced by expression of SNAP-25_(1–197). These effects are abolished by the Ca_V1.2_(753–893) peptide. In sharp contrast, L-type Ca_V currents in untransfected cells are significantly attenuated by intracellular application of SNAP-25_(198–206). The inhibitory effect of SNAP-25_(198–206) on L-type Ca_V currents is greater than that of the stimulatory effect of SNAP-25_(1–197) on these currents. Taken together, it is clear that the SNARE protein SNAP-25 possesses distinct inhibitory and stimulatory domains that act on the Ca_V1 channel (337).

Overall, the ß-cell Ca_V1 channel complexes with the exocytotic machinery to form a functional molecular network. This network serves both as a fine-tuning mechanism of ß-cell Ca_V1 channel function and as an anchoring machinery to optimally organize this channel at the site of insulin exocytosis (Fig. 6).

2. Ca_V ß3 subunit-intracellular Ca²⁺ store network.
The plasma membrane Ca_V channels and intracellular InsP₃ receptors share a common function, i.e., intracellular Ca²⁺ supply, genuinely important to the cell (3, 338). Integration of Ca²⁺ influx through Ca_V channels with Ca²⁺ mobilization from intracellular stores, mediated by InsP₃ receptors, establishes the time course and spatial arrangement of the intracellular Ca²⁺ signal, which serves as a ubiquitous second messenger to control Ca²⁺-dependent protein-protein interactions and enzymatic responses in the cell (5, 6). Interestingly, our recent data suggest that the Ca_Vß3 subunit is not a required building blocker of ß-cell Ca_V channels. Instead, it interacts with the intracellular Ca²⁺ release machinery to form a Ca_Vß3 subunit-intracellular Ca²⁺ store network (Fig. 6) (295). Patch-clamp analysis showed that the Ca_Vß3 subunit knockout (ß₃^–/–) does not affect the activity and gating properties of Ca_V channels at both the single channel and whole-cell level. However, it markedly increases InsP₃-induced Ca²⁺ release and strikingly fastens glucose-induced [Ca²⁺]_i oscillations in ß-cells. Furthermore, ß₃^–/– mice exhibit a more efficient glucose homeostasis. The intact Ca_Vß3^–/– islets show a significant increase in glucose-induced insulin secretion, whereas permeabilized Ca_Vß3^–/– islets display no change in Ca²⁺-evoked insulin secretion. The enhanced insulin secretion is blocked by restoration of the Ca_Vß3 subunit. The molecular mechanisms responsible for the enhanced insulin secretion by genetic deletion of the Ca_Vß3 subunit have been extensively explored. Expression of the Ca_Vß3 subunit in COS-7 cells, which do not possess endogenous Ca_V channels, significantly decreases Ca²⁺ release from InsP₃-sensitive stores. The endogenous Ca_Vß3 subunit in pancreatic ß-cells and expressed Ca_Vß3 subunit in COS-7 are observed predominantly in intracellular compartments resembling ER where InsP₃ receptors localize. Furthermore, Ca_Vß3 subunits partially colocalize with type 3 InsP₃ receptors in ß-cells under basal conditions (295).

The InsP₃ receptor subunit consists of about 2,700 amino acid residues (human type 1 InsP₃ receptor, 2,695; human type 2 InsP₃ receptor, 2,701; and human type 3 InsP₃ receptor, 2,671) (339, 340). Each InsP₃ receptor subunit possesses an N-terminal InsP₃ binding site, a middle modulatory domain, and a C-terminal channel region comprising six membrane-spanning helices. Actually, the InsP₃ receptor subunit only uses about 5% of its residues to form the Ca²⁺ conducting pore. The large cytoplasmic region of this subunit harbors numerous recognition sites for a variety of small molecules and proteins including InsP₃, Ca²⁺, nucleotides, protein kinases and phosphatases, calmodulin, apoptotic proteins, transient receptor potential channels, G protein ß subunits, etc. Therefore, the InsP₃ receptor-mediated intracellular Ca²⁺ release is exquisitely modulated (338). Taken together with our data, it is highly attractive to speculate that the Ca_Vß3 subunit reversibly interacts with InsP₃ receptors to function as a brake on InsP₃ receptor-mediated Ca²⁺ mobilization from intracellular stores in the pancreatic ß-cell. It is intriguing to examine dynamic association of Ca_Vß3 subunits with InsP₃ receptors in response to glucose, interaction sites on both Ca_Vß3 subunits and InsP₃ receptors, and regulation of InsP₃ receptor activity by the Ca_Vß3 subunit.

	IV. Role of CaV Channels in ß-Cell Pathophysiology

Top Abstract I. Introduction II. General Aspects of... III. Role of CaV... IV. Role of CaV... V. ß-Cell CaV Channel... VI. Future Perspectives References

 
An inherited or acquired defect in ß-cell Ca_V channels is a key determinant in ß-cell pathophysiology. Generally speaking, such a defect encompasses exaggerated up- and down-regulation, disappearance and appearance, and inadequate redistribution of Ca_V channels in the ß-cell. These abnormal changes can cause excessive or insufficient insulin output and even ß-cell death. Therefore, these pathophysiological circumstances are considered in this review.

A. Disorder of ß-cell Ca_V channels in diabetic milieu
Disorder of ß-cell Ca_V channels has been observed in diabetic animal models and diabetic patients (our unpublished observations) (17, 18, 19, 20). Pancreatic ß-cells isolated from neonatal streptozocin-induced diabetic (STZ) rats show drastic up-regulation of both Ca_V1 and Ca_V3 channel activity. This effect on the Ca_V1 channel is most likely due to an increase in the activity rather than in the number of Ca_V channels, because DHP binding sites in the diabetic ß-cell even decrease (18). Furthermore, STZ islets show a significant decrease in the expression of Ca_V channel subunit genes (17). Indeed, direct depolarization with 20 mM arginine induces a more pronounced [Ca²⁺]_i elevation in diabetic ß-cells in comparison with control ß-cells (341). However, the diabetic rat islets exhibit blunted [Ca²⁺]_i elevation and insulin secretion after glucose stimulation due to impaired glucose sensitivity of ß-cell K_ATP channels (18, 341, 342). It seems that in vivo hyperactivation of Ca_V channels in the STZ ß-cell can hardly occur. However, it cannot be ruled out that in vivo long-lasting severe hyperglycemia may hyperactivate ß-cell Ca_V channels because both Ca_V1 and Ca_V3 channel activity in the STZ ß-cell are already significantly enhanced when the cells are depolarized to potentials within the physiological range (–20 mV) (18).

Similar changes in ß-cell Ca_V channel activity have also been visualized in the Goto-Kakizaki rat, a non-insulin-dependent diabetic model (19). However, the enhancement of Ca_V1 channel activity in cultured Goto-Kakizaki rat ß-cells somewhat differs from that in freshly isolated ones. Like in STZ rats, cultured Goto-Kakizaki rat ß-cells show larger L-type Ca_V currents when the cells are depolarized within the range from –20 mV to +40 mV, in comparison to cultured control rat ß-cells. In contrast, the increase in L-type Ca_V currents in freshly isolated Goto-Kakizaki rat ß-cells takes place in a narrower range of depolarizations from +10 to +20 mV. This makes it more difficult to explain the functional significance of the enhanced Ca_V1 channel activity in the Goto-Kakizaki rat ß-cell because depolarizations positive to +10 mV hardly occur in vivo. It is worthwhile to note that a relatively high concentration of Ba²⁺ was used as the charge carrier during whole-cell Ca_V current recordings. It is well-known that the high concentration of Ba²⁺ shifts the current-voltage relationship of HVA Ca²⁺ channels to more positive voltages (343). Therefore, the enhanced Ca_V1 channel activity in the Goto-Kakizaki rat ß-cell may be present in the in vivo milieu containing physiological concentration of Ca²⁺. Furthermore, the mechanism responsible for the hyperactivation of Ca_V1 channels in Goto-Kakizaki rat ß-cells was clarified. Hyperglycemia was suggested to be the cause because 16.7 mM glucose significantly increased L-type Ca_V currents in the control ß-cell but failed to alter Ca_V1 channel activity in the Goto-Kakizaki rat ß-cell due to the preexisting action of hyperglycemia (19).

In sharp contrast to STZ and Goto-Kakizaki rats, ß-cell Ca_V channels are altered in a completely different way in Zucker diabetic fatty (ZDF) rats, a type 2 diabetic animal model (20). Quantitative reverse transcription-PCR analysis reveals that overtly diabetic ZDF islets display 45 and 53% reductions in the levels of mRNA encoding Ca_V1.2 and Ca_V1.3 subunits, respectively. Moreover, Ca_V1.2 and Ca_V1.3 subunit mRNA levels are already decreased by 28 and 38%, respectively, in the prediabetic stage of this animal model. Electrophysiological analysis clearly shows a functional relevance of the reduction in Ca_V1.2 and Ca_V1.3 subunit mRNAs. ZDF ß-cells have almost completely lost Ca_V currents. The loss of Ca_V channels severely damages ZDF ß-cell function as demonstrated by assessments of [Ca²⁺]_i and insulin secretion dynamics. Repeated stimulation with either 20 mM K⁺ or 12 mM glucose generates a corresponding wave of [Ca²⁺]_i in control islets but gives rise to little effect on [Ca²⁺]_i in ZDF islets. Moreover, the effect of Ca_V channel opener BAY K8644 on [Ca²⁺]_i also disappears in ZDF islets. However, carbachol-induced [Ca²⁺]_i mobilization remains intact in ZDF islets. The pulsatile insulin release induced by oscillatory changes of glucose concentration, generated by computer-controlled peristaltic pumps, has also been examined in ZDF islets. Interestingly, the oscillatory glucose stimulation only induces in-phase insulin response in control islets. ZDF islets are inert to such oscillatory glucose stimulation. More interestingly, both glucose-induced [Ca²⁺]_i responses and insulin secretion are evidently blunted in prediabetic ZDF islets. Unfortunately, electrophysiological evaluation of Ca_V channel activity has not been carried out at the prediabetic stage. Therefore, it is difficult to conclude whether both the genetic alteration and the diabetic milieu, such as hyperglycemia, result in the reduction of Ca_V channels in ZDF ß-cells. Nevertheless, the reduced expression and decreased activity of Ca_V channels definitely contribute to the dysfunction of ZDF ß-cells, exemplified by blunted stimulus-secretion coupling (20).

Another type 2 diabetic model, Otsuka Long-Evans Tokushima Fatty (OLETF) rat, also displays quantitative alteration in ß-cell Ca_V channel subunits (17). Quantification of Ca_V subunit mRNAs revealed that Ca_V1.3, Ca_Vß2 and ß3 subunit mRNAs from diabetic OLETF rats are drastically reduced in comparison with age and strain-matched Long-Evans Tokushima Otsuka rats and prediabetic OLETF rats. Correspondingly, diabetic OLETF islets secrete less insulin after glucose stimulation compared with islets from prediabetic OLETF and other control rats. Interestingly, diabetic OLETF islets exposed to the Ca_V channel opener BAY K8644 release more insulin than prediabetic OLETF islets do. It is speculated that the elevation of BAY K8644-induced insulin release from diabetic OLETF islets results from an increase in Ca_V channel activity although there is a decrease in Ca_V channel subunit gene expression. Regrettably, Ca_V channel activity has not been directly evaluated (17).

In summary, there is no doubt that ß-cell Ca_V channel disorder occurs in diabetic subjects. However, the inconsistent direction of the changes in ß-cell Ca_V channels in this polygenetic disease generates quite a few dilemmas. For example, does a specific pathogenesis cause distinct changes in ß-cell Ca_V channels? Do ß-cell Ca_V channels display different patterns of disorders during the progression of diabetes? How does the down-regulation of Ca_V channel subunit genes go together with the enhancement of Ca_V channel activity in STZ ß-cells?

B. Phenotypic switch of ß-cell Ca_V channels in diabetes
A series of studies have verified that Ca_V3 channels are absent in the normal mouse pancreatic ß-cell (2, 219, 220, 227, 236). Interestingly, NOD mouse pancreatic ß-cells abnormally express Ca_V3 channels (16). Patch-clamp analysis reveals that the depolarized ß-cells from 8- to 10-wk-old NOD mice display an abnormal Ca_V current characterized by a fast inactivation (20- to 30-msec time constants at –20 mV), a lower threshold for activation (–50 mV), and a maximal activation at –20 mV. ß-Cells isolated from the strain-matched control mice do not show such a type of LVA Ca²⁺ currents. Further characterization indicates that NOD mouse ß-cell Ca_V3 channels contribute to the elevated basal [Ca²⁺]_i, resulting in apoptosis of these cells (16, 148).

The NOD mouse is an animal model of human type 1 diabetes. Most NOD mice show obvious insulitis at about 4 wk of age when mixed leukocytes including macrophages, CD4+ and CD8+ T cells, and B cells infiltrate pancreatic islets. Subsequently, selective ß-cell destruction occurs, ß-cell mass decreases, and diabetes develops starting at approximately 14 wk of age. Eventually, 60–90% of the female mice and 10–40% of the male mice suffer from overt diabetes after 20–30 wk of age (344, 345). It is believed that inflammatory cytokines, produced by the immune cells in the periislet and intraislet infiltrate, operate in concert with excessive [Ca²⁺]_i to kill ß-cells through apoptosis. Investigations of the underlying pathogenic mechanism raised the intriguing question of whether the abnormally expressed Ca_V3 channel in NOD ß-cells results from the cytotoxic effect of the cytokine. Indeed, chronic treatment with a cytokine cocktail containing interferon- and IL-1ß initiates expression of Ca_V3 channels in ß-cells from Swiss-Webster mice, a compatible control strain for NOD mice. This Ca_V channel resembles the Ca_V3 channel visualized in NOD mouse ß-cells and is blocked by the inorganic Ca_V3 channel blocker NiCl₂. Furthermore, pharmacological manipulation clearly demonstrates that Ca²⁺ influx through this newly emerged ß-cell Ca_V3 channel contributes to a 3-fold increase in basal [Ca²⁺]_i. Interestingly, like Swiss-Webster mouse ß-cells, ßTC3 cells, a mouse insulin-secreting cell line, also display clear Ca_V3 currents concomitant with apoptotic ß-cell death characterized by DNA fragmentation after cytokine treatment. More interestingly, the cytokine-induced apoptosis is effectively reduced by the Ca_V3 channel blockers NiCl₂, amiloride, and mibefradil, but not by the Ca_V1 channel blocker nifedipine. In striking contrast, -TC cells, a glucagon-secreting cell line, are invulnerable to cytokine treatment (148).

Although ß-cell Ca_V channel types are genetically well defined, their phenotypes can change under pathophysiological conditions. The above discussed studies clearly demonstrate that cytokines are capable of altering ß-cell Ca_V channel phenotypes. It is of interest to investigate how cytokines selectively turn on expression of ß-cell Ca_V3 channels in the type 1 diabetic situation. Do they switch on gene expression of ß-cell Ca_V3 channels or just expose genetically defined but hidden Ca_V3 channel in ß-cells?

C. Ca_V channel mutation and inadequate insulin secretion
Recently, a missense mutation in the Ca_V1.2 subunit has been identified in the Timothy syndrome, a lethal arrhythmia disorder associated with multiorgan dysfunction including webbing of fingers and toes, congenital heart disease, immune deficiency, intermittent hypoglycemia, cognitive abnormalities, and autism (21). DNA sequence analysis has revealed that a single nucleotide G>A transition occurs at position 1216 in the Ca_V1.2 subunit gene from these patients. The G1216A transition causes a substitution of glycine with arginine at residue 406 (G406R), which is localized at the C terminus of the IS6 segment. The pore-lining segment IS6 plays an important role in voltage-dependent inactivation. Therefore, the substitution of glycine with arginine in the IS6 segment is supposed to alter Ca_V1.2 channel inactivation. Indeed, the mutant Ca_V1.2 subunit coexpressed with other wild-type Ca_V channel auxiliary subunits in both Xenopus oocytes and CHO cells almost completely lost voltage-dependent inactivation. Interestingly, 36% of the patients showed episodic hypoglycemia, strongly indicating that insulin secretion was inadequately enhanced by the elevated Ca²⁺ influx through the mutant Ca_V1.2 channels in the pancreatic ß-cell. This inadequate insulin secretion has resulted in the death of some affected individuals (21).

D. Ca_V channel gene polymorphism and diabetes
Massive efforts have been made to unravel the complex pathogenesis and inheritance of type 2 diabetes. Clinical and genetic studies have revealed that type 2 diabetes is a complex polygenic trait. This polygenic disease is characterized by both impaired insulin secretion and insulin resistance (346). Therefore, genes critical for stimulus-secretion coupling, such as Ca_V channel genes, have been regarded as obvious candidates for type 2 diabetes susceptibility (347). Unfortunately, it is difficult to evaluate correlation between Ca_V channel gene mutations and type 2 diabetes. In striking contrast to the neuron, pancreatic ß-cell seems to be invulnerable to Ca_V channel gene polymorphisms, although they express almost equal types of Ca_V channels (2, 157). This may be due to a marked difference in Ca_V channel localization between the two types of cells. Each type of neuronal Ca_V channel exerts a more defined function because of its distinct localization, such as axonal terminals, dendrites, and soma (157). However, the pancreatic ß-cell has no such characteristic morphology. All types of ß-cell Ca_V channels can contribute to stimulus-secretion coupling, although Ca_V1 channels dominate over the other types under physiological conditions (2, 219, 220). Consequently, a key question is why nature arranges such a redundancy. The fact that the body only uses the pancreatic ß-cell to efficiently secrete insulin for maintaining glucose homeostasis could be the explanation. Nature endows the pancreatic ß-cell with multiple types of Ca_V channels to avoid disaster. It is attractive to speculate that when polymorphism occurs in one type of Ca_V channel, the compensatory change in other types of Ca_V channels may result in the lack of mutation-caused dysfunctions in the ß-cell, but not in the neuron. Although tottering mice carrying a missense mutation (P601L) in the Ca_V2.1 subunit exhibit significant up-regulation of the Ca_V1.2 subunit expression in cerebellar Purkinje cells, the up-regulated Ca_V1.2 channel cannot compensate for the lost function of the mutant Ca_V2.1 channel, but instead significantly contributes to the phenotype of dystonic episodes (348, 349). It is worthwhile to note that a plethora of factors, e.g., age, obesity, and lifestyle in type 2 diabetes, can influence the phenotypical manifestation of genetic defects (350).

Three human Ca_V1 subunit genes can undergo a series of polymorphisms that cause several inherited diseases. Mutations in the Ca_V1.1 subunit gene (CACNA1S) result in malignant hyperthermia and hypokalemic periodic paralysis. Mutations in Ca_V1.4 subunit gene (CACNA1F) are responsible for incomplete congenital stationary night blindness. Mutations in Ca_V2.1 subunit gene (CACNA1A) underlie familial hemiplegic migraine, episodic ataxia type 2, and spinocerebellar ataxia type 6 (9). Recently, it has been revealed that nonsynonymous single nucleotide polymorphisms of Ca_V3.2 subunit gene (CACNA1H) are highly associated with childhood absence epilepsy (351, 352). As mentioned earlier, the human pancreatic ß-cell does not express Ca_V1.1 and Ca_V1.4 subunits, but indeed harbors Ca_V2.1 subunits (2, 219). Interestingly, a Japanese family with spinocerebellar ataxia type 6 caused by Ca_V2.1 subunit gene mutations is highly associated with type 2 diabetes. Thirteen members in this five-generation family are diagnosed as spinocerebellar ataxia type 6. Three of the five patients examined suffer from overt type 2 diabetes. It is unclear whether the other two patients are diabetic or not (353). This Ca_V2.1 subunit mutant carries abnormal CAG repeat expansion, which encodes a polyglutamine tract. Biophysical properties and surface expression of this mutant have been evaluated in a mammalian heterologous expression system (354, 355). However, results are inconsistent. One group found that the polyglutamine-containing Ca_V2.1 subunit expressed in HEK 293 cells shows a negative shift of voltage-dependent inactivation, which results in reduced Ca²⁺ influx. The reduced Ca²⁺ influx mediated by the mutant Ca_V2.1 subunit is believed to underlie degeneration of cerebellar Purkinje cells (354). In striking contrast, another group showed that the surface expression of the polyglutamine-containing Ca_V2.1 subunit is more abundant than that of the wild-type Ca_V2.1 subunit in HEK 293 cells. Consequently, higher Ca_V current density is observed in cells transfected with the mutant Ca_V2.1 subunit that did not show altered biophysical properties. Therefore, Ca²⁺ overload through the mutant Ca_V2.1 channels is attributed to damage of cerebellar Purkinje cells (355). It is well known that Ca_V2.1 channels are present in the human ß-cell and play a prominent role in insulin exocytosis (219). Therefore, it is not surprising that a high incidence of type 2 diabetes is observed in patients with spinocerebellar ataxia type 6 caused by Ca_V2.1 subunit gene mutations (353). It is attractive to speculate that the mutant Ca_V2.1 channel may lead to Ca²⁺ overload-induced ß-cell death. Hence, biophysical properties, surface expression of ß-cell Ca_V2.1 channels, as well as insulin secretion and ß-cell mass in spinocerebellar ataxia type 6 patients should be investigated in greater detail.

Human Ca_V1.3 subunit gene polymorphisms have been examined in 918 Japanese type 2 diabetics and 336 control subjects (356). Interestingly, an ATG repeat expansion in the human Ca_V1.3 gene is only found in type 2 diabetics (356). However, the frequency of this mutation is low and not associated with the development of common type 2 diabetes. Nevertheless, it may be involved in the pathogenesis of a subgroup of this polygenic disease (356). Recently, a genomewide linkage analysis of a large consanguineous family with autosomal recessively inherited neonatal diabetes has identified a novel neonatal diabetes locus, which is mapped to chromosome 10p12.1-p13 (357). Intriguingly, this region contains the Ca_Vß2 subunit gene (48). In this family, all affected individuals have low to undetectable levels of insulin (357). Other studies have also demonstrated that insulin secretory failure occurs in the early postnatal period of permanent neonatal diabetes, and both the insulin secretory defect and the developmental insulin production disorder occur in transient neonatal diabetes (358, 359). Therefore, the gene encoding Ca_Vß2 subunit, a predominant isoform of Ca_V channel ß subunits in the pancreatic ß-cell, should be one of the potential susceptibility genes for neonatal diabetes. Furthermore, some studies have manifested that individuals with transient neonatal diabetes are predisposed to type 2 diabetes (358, 360). Therefore, it is reasonable to look into possible polymorphisms in the Ca_Vß2 subunit gene in type 2 diabetics.

E. Ca_V channels and ß-cell death
Dynamics and homeostasis of [Ca²⁺]_i in the ß-cell critically depend on the Ca²⁺ handling molecular network, where Ca_V channels take center stage (361). Glucose metabolism-derived signals activate the Ca²⁺ handling molecular network in the ß-cell to generate complex [Ca²⁺]_i signals, which control numerous cellular events including cell viability (24, 362). The cell can only survive in an appropriate range of [Ca²⁺]_i. When [Ca²⁺]_i goes either above or below this range, a set of molecules responsible for cell survival can no longer function and another set of molecules being in charge of cell demise becomes activated. Therefore, [Ca²⁺]_i serves as an important signal for cell viability (4, 5, 6, 7). Under physiological conditions, the Ca²⁺ handling molecules act in concert to compose adequate [Ca²⁺]_i signals maintaining ß-cell growth, proliferation, and differentiation. On the contrary, pathophysiological changes in the extracellular and/or intracellular milieu disturb the Ca²⁺ handling molecular network leading to chaos in [Ca²⁺]_i, which drives the ß-cell to death (Fig. 7) (24, 25, 148, 363).

Hyperactivated Ca_V channel-mediated Ca²⁺ overload can trigger apoptotic and necrotic ß-cell death through various Ca²⁺-sensitive enzymes, e.g., calcineurin, endonucleases, transglutaminase, and calpains (362, 363, 364, 365). Calcineurin is a serine- and threonine-specific protein phosphatase (PP). This enzyme is conserved in all eukaryotes and senses Ca²⁺ through its activation by calmodulin. It takes center stage in cell death signaling. Activated calcineurin dephosphorylates the Bcl-2 family member Bad to exert its proapoptotic effect (7). Indeed, calcineurin integrates the Ca²⁺ influx through ß-cell Ca_V1 channels with the cytokine signal transduction cascade to drive ß-cells to death. Exposure to IL-1ß makes pancreatic ß-cells undergo apoptosis. This apoptotic event is largely blocked by either the Ca_V1 channel inhibitor D-600 or the calcineurin inhibitor deltamethrin (365). Additionally, hyperactivated Ca_V channels can initiate activation of calpain by overloading insulin-secreting MIN6N8 cells with Ca²⁺, and then activated calpain turns on calcineurin. Subsequently, activated calcineurin dephosphorylates Bad, making these insulin-secreting cells undergo apoptosis (363).

Ca²⁺-dependent endonucleases cleave chromosomal DNA into oligonucleosomal size fragments leading to programmed cell death or apoptosis (7). It has been demonstrated that excessive Ca²⁺ influx through Ca_V1 channels activates Ca²⁺-dependent endonucleases causing pancreatic ß-cell apoptosis. Our group demonstrated that 40-h exposure to glucose (11–27 mM) induces a concentration-dependent ß-cell death. The effect is mimicked by depolarization with the K_ATP channel blocker tolbutamide and blocked by either hyperpolarization with the K_ATP channel opener diazoxide or the Ca_V1 channel blocker D-600. The dead cells display apoptosis-characteristic DNA ladders. Therefore, this glucose-induced ß-cell apoptosis is Ca²⁺-dependent. Furthermore, the glucose-induced apoptosis is effectively prevented by the endonuclease inhibitor aurintricarboxylic acid (362).

Transglutaminases are a widely distributed group of enzymes catalyzing Ca²⁺-dependent posttranslational modification of proteins. Transglutaminase 2, also called tissue transglutaminase, is ubiquitously expressed in mammalian cells including ß-cells. This enzyme is regulated by both Ca²⁺ and GTP. Upon activation by Ca²⁺, transglutaminase 2 extensively polymerizes various cytoskeletal proteins including microtubule protein tau, ß-tubulin, actin, myosin, spectrin, thymosin ß, troponin T, and vimentin. This Ca²⁺-dependent cross-linking of cytoskeletal proteins constitutes the final steps of apoptosis (366). Transglutaminase 2-catalyzed modification of nuclear proteins, such as core histones, has also been demonstrated. This leads to the speculation that transglutaminase 2 may catalyze histone cross-linking to mediate the chromatin condensation in apoptosis (367). Additionally, activation of transglutaminase 2 also initiates apoptotic cell death in a Ca²⁺-dependent manner (7). For example, depletion of GTP in insulin-secreting HIT-T15 cells with mycophenolic acid significantly increases transglutaminase 2 activity and in turn makes these cells undergo apoptosis. Interestingly, this apoptotic ß-cell death induced by activation of transglutaminase 2 is markedly reduced by lowering extracellular Ca²⁺ concentrations (364). This suggests that transglutaminase 2 is a potential decoder of hyperactivated Ca_V channel-mediated Ca²⁺ entry in ß-cell apoptosis.

A family of Ca²⁺-activated neutral cysteine proteases, calpains, has been identified. Two ubiquitous isoforms, µ- and m-calpain, have been studied extensively. As indicated by their names, µ- and m-calpains are activated in vitro by concentrations of Ca²⁺ in micromolar and millimolar ranges, respectively. Consequently, activated µ- and m-calpains proteolyze a wide range of cytoskeletal, membrane-associated, and regulatory proteins participating in a variety of cellular processes, particularly necrosis and apoptosis (368, 369). A recent study revealed that an apoptotic cascade operates in the order of cytokine, PKC, Ca_V channels, calpain, calcineurin, Bad, cytochrome c, and caspases in insulin-secreting MIN6N8 cells (363). Coapplication of interferon- and TNF- drastically increases HVA Ca²⁺ currents, markedly raises [Ca²⁺]_i, and consequently makes this insulin-secreting cell line undergo apoptotic death. The effects are significantly blocked by treatment with the PKC inhibitor chelerythrine. This indicates that PKC phosphorylation makes HVA Ca²⁺ channels hyperactivated. Excessive Ca²⁺ influx through these channels triggers apoptosis. Further characterization demonstrated that this excessive Ca²⁺ influx first turns on the Ca²⁺-activated protease calpain. Subsequently, the activated calpain initiates activation of calcineurin, which in turn dephosphorylates Bad at S112. The cytochrome c moves from the mitochondria to the cytoplasm after Bad dephosphorylation. Eventually, cleavage of caspases 9, 3, and 7 occurs, leading to apoptosis (363).

Generally speaking, ß-cell Ca_V channels can participate in apoptosis in two ways. In some cases, the channel becomes hyperactivated and thereby conducts large amounts of Ca²⁺ into the ß-cell. The extremely high [Ca²⁺]_i resulting from this extraordinarily excessive Ca²⁺ influx can directly activate apoptotic cascades in the ß-cell. A good example is calpain-mediated ß-cell apoptosis triggered by the hyperactivated Ca_V channel-mediated Ca²⁺ entry (363). In other situations, physiological Ca²⁺ influx through ß-cell Ca_V channels can be a necessary factor to allow other apoptotic challenges to exert their effects. For example, IL-1ß cannot induce ß-cell apoptosis without Ca²⁺ influx through Ca_V1 channels. However, this cytokine does not alter ß-cell Ca_V channel activity (365).

Indeed, we have in great detail investigated the role of Ca_V channels in ß-cell apoptosis and necrosis in vitro (24, 25, 362, 365). Although it is of utmost importance, we have not yet started to examine the role of ß-cell Ca_V channels in apoptosis and necrosis in vivo during the development of diabetes. The loss of ß-cells occurs in both type 1 and type 2 diabetes. Type 1 diabetes is characterized by the absolute loss of pancreatic ß-cells. Type 2 diabetes is defined by not only the progressive loss of ß-cell function but also increased ß-cell apoptosis (370). It is well known that hyperactivation of ß-cell Ca_V channels plays an important role in ß-cell apoptosis (24, 25, 148, 363). Therefore, ß-cell Ca_V channels are potential therapeutic targets with regard to the prevention of ß-cell apoptosis and necrosis during the development of diabetes.

F. Ca_V1 channels and type 1 diabetic serum-induced ß-cell apoptosis
In a diabetic milieu, a number of extracellular and intracellular factors selectively drive ß-cells into death and thereby aggravate diabetes. Such factors appear to be present in type 1 diabetic serum. They compel unphysiological amounts of Ca²⁺ to enter pancreatic ß-cells through hyperactivated ß-cell Ca_V1 channels resulting in ß-cell apoptosis (Fig. 7) (24, 25, 148, 363). Single channel recordings show that treatment with type 1 diabetic serum makes ß-cell Ca_V1 channels open more frequently. Correspondingly, whole-cell patch-clamp analysis revealed a massive increase in the amplitude of L-type Ca²⁺ currents. Consequently, this abnormal Ca²⁺ entry through the hyperactivated Ca_V1 channels gives rise to a Ca²⁺ overload in the ß-cell, which can be clearly manifested by measurements of [Ca²⁺]_i. Indeed, the Ca²⁺ overload, in turn, causes typical ß-cell apoptosis characterized by DNA fragmentation. The contribution of the hyperactivated Ca_V1 channels to the apoptotic effect of type 1 diabetic serum is further confirmed by the counteraction of the ß-cell apoptosis by the Ca_V1 channel blocker verapamil (24).

Experimental evidence suggests that multiple factors in type 1 diabetic serum can attack ß-cell Ca_V channels (24, 25, 148, 363). A study performed in neuroblastoma cells proposed that the Fas-specific antibodies in type 1 diabetic serum possibly act as another factor involved in the hyperactivation of ß-cell Ca_V channels (371). N1E-115 murine neuroblastoma cells exhibit a progressive increase in [Ca²⁺]_i after exposure to type 1 diabetic serum. However, no effect on [Ca²⁺]_i occurred in these cells after administration of serum from healthy subjects. Treatment with type 1 diabetic serum also causes neuroblastoma cell apoptosis characterized by condensed chromatin, shrunken cytoplasm, and DNA fragmentation. Interestingly, immunofluorescence labeling reveals that the death receptor protein Fas distributes at the neuroblastoma cell surface and mediates type 1 diabetic serum-induced apoptosis (371). ß-Cells are also equipped with the Fas signaling pathway mediating ß-cell apoptosis (372). Therefore, it is important to examine whether Fas-specific antibodies in type 1 diabetic serum hyperactivate ß-cell Ca_V1 channels, causing ß-cell apoptosis through activation of the Fas signaling pathway.

We are still ignorant of the mechanisms whereby type 1 diabetic serum hyperactivates ß-cell Ca_V channels and brings about ß-cell apoptosis. However, some clues have emerged from studies with serum from the type 1 diabetic animal model, Bio Bred/Worchester diabetic (BBW) rat. The effect of the BBW rat serum on neuronal Ca_V channels highly resembles that of the human type 1 diabetic serum on ß-cell Ca_V channels. Ca_V channel activity in nondiabetic rat dorsal root ganglion neurons is drastically increased after incubation with the BBW rat serum. This effect is tightly associated with impaired regulation of the inhibitory G protein-Ca_V channel complex (373). Moreover, dorsal root ganglion neurons from the BBW rat also exhibit an enhancement of both HVA and LVA Ca²⁺ currents, which is attributed to a decrease in opiate-mediated inhibition of pertussis toxin-sensitive, G protein-coupled Ca_V channels (374, 375, 376). Inhibitory G proteins also reside in the ß-cell and down-regulate ß-cell Ca_V channel activity (377, 378). It would be interesting to look into a possible involvement of inhibitory G proteins in the hyperactivation of ß-cell Ca_V channels by type 1 diabetic serum. Recently, we revealed that incubation with type 1 diabetic serum facilitates the expression of Ca_V3 channels in a particular type of neurons with triangular soma in cerebellar granule cell cultures (379). However, type 1 diabetic serum does not affect the Ca_V 3 channel in RINm5F cells, an insulin-secreting cell line (24). This may reflect the fact that the affected neuronal Ca_V3 channel is a subtype different from the RINm5F cell Ca_V3 channel.