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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.