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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 |
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1 subunits, including CaV1.2, CaV1.3, CaV2.1, CaV2.2, CaV2.3, and CaV3.1, 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 CaV currents, respectively. ß-Cell CaV channels take center stage in insulin secretion and play an important role in ß-cell physiology and pathophysiology. CaV3 channels become expressed in diabetes-prone mouse ß-cells. Point mutation in the human CaV1.2 gene results in excessive insulin secretion. Trinucleotide expansion in the human CaV1.3 and CaV2.1 gene is revealed in a subgroup of patients with type 2 diabetes. ß-Cell CaV 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 CaV channels causes ß-cell dysfunction and even death manifested in both type 1 and type 2 diabetes. This review summarizes current knowledge of CaV channels in ß-cell physiology and pathophysiology.
| I. Introduction |
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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 CaV channels take center stage in this process (2, 11). In addition to controlling insulin secretion, ß-cell CaV 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 CaV channels (16, 17, 18, 19, 20). Point mutation of human CaV1.2 channels makes the ß-cell secrete insulin excessively (21). ß-Cell CaV 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 CaV channel activity and/or density results in enhanced insulin exocytosis and more efficient glucose homeostasis (2). Moreover, down-regulation of CaV 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 CaV 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 CaV channels leads to ß-cell death (24, 25).
This review focuses on the role of CaV channels in pancreatic ß-cell physiology and pathophysiology.
| II. General Aspects of CaV Channels |
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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 CaV channel gene products (33). The gene nomenclature, such as CACNA1 and CACNB, was employed to depict CaV channel genes (34). These multiple nomenclatures of CaV channels have given rise to obvious communication problems. To avoid the problems, a group of leading scientists in the area of CaV channel research recommended a comprehensive nomenclature of CaV channels based on sequence analysis in 2000 (29). Primary structure analysis of CaV
1 subunits categorizes CaV channels into three families, CaV1, CaV2, and CaV3, consisting of closely related members (29). This structure-based nomenclature has been widely accepted to describe CaV channel proteins and mRNAs. However, it is hardly applied to the description of CaV currents recorded from native cells expressing complex mixtures of CaV channel subunits. For example, both CaV1.2 and CaV1.3 subunits conduct the ß-cell L-type CaV current. There is no way to discriminate the current flowing through CaV1.2 from that through CaV1.3 subunits. In this case, L-type may be the best description. In this article, we employ the phenomenological nomenclature to describe CaV currents and the structural nomenclature to depict CaV channel proteins and mRNAs. Table 1
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2. CaV2 channels.
The CaV2 channel family includes three members, CaV2.1, CaV2.2, and CaV 2.3 channels (2, 3, 29). CaV2.1 channels mediate P/Q-type CaV currents. Biophysical features of P/Q-type CaV currents conducted by CaV2.1 channels are very similar to those of N-type CaV currents mediated by CaV2.2 channels. P/Q-type CaV currents are indistinguishable from N-type CaV currents without peptide blockers (38). P-type CaV currents were first identified using
-agatoxin IVA (
-Aga IVA) in cerebellar Purkinje cells (39). Subsequently, Q-type CaV currents were revealed in cerebellar granule cells (38). Both the P-type and the Q-type CaV currents are blocked by
-Aga IVA. However, the P-type CaV current is more sensitive to this blocker and does not inactivate during 0.1-sec depolarizations. The Q-type CaV 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 CaV currents are conducted by the same pore-forming subunit CaV2.1 (2, 3, 29). Hence, they are now combined as P/Q-type CaV currents (2, 3, 29). CaV2.1 channels play a key role in neurotransmitter release (Table 1
) (2, 3).
CaV2.2 channels conduct N-type CaV currents, which fall in between the T- and L-type CaV currents in terms of biophysical properties. They display smaller unitary Ba2+ conductance, lower activation threshold, and faster inactivation rate than L-type CaV currents, but larger unitary Ba2+ conductance, higher activation threshold, and slower inactivation rate than T-type CaV currents (30). Apparently, such currents are neither T nor L. Additionally, they were only found in neurons in the earliest studies on CaV channels. Therefore, these currents were classified as a distinct type named N-type. N-type CaV currents are blocked by the peptide blocker
-conotoxin GVIA (
-CTX GVIA), but are insensitive to the organic CaV channel antagonists DHPs, phenylalkylamines, and benzothiazepines (30). CaV2.2 channels mainly mediate Ca2+ influx in active zones to trigger neurotransmitter release (Table 1
) (2, 3).
CaV2.3 channels were discovered in cerebellar granule cells subjected to combined treatment with the multiple CaV channel blockers nimodipine,
-CTX GVIA, and
-Aga IVA. The treated cerebellar granule cells showed a residual CaV current resistant to these CaV channel blockers (38). Therefore, this residual current resistant to all available CaV channel blockers was termed the R-type. The R-type CaV current is now no longer resistant to "everything." A novel toxin, SNX-482, with high affinity for CaV2.3 channels has been purified (40). However, it is worthwhile to note that SNX-482 is capable of blocking other types of CaV channels as well (11, 41). Therefore, caution should be exercised when using this CaV channel blocker to dissect native R-type CaV currents in cells containing multiple types of CaV channels. The R-type current is characterized by faster inactivation rate in comparison with other HVA Ca2+ channels. The CaV2.3 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. CaV3 channels.
CaV3 channels are categorized into CaV3.1, CaV3.2, and CaV3.3 channels according to their pore-forming subunits (2, 3, 29). These channel-mediated CaV 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 CaV currents (30). Like CaV1 channels, CaV3 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 CaV channels
1. Molecular composition of CaV channels.
During the past two decades, great progress has been made toward elucidating the molecular entities responsible for CaV currents. The Catterall group (31) has pioneered the field of CaV channel biochemistry. Initially, this group found that the skeletal muscle CaV channel consists of the CaV
1, CaVß, and CaV
subunits (31). Shortly thereafter, this group and several other groups demonstrated that the skeletal muscle CaV channel is composed of not only the CaV
1, CaVß, and CaV
subunits but also the CaV
2
subunit (32, 43, 44, 45, 46, 47). Accordingly, the Catterall group proposed a widely accepted model of the subunit structure of CaV channels on the basis of the systematic biochemical analysis of CaV1.1 channels (3, 32). In their experiments, the molecular mass, DHP binding site, PKA phosphorylation site, glycosylation, hydrophobicity, disulfide-bridge heterodimer, and noncovalent association of CaV1.1 channel subunits were carefully characterized. These biochemical properties demonstrated that hydrophobic CaV1.1 subunits noncovalently associate with a disulfide-linked CaV
2
dimer, an intracellular phosphorylated CaVß subunit, and a transmembrane CaV
subunit. In this complex, only the CaV1.1 subunit is large enough to contain four homologous transmembrane domains forming a Ca2+ conducting pore (3, 32). Other subunits, CaVß, CaV
, and CaV
2
subunits, modulate CaV 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 CaV channel subunits laid a solid foundation for the first-ever cloning of a CaV channel gene, CaV1.1 gene. In cloning this gene, the Numa group first isolated tryptic peptides derived from the purified CaV1.1 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 CaV1.1 gene (52). Thereafter, numerous CaV channel genes and their products have been extensively characterized (3, 29). The molecular composition of CaV channels has thereby become clearer (Fig. 1
).
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1 subunits.
1 genes: CACNA1S (CaV1.1), CACNA1C (CaV1.2), CACNA1D (CaV1.3), CACNA1F (CaV1.4), CACNA1A (CaV2.1), CACNA1B (CaV2.2), CACNA1E (CaV2.3), CACNA1G (CaV3.1), CACNA1H (CaV3.2), and CACNA1I (CaV3.3). Their chromosomal locations have been revealed in some species. In humans, these genes (in the order described above) have been mapped to 1q31-q32, 12p13.3, 3p14.3, Xp11.23, 19p13, 9q34, 1q25-q31, 17q22, 16p13.3, and 22q12.3-q13.2, respectively (29). Gene structure analysis has revealed that the large size of CaV
1 genes spans at least 250 kb in the human genome and contains up to 50 exon-intron boundaries. A number of these intron transcripts in pre-mRNAs of CaV
1 subunits contain alternative splicing sites (53). Alternative splicing results in diverse mRNAs with different insertion, deletion, truncation, and alternative sequence. One has proposed that each CaV
1 gene may contain at least 10 alternative splicing sites. If these 10 alternative splicing sites are regulated independently, alternative splicing could produce more than 1000 distinct mRNAs from each CaV
1 gene and in turn give rise to the corresponding number of splice variants at the protein level (53). A variety of CaV
1 subunit isoforms, resulting from alternative splicing, should account for diverse CaV channel-mediated signaling pathways.
Molecular characterization demonstrates that the CaV
1 subunit is the primary determinant of CaV channel biophysics. The CaV
1 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 (LI-II, LII-III, and LIII-IV), and N- and C-termini (Fig. 1
). This four homologous repeat structure endows the CaV
1 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 CaV 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 CaV channels (63).
The voltage-dependent allosteric change of the Ca2+-conducting pore is a fundamental process of CaV channels. To be able to experience voltage alteration, the CaV 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 CaV 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 CaV 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 CaV currents implies that the CaV channel is closed at both ends of the Ca2+-conducting pore (67). However, the structural basis of the CaV channel activation gate remains to be explored. During membrane depolarization, opened CaV channels become less permeable. This process is termed inactivation. In general, CaV 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 CaV
1 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 CaV
1 subunits although it is modulated by other auxiliary subunits (68). Molecular structures underlying CaV channel inactivation, i.e., CaV channel inactivation gates, have been investigated extensively. Inactivation analysis of point-mutated CaV
1 subunits and chimeric CaV
1 subunits has demonstrated that the CaV
1 subunit is equipped with multiple structural determinants of the voltage-dependent inactivation. These inactivation determinants are mainly localized in the LI-II, S5, and S6 segments and their adjacent P-loops (68).
A striking biochemical feature of the CaV
1 subunit is that this subunit contains multiple potential protein phosphorylation sites. For example, NetPhos 2.0 predicts that the rabbit CaV1.1 subunit carries 27 serine, seven threonine, and seven tyrosine residues with high phosphorylation potentials (Fig. 2
). Early studies showed that purified CaV1.1 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 CaV1.1 subunit (Fig. 2
) (74, 75, 76). PKA phosphorylation at S1757 and S1854 of the rabbit CaV1.1 subunit has also been demonstrated in intact cells (77). Furthermore, both in vitro and in vivo experiments have demonstrated that the rabbit CaV1.2 subunit S1928 is a substrate of PKA (78). In vitro phosphorylation at S1627 and S1700 of the bovine CaV1.2 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 CaV1.1 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 CaV1.2 subunit can be phosphorylated at S1928 by PKA, but the corresponding site of the bovine CaV1.2 subunit cannot (78, 79). PKC phosphorylation sites at the CaV1.2 subunit have not been biochemically identified. Electrophysiological analysis reveals that the CaV1.2 channel mutated at either T27 or T31 can no longer respond to PKC activation. This indicates that the CaV1.2 T27 and T31 must be phosphorylated by this protein kinase (81, 82). Furthermore, all known CaV
1 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 CaV1.1 and CaV1.2 subunits (32, 90). Interestingly, a short form of the CaV2.1 subunit, containing approximately the first half of the full-length CaV2.1 subunit, is glycosylated (91).
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1 subunit (3, 28). The CaVß subunits can be divided into five domains (D15) in terms of the similarities of their amino acid sequences. N-terminal D1, middle D3, and C-terminal D5 domains are significantly variable. D2 and D4 domains, connecting D1, D3, and D5, are highly conserved (48). Secondary structure prediction suggested that the CaVß subunit peptide is organized into PDZ-like, Src homology 3 (SH3), and guanylate kinase (GK) domains (96). This prediction has been verified by crystallographic analysis (for details, see Section II.B.2.a) (97, 98, 99). Although the ß-interaction domain (BID) in the CaVß subunit has long been considered as a crucial site interacting with the
1-interaction domain (AID) in the CaV
1 subunit, the crystal structure of CaVß subunits challenges this prevailing view. It seems that the AID cannot easily get access to the BID (see Section II.B.2.a). In addition to BID, a C-terminal region can also physically associate with the CaV
1 subunit (100). A recent study showed that two newly identified splice variants of the human cardiac CaVß2 subunit completely lack BID and
1-subunit binding pocket, but still efficiently interact with the CaV1.2 subunit to modulate its trafficking and biophysical properties (94). Generally speaking, the CaVß subunit exerts two major functions, i.e., enhancement of plasma membrane trafficking of the CaV
1 subunit and regulation of biophysical properties of CaV channels through its interaction with the CaV
1 subunit (28). However, distinct CaVß subunits can exhibit opposite effects, especially on inactivation kinetics. For example, coexpression of the CaVß2 subunit confers slower inactivation. On the contrary, coexpression of the CaVß3 subunit makes inactivation significantly faster (28, 101). Regarding the effects of CaVß subunits on the CaV channel trafficking, a detailed description will be given in Section II.B.3.b. The CaVß 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 CaVß1a subunit (102, 103). However, it is not clear whether phosphorylation of these two residues contributes to the regulation of channel activity (104). The CaVß2a 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 CaVß2a subunit are critical for the regulation of the activity of truncated CaV1.2 channels (108). Recently, the CaVß subunit has also been demonstrated to mediate MAPK modulation of CaV2.2 channels (109). Palmitoylation of the rat CaVß2a subunit has been extensively studied. The two N-terminal cysteines are palmitoylation sites (104, 110, 111). Functional effects of posttranslational modifications of CaVß2a subunits will be discussed in Section II.B.3.b.
c. CaV
subunits.
Originally, the CaV
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 CaV
subunit gene. Therefore, this gene was named CaV
2, and the first-identified skeletal muscle CaV
subunit gene was termed CaV
1 (112). Soon, six other CaV
subunit isoforms (CaV
38) were identified by searching the DNA database in terms of sequence homology to CaV
1 and CaV
2 genes (113, 114, 115, 116, 117). The chromosomal locations of human CaV
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 CaV
18 (114, 116). The CaV
5 and CaV
7 genes comprise five exons, but other isoforms consist of four exons (50, 117, 118). The CaV
1 is tightly associated with the CaV1.1 subunit and copurified with the CaV1.1 subunit (31, 32, 44). Amino acid sequence analysis indicates that the CaV
subunit is composed of a conserved four-transmembrane domain topology together with intracellular N and C termini (Fig. 1
). All known CaV
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, CaV
25 and CaV
78 bear a PDZ binding or related motif at the C terminus (117). CaV
subunits inhibit CaV channel activity and modulate its activation and inactivation kinetics (28). CaV
subunits have little effect on CaV channel trafficking (see Section II.B.3.b). However, the CaV
2 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. CaV
2
subunits.
Like other CaV channel subunits, the CaV
2
subunit was first copurified with the CaV1.1 subunit from the skeletal muscle preparation (32, 45, 46, 47). Subsequently, the CaV
2
subunit was found to associate with other CaV
1 subunits from other tissues (119, 120). In 1988, molecular cloning of the CaV
2
subunit was accomplished (121). Since then, no further CaV
2
subunit genes were uncovered for about 10 yr. After the successful identification of multiple CaV
subunit genes by searching the sequence database, the same strategy was applied to disclose novel CaV
2
subunit genes (122). It turned out that in the database, two novel genes encoding proteins with significant similarities to the previously identified CaV
2
subunit were identified (122). Thereafter, the previously identified CaV
2
subunit was referred to as the CaV
2
1, and the other two were known as the CaV
2
2 and CaV
2
3 (122). Recently, the human CaV
2
4 gene has been isolated and characterized (123). Human CaV
2
1, CaV
2
2, CaV
2
3, and CaV
2
4 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 CaV
2
1 subunits. The CaV
2
1 subunit exists in five splice variants within mouse tissues: CaV
2
1a, CaV
2
1b, CaV
2
1c, CaV
2
1d, and CaV
2
1e. Only the single isoforms CaV
2
1a and CaV
2
1b express in skeletal muscle and brain, respectively. Heart and smooth muscle tissues contain multiple isoforms of CaV
2
1 subunits (124). Two alternative spliced regions have been revealed in the human CaV
2
2 subunit. Three isoforms of this subunit, CaV
2
2a, CaV
2
2b, and CaV
2
2c subunit, have been found in human tissues. The human heart only expresses CaV
2
2a, whereas a human medullary thyroid carcinoma cell line contains all three isoforms, CaV
2
2a, CaV
2
2b, and CaV
2
2c (125). The human CaV
2
3 gene produces a full-length and a truncated transcript. No additional splice variants of the human CaV
2
3 gene are detected (126). The gene structure of the human CaV
2
4 is best characterized in the CaV
2
family. There are 39 exons in the human CaV
2
4 gene. Exons 237 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 CaV
2
4 subunit. Therefore, four splice variants, CaV
2
4a, CaV
2
4b, CaV
2
4c, and CaV
2
4d have been expected. However, these splice variants remain to be identified (123).
The CaV
2
subunit is a most highly glycosylated protein among CaV channel subunits (32, 127, 128). The premature CaV
2
subunit is a single polypeptide encoded by a single CaV
2
gene (121, 129). The posttranslational cleavage and disulfide linkage make up the mature form of the CaV
2
subunit, a two-peptide dimer linked by disulfide bonds, from the single precursor CaV
2
subunit (129, 130). CaV
2
subunits contain numerous glycosylation sites, cysteine residues, and hydrophobic sequences (121, 122, 123). Topological analysis indicates that CaV
2 is an entirely extracellular polypeptide and CaV
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 CaV
polypeptide in the plasma membrane. Interestingly, neither the transmembrane domain nor the intracellular part of the CaV
polypeptide is involved in the interaction of the CaV
2
subunit with the CaV
1 subunit. By contrast, both the entire CaV
2 and CaV
extracellular portion are responsible for their association with the CaV
1 subunit (131). The CaV
2 polypeptide functions as a crucial determinant required for stimulation of the current amplitude, whereas the CaV
domain mainly modulates voltage-dependent activation and steady-state inactivation (132). Distinct CaV
2
subunits have slightly different contributions to channel function (28, 132, 133). For example, coexpression of the CaV
2
1 subunit promotes CaV
1 subunit trafficking to the plasma membrane (also see Section II.B.3.b), increases current amplitude, and fastens activation and inactivation (132). The CaV
2
2 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 CaVß subunit, whereas others do not require the coexpression of the CaVß subunit (122, 127).
2. Molecular architecture of CaV channels.
Lack of knowledge about atomic level structures of CaV channel subunits and in particular CaV subunit complex prevents us from understanding a large number of critical issues concerning CaV channel subunit assembly, biophysical properties, and modulation mechanisms. The large size and multiple transmembrane segments of pore-forming CaV
1 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 CaV
1 subunits from heterologous expression systems including yeasts, bacteria, and mammalian cells. Therefore, CaV
1 subunits have not yet been crystallized and visualized at the atomic level. Although CaV
2
and CaV
subunits are smaller in size and they are either entirely extracellular (CaV
2 subunits) or equipped with fewer transmembrane segments (CaV
and CaV
subunits) than CaV
1 subunits, there is still no published information about crystal structures of these subunits.
a. Crystallographic structures of AID-CaVß subunit complexes.
Recently, a breakthrough has been made in understanding the structure of the AID-CaVß subunit complex at the atomic level. High-resolution x-ray crystallographic analysis not only allows us to understand the atomic disposition within the AID-CaVß subunit complex but also provides an insight into the molecular mechanisms of CaV channel regulation. The studies done by three different groups present several novel findings (97, 98, 99). First, the CaVß 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 CaVß subunit SH3 domain mainly consists of five ß-strands and two
-helices. The CaVß subunit GK domain primarily comprises five ß-strands and seven
-helices. In the AID-CaVß 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 CaVß 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 CaVß subunit GK domain (97, 98, 99). Second, the CaVß subunit directly modulates the movement of the IS6 segment in the CaV
1 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 CaVß subunit can perform a diverse range of tasks in terms of its common protein interaction domains (97, 98, 99).
b. Electron-microscopic structures of CaV channel subunit complexes.
As mentioned before, so far it has not been possible to crystallize CaV
1 subunits. Electron microscopy as an alternative approach has been used to visualize the molecular architecture of CaV channels. In the early 1980s, the two-dimensional electron microscopic structure of CaV1.1 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 CaV1.1 channels (138). Typically, four CaV1.1 channels are disposed in the corners of a small square. Therefore, this profile of CaV1.1 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 CaV1.1 channels and ryanodine receptors in excitation-contraction coupling (139). Successful purification of CaV1.1 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 CaV1.1 channels by freeze-dried, rotary shadow electron microscopy. They revealed that the purified CaV 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 CaV1.1 channels demonstrated that the ovoidal particle corresponds to a CaV channel complex composed of CaV
1, CaVß, CaV
, and CaV
2
subunits (140, 141).
All of the above two-dimensional structures of a CaV channel subunit complex were obtained in the 1980s. There were few documents about electron microscopic structures of CaV 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 CaV channel ultrastructure. The single particle analysis of cryoelectron microscopic images can reconstruct the refined 3D structure of a CaV1.1 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 CaV1.1 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 CaV1.1 and a CaVß subunit. However, the CaVß subunit does not completely cover the intracellular edge of the CaV1.1 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 CaV1.1 subunit, form a 2-nm central ion-conducting pore. The ball was identified as the CaV
2 subunit (145). Shortly afterward, the 3D structure of the CaV1.1 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 CaV1.1 subunit associated with the CaVß and CaV
subunits. However, they did not observe any ion-conducting pore feature within the heart-shaped region. The handle-shaped region was regarded as the CaV
2
complex, representing approximately 35% of total volume equal to about 140 kDa (142). Recently, the 3D structure of the CaV1.1 channel complex has been visualized at a 23-Å resolution, the highest resolution of an electron microscopic structure of the CaV1.1 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 CaV1.1 channel complex structure. The reconstructed 3D structure with an estimated total molecular mass of 550 kDa corresponds approximately to the assembly of the five CaV channel subunits, i.e., the CaV1.1, CaVß, CaV
, and CaV
2
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 CaV1.1 subunit. Antibody labeling and the calculated molecular mass indicate that the smaller subvolume contains both the CaVß and CaV
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-CaV
2 antibody. This indicates that the CaV
subunit is adjacent to or partially embedded within the CaV1.1 subunit (the larger subvolume) because the CaV
2 subunit is linked to the CaV
subunit by a disulfide bond (143). Additionally, Wang et al. (144, 146) reported a ring-shaped dimer of both CaV1.1 and CaV1.2 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 CaV channels.
3. Expression and trafficking of CaV channels
a. Expression and posttranslational modification of CaV channels.
To date, molecular cloning has documented 26 distinct genes encoding different CaV channel subunits (3, 28). CaV channel gene expression is controlled by the interaction of the CaV 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 CaV subunit genes will be expressed (147). However, it should be noted that CaV 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 CaV3 channels in mouse ß-cells (for further details, see Section IV.B) (148). Before innervation, myotubes are characterized by the expression of CaV3 channels. The CaV channel expression switches from CaV3 to CaV1.1 resembling the adult pattern shortly after myotubes are innervated. On the contrary, denervated muscles display the embryonic profile of CaV channel expression. However, these CaV channel phenotype switches depend on electrical activity rather than the presence of nerves because electrical stimulation suffices to keep the adult pattern of CaV channel expression in the denervated muscles (64).
Another important process is splicing of CaV 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 CaV channel subunit (149, 150). One has estimated that alternative splicing could give rise to over 1000 distinct mRNAs from each CaV
1 gene in terms of possible number of alternative exons (see Section II.B.1.a) (53).
The CaVß subunit as an intracellular protein is synthesized by free ribosomes. In contrast, CaV
1 and CaV
subunits as well as the precursor polypeptide of CaV
2
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 CaV 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 CaV 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 CaV 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 35 amino acids per second at 37 C. It probably takes about 10 min for a ribosome to synthesize a 2000-residue CaV
1 subunit (64).
Before a newly synthesized CaV 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 CaVß subunits (32). However, these subunits can be reversibly phosphorylated (48). The rat CaVß2a subunit, a particular isoform of CaVß2 subunits, is also reversibly palmitoylated at its N-terminal dicysteine motif (104, 110, 111). The transmembrane subunits CaV
and CaV
2
are permanently glycosylated (28, 32, 50, 127). Both in vitro and in vivo phosphorylation of CaV
1 have been experimentally demonstrated (3, 81). Several potential sites for serine or threonine phosphorylation have been revealed in most of the CaV
subunits (51, 115). Disulfide bridges between CaV
2 and CaV
are built in the ER lumen by protein disulfide isomerase (153, 154). Two other important processes in the maturation of CaV subunits are proper folding and assembly. Like other proteins, CaV subunits begin to fold during their synthesis. Eventually, molecular chaperones and posttranslational alterations help the CaV subunit molecule fold up into its unique 3D conformation. The proper folding and assembly of the transmembrane CaV
1, CaV
, and CaV
subunits, the intracellular CaVß subunit, and the extracellular CaV
2 subunit in the ER are prerequisites for CaV channel trafficking to the plasma membrane. The assembly of the CaV
1/ß complex in ER has been indicated by a heterologous expression system (155). The CaV1.1 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 CaV currents. In contrast, when CaVß1a is expressed alone in these cells, its distribution pattern is quite different from that of the expressed CaV1.1. The expressed CaVß1a is evenly distributed all over the cytoplasm without association with any cytoplasmic structure. However, the CaVß1a becomes colocalized with the coexpressed CaV1.1 in ER. Most importantly, coexpression of these two subunits endows the transfected cell with genuine CaV1.1 channels. This demonstrates that assembly of the CaV
1/ß complex in ER is a prerequisite for the maturation of CaV channels (155).
b. Trafficking and targeting of CaV channels.
CaV channel trafficking and targeting enable distinct areas of the cell to be equipped with particular types and densities of CaV channels for specific functions (156, 157, 158, 159, 160). Similar to other ion channels, newly synthesized CaV channel subunits are properly folded and assembled in the ER to build up CaV channel subunit complexes. Subsequently, these CaV 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 CaV channel subunit complex separately bud off from the trans-Golgi network using sorting mechanisms. Subsequently, these vesicles carry CaV 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 CaV channel subunit folding and assembly but also in CaV 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 CaV channel trafficking (127, 153). PKA phosphorylation significantly facilitates budding off constitutive secretory vesicles from the trans-Golgi network (162). Interactions among CaV channel subunits themselves and between CaV channel subunits and other nonchannel proteins take center stage in CaV channel trafficking, targeting, and anchoring (161, 163, 164). The section below discusses this aspect in detail.
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