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MINIREVIEW

-Cell Ca_V channel regulation in physiology and pathophysiology

The Rolf Luft Center for Diabetes Research, Karolinska Diabetes Center, Department of Molecular Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden

Submitted 29 January 2004 ; accepted in final form 17 September 2004

	ABSTRACT

TOP ABSTRACT PHYSIOLOGICAL TYPES OF CaV... MOLECULAR STRUCTURE OF Cav... {beta}-CELL CaV CHANNELS AND... {beta}-CELL CaV CHANNELS AND... CUSTOMARY MECHANISMS OF {beta}... REGULATION OF {beta}-CELL L-TYPE... REGULATION OF {beta}-CELL L-TYPE... REGULATION OF {beta}-CELL L-TYPE... CONCLUSIONS GRANTS REFERENCES

 
The -cell is equipped with at least six voltage-gated Ca²⁺ (Ca_V) channel ₁-subunits designated Ca_V1.2, Ca_V1.3, Ca_V2.1, Ca_V2.2, Ca_V2.3, and Ca_V3.1. These principal subunits, together with certain auxiliary subunits, assemble into different types of Ca_V channels conducting L-, P/Q-, N-, R-, and T-type Ca²⁺ currents, respectively. The -cell shares customary mechanisms of Ca_V channel regulation with other excitable cells, such as protein phosphorylation, Ca²⁺-dependent inactivation, and G protein modulation. However, the -cell displays some characteristic features to bring these mechanisms into play. In islet -cells, Ca_V channels can be highly phosphorylated under basal conditions and thus marginally respond to further phosphorylation. In -cell lines, Ca_V channels can be surrounded by tonically activated protein phosphatases dominating over protein kinases; thus their activity is dramatically enhanced by inhibition of protein phosphatases. During the last 10 years, we have revealed some novel mechanisms of -cell Ca_V channel regulation under physiological and pathophysiological conditions, including the involvement of exocytotic proteins, inositol hexakisphosphate, and type 1 diabetic serum. This minireview highlights characteristic features of customary mechanisms of Ca_V channel regulation in -cells and also reviews our studies on newly identified mechanisms of -cell Ca_V channel regulation.

exocytotic proteins; inositol hexakisphosphate; pancreatic -cell; type 1 diabetic serum; voltage-gated Ca²⁺ channels

A mission of paramount importance for -cell Ca_V channels is to trigger insulin exocytosis. When plasma glucose levels rise, resultant increases in glucose uptake into and metabolism by the pancreatic -cell lead to an increase in the ATP-to-ADP ratio, inhibiting -cell ATP-sensitive K⁺ channels and consequently depolarization of the plasma membrane. The membrane depolarization opens -cell Ca_V channels to mediate Ca²⁺ influx, thereby stimulating insulin secretion (9). -Cell Ca_V channel activity and/or density is regulated by a variety of mechanisms, such as cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i), protein phosphorylation, translocation, interaction with other proteins, and so on. Up- and downregulation of Ca_V channel activity and/or density result in more or less insulin exocytosis, respectively (1, 4, 41, 47, 51, 75, 79, 113, 116). Another important task of -cell Ca_V channels is to regulate -cell fate by controlling [Ca²⁺]_i dynamics (48, 49). Hyperactivation of -cell Ca_V channels leads to -cell death (48, 49). Recently, novel mechanisms of -cell Ca_V channel regulation have been revealed under physiological and pathological conditions. In this review, we will briefly summarize current views on the biophysical feature, structure, and function of -cell Ca_V channels. We will then highlight and discuss recent findings concerning molecular mechanisms of -cell Ca_V channel regulation.

Electrophysiological recording, pharmacological manipulation, biochemical purification, and molecular cloning have identified diverse Ca_V currents, Ca_V channel proteins, and genes. The researchers in these different fields used to employ different terminologies to describe these entities (25). Physiologists depicted Ca_V currents phenomenologically (such as long lasting and large conductance for L-type Ca_V currents). Biochemists named Ca_V channel proteins with Greek letters (₁-, -, -, and ₂-subunits). Molecular biologists described Ca_V channel mRNAs using the terminology class A, class B, etc. This made the nomenclature of Ca_V channels confusing. Therefore, a comprehensive nomenclature of Ca_V channels was proposed in 2000 on the basis of sequence analysis (25). It classifies Ca_V channels into three families Ca_V1, Ca_V2, and Ca_V3, consisting of closely related members. This nomenclature, known as the structural 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. In this review, the phenomenological nomenclature is used to describe Ca_V currents, and the structural nomenclature is used to describe Ca_V channel proteins and mRNAs. To comprehend the literature cited, some old terms are mentioned and followed by the structural nomenclature in brackets.

	PHYSIOLOGICAL TYPES OF CAV CHANNELS

TOP ABSTRACT PHYSIOLOGICAL TYPES OF CaV... MOLECULAR STRUCTURE OF Cav... {beta}-CELL CaV CHANNELS AND... {beta}-CELL CaV CHANNELS AND... CUSTOMARY MECHANISMS OF {beta}... REGULATION OF {beta}-CELL L-TYPE... REGULATION OF {beta}-CELL L-TYPE... REGULATION OF {beta}-CELL L-TYPE... CONCLUSIONS GRANTS REFERENCES

 
Our understanding of the diversity of Ca_V channels began with electrophysiological recordings. In 1975, Hagiwara et al. (30) recorded two types of Ca_V currents with distinct activation and inactivation from fertilized starfish eggs. They made the earliest classification of Ca_V channels, channel I (low-voltage activated, LVA) and channel II (high-voltage activated, HVA), on the basis of biophysical properties. Six years later, the LVA and HVA Ca²⁺ currents were also found in central neurons (60). Application of the patch clamp technique in combination with selective Ca_V channel blockers has been crucial in the further classification of Ca_V channels. The Tsien group has made a number of landmark studies on Ca_V channel classification using patch clamp analysis in combination with pharmacological manipulation. This group and others (59, 81, 104) identified five or six types of Ca_V currents, L-, P/Q-, N-, R-, and T-types, and proposed the presence of the corresponding types of Ca_V channels. The biophysical and pharmacological properties as well as localizations and functions of Ca_V channels are summarized in Table 1.

The L-, P/Q-, N-, and R-type Ca_V channels are opened by large depolarizations and belong to the HVA Ca²⁺ channel family. Its members can be discriminated according to their biophysical and pharmacological properties. L-type Ca_V channels are sensitive to dihydropyridines (DHPs), are characterized by a large unitary conductance, and conduct a long-lasting current. Therefore, these channels are named L-type Ca_V channels. This type of Ca_V channel is widely distributed in all excitable and some nonexcitable cells. They play important roles in excitation-contraction coupling, hormone secretion, [Ca²⁺]_i homeostasis, and gene regulation (15, 104).

N-type Ca_V channels display some biophysical properties, such as single-channel conductance and inactivation rate, intermediate between those of the T- and L-type Ca_V channels. These channels are neither T nor L and are blocked by -conotoxin GVIA. Originally they were found only in neurons and play a key role in neurotransmitter release. Accordingly, they are termed N-type Ca_V channels (15, 104).

P-type Ca_V channels were first identified in cerebellar Purkinje cells (59). Subsequently, Q-type Ca_V channels were discovered in cerebellar granule cells (81). Initially, the P- and Q-types were regarded as two distinct types on the basis of differences in their inactivation rate and sensitivity to -agatoxin IVA. These two types are now combined as P/Q-type Ca_V channels because both of them use the same principal subunit, Ca_V2.1, to conduct currents. The biophysical properties and functions of P/Q-type Ca_V channels highly overlap with those of N-type Ca_V channels (15, 104).

R-type Ca_V channels were found in the same experiment where Q-type Ca_V channels were characterized (81). In cerebellar granule cells, a residual Ca_V current was shown to be resistant to DHPs and N- and P/Q-type Ca_V channel toxins. A novel toxin, SNX-482, with high affinity for R-type Ca_V channels has been purified (71). Fast inactivation is a major biophysical difference between R-type and other HVA Ca²⁺ channels. The R-type Ca_V channel is a key player in generation of Ca²⁺-dependent action potentials and plays a role in neurotransmitter release (15, 104).

	MOLECULAR STRUCTURE OF CAv CHANNEL SUBUNITS

TOP ABSTRACT PHYSIOLOGICAL TYPES OF CaV... MOLECULAR STRUCTURE OF Cav... {beta}-CELL CaV CHANNELS AND... {beta}-CELL CaV CHANNELS AND... CUSTOMARY MECHANISMS OF {beta}... REGULATION OF {beta}-CELL L-TYPE... REGULATION OF {beta}-CELL L-TYPE... REGULATION OF {beta}-CELL L-TYPE... CONCLUSIONS GRANTS REFERENCES

 
Advanced molecular biology, protein chemistry, and X-ray crystallography enabled us to learn a great deal about Ca_V channels. The Catterall group (18) made the first solubilization and purification of Ca_V channel proteins from the transverse tubule membranes of skeletal muscle. Initially, they found that the skeletal muscle Ca_V channel consists of ₁-, -, and -subunits and later revealed an additional ₂-subunit (15). The Numa group (103) subsequently cloned the skeletal muscle _1S-subunit (Ca_V1.1) cDNA, which is the first cloned cDNA of Ca_V channels. Two years later, this group isolated the complete cDNA clone of the _1C-subunit (Ca_V1.2) from rabbit cardiac muscle and succeeded in functional expression of Ca_V1.2 channels in Xenopus oocytes (66). Shortly thereafter, the cDNA of the _1D-subunit (Ca_V1.3) was isolated from human pancreatic islets (95). Recently, the cDNA and amino acid sequences of _1G (Ca_V3.1) from the insulin-secreting cell line INS-1 have been determined (119). Until now, the primary structures of ten distinct ₁ and numerous auxiliary subunits have been identified by cDNA cloning and sequencing (Fig. 1) (3, 15, 25).

View larger version (48K): [in this window] [in a new window]   Fig. 1. A: molecular organization of voltage-gated Ca2+ (CaV) channel subunits in the plasma membrane. B: predicted topology of CaV channel subunits. C: nomenclature of CaV channel subunits.

-Subunits and other subunits are not directly involved in the formation of the Ca²⁺-conducting pore and thus are called auxiliary subunits. However, they do play important roles in the regulation of surface expression, gating properties, and voltage dependence of Ca_V channels (3, 15). Four distinct -subunits have been identified. The -subunit is entirely cytosolic and associates with the ₁-subunit. Importantly, this subunit is a substrate of protein kinase A. Modulation of Ca_V channel activity can result from -subunit phosphorylation. The -subunit has two major functions, i.e., enhancement of plasma membrane trafficking of the ₁-subunit and regulation of biophysical properties of Ca_V channels. However, distinct -subunits can exhibit opposite effects, especially on inactivation kinetics. For example, coexpression of the ₂-subunit makes inactivation slower. On the contrary, coexpression of the ₃-subunit significantly accelerates inactivation (3, 42). Molecular cloning has revealed four distinct ₂-subunits (₂₁–₂₄) (3). The ₂-subunit comes from the same gene and is a two-peptide dimer linked by disulfide bounds (3). Although ₂ is entirely extracellular and possesses a single transmembrane region, ₂ interacts with the ₁-subunit (3). Distinct ₂-subunits have slightly different contributions to channel function (3). Basically, coexpression of the ₂-subunits promotes ₁-subunit trafficking to the plasma membrane and increases current amplitude. In addition, channel activation and inactivation are also affected by the co-expression of some ₂-subunits (3). Some of these effects occur in the presence of the -subunit, whereas others do not require the co-expression of the -subunit (3). The -subunit was originally identified only in skeletal muscle. Recently, several -subunits (₁–₈) have been found within a wide variety of tissues (3). All the identified -subunits have no effect on channel trafficking (3); however, they inhibit channel activity and modulate activation and inactivation kinetics (Fig. 1) (3).

-Cell Ca_V channels are heterogeneous. Multiple ₁-subunit mRNAs and proteins have been revealed in insulin-secreting cells or islets (Table 2). All studies indicate that the Ca_V1.2, and in particular Ca_V1.3, subunit mRNAs and proteins are predominant in all tested insulin-secreting cells and islets (36, 46, 86, 93, 95, 116). The -cell from any tested species carries Ca_V1.2 and Ca_V1.3 subunit mRNAs (Table 2). The Ca_V1.2 subunit protein has been identified in mouse islet -cells as well as in HIT-15T, RINm5F, MIN6, and TC-3 cells (Table 2). The Ca_V1.3 subunit protein has been detected in mouse islet -cells and in INS-1 and RINm5F cells (Table 2). More interestingly, expansion of the ATG trinucleotide repeat in the human Ca_V1.3 gene (CACNL1A2) from seven to eight was found only in type 2 diabetics. Although the frequency of this mutation is low and not associated with the development of common type 2 diabetes, it may be involved in the pathogenesis of a subgroup of this polygenic disease (114, 115). A Japanese family with spinocerebellar ataxia type 6 caused by mutations of the Ca_V2.1 gene (CACNL1A4) is highly associated with type 2 diabetes (100). For the rest of the ₁-subunits, there are obvious interspecies differences (Table 2). ₁-Subunit mRNAs and proteins in islet -cells and insulin-secreting cell lines from different species are listed in Table 2. The ₂₂-subunit mRNA has been revealed in the human pancreas (26). The ₂- and ₃-subunit mRNAs have been found in rat pancreatic islets. Competitive RT-PCR shows that the ₂-subunit mRNA level is much higher than the ₃-subunit mRNA level, suggesting that the ₂-subunit is predominant in rat pancreatic islets (45). We have revealed both ₂- and ₃-subunits at the mRNA and protein levels in mouse islets (Berggren P-O, unpublished observations). It is unknown whether the -subunit is expressed in the -cell. Although some -cell Ca_V channel mRNA and protein isoforms were identified using fluorescence-activated cell sorting and immunocytochemical approaches, others were manifested in islet tissues. Therefore, caution is needed in interpretation of the results from the islet tissue, as it contains four types of endocrine cells as well as nerve endings and capillaries. Application of optical tweezers, fluorescence-activated cell sorting, and single-cell PCR will provide more detailed information on -cell Ca_V channel mRNA and protein isoforms.

	-CELL CAV CHANNELS AND INSULIN SECRETION

TOP ABSTRACT PHYSIOLOGICAL TYPES OF CaV... MOLECULAR STRUCTURE OF Cav... {beta}-CELL CaV CHANNELS AND... {beta}-CELL CaV CHANNELS AND... CUSTOMARY MECHANISMS OF {beta}... REGULATION OF {beta}-CELL L-TYPE... REGULATION OF {beta}-CELL L-TYPE... REGULATION OF {beta}-CELL L-TYPE... CONCLUSIONS GRANTS REFERENCES

Physiological and pharmacological studies have revealed that the L-type Ca_V channel is expressed in all the islet -cells and insulin-secreting cell lines from any species tested (Table 2). The -cell L-type Ca_V channel has the same biophysical features as the neuronal L-type Ca_V channel: normally large single-channel conductance, long-lasting kinetics, and high sensitivity to DHPs. There is now consensus that the L-type Ca_V channel is the major Ca_V channel type playing a predominant role over other types of Ca_V channels in Ca²⁺-triggered insulin exocytosis (5, 89, 96). However, the proportion of L-type Ca_V currents to total Ca_V currents in the -cell varies among species. The majority of Ca_V currents in the mouse islet -cell flow through the L-type Ca_V channel. Therefore, the mouse pancreatic -cell was thought to be equipped with only L-type Ca_V channels (5). However, later studies revealed the presence of other types of Ca_V channels as well in the mouse pancreatic -cell (94). Also in the rat islet -cell, the L-type Ca_V channel dominates, although this cell carries more types of Ca_V channels than the mouse -cell (Table 2). A smaller number of Ca_V channel studies have been performed in human -cells. However, data available clearly demonstrate that L-type Ca_V channels underlie the principal HVA Ca²⁺ currents of human -cells (96). Pharmacological experiments demonstrate that 60–80% of glucose-induced insulin secretion from mouse, rat, and human -cells, equipped with various types of Ca_V channels, is attributed to Ca²⁺ influx through the L-type Ca_V channel (19, 72, 94). Hence, although insulin-secreting cell lines use various types of Ca_V channels to trigger insulin exocytosis, L-type Ca_V channels still play a major role (89, 91, 96, 105).

There are two subtypes of -cell L-type Ca_V channels: Ca_V1.2 and Ca_V1.3 channels (69, 94, 113, 116). The distinct contribution of Ca_V1.2 and Ca_V1.3 subtypes to insulin exocytosis has not been thoroughly studied in different species and remains controversial. L-type Ca_V channel subtype-specific regulation of insulin secretion has not been examined in human islet -cells. In rat -cells, the level of _1D-subunit mRNA is 2.5 times higher than that of _1C-subunit mRNA (46). Recently, -cell Ca_V1.2-specific knockout mice have been created. Mouse -cells lacking Ca_V1.2 subunit exhibit a decrease in Ca_V currents by 45%, an inhibition of first-phase insulin secretion by 80%, and glucose intolerance (94). L-type Ca_V channel blockers had no effect on Ca_V channel currents and insulin release from -cells lacking Ca_V1.2 subunits. Furthermore, previous studies showed negative Ca_V1.3 subunit-like immunoreactivity in mouse pancreatic -cells (7, 94). Those results led to the conclusion that only Ca_V1.2 subunits conduct L-type Ca_V currents in mouse pancreatic -cells and play a crucial role in stimulus-secretion coupling. However, the presence of Ca_V1.3 subunit mRNAs and proteins in mouse pancreatic -cells has been clearly demonstrated by other groups (69, 116). Additionally, Ca_V1.3 subunit knockout mice displayed the compensatory overexpression of Ca_V1.2 subunit proteins in -cells (69). Electrophysiological analysis showed that there was no difference in either total voltage-gated Ba²⁺ current density or L-type current density between mutant and control cells. However, biophysical properties of L-type Ca_V currents in Ca_V1.3 subunit-deficient -cells were significantly altered. The current-voltage relationship of the mutant -cells was shifted by 10 mV toward more positive potentials at the lower voltage range. Furthermore, mutant islets secreted less insulin than control islets in the presence of 3 mM glucose. However, insulin secretion from mutant islets was similar to that from control islets when subjected to 6 mM or higher concentrations of glucose. This indicates that overexpression of Ca_V1.2 subunits indeed compensates for the loss of Ca_V currents conducted by Ca_V1.3 subunits and thereby maintains insulin secretion capacity. Hence, -cell Ca_V1.3 subunits in wild-type mouse -cells are likely to play an important role in basal insulin secretion and also stimulus-secretion coupling at the lower range of glucose concentrations (69). The compensatory responses in -cells may differ between Ca_V1.2 and Ca_V1.3 subunit-deficient mice. Apparently, the distinct contribution of Ca_V1.2 and Ca_V1.3 subtypes to insulin exocytosis remains to be elucidated.

The selective N-type Ca_V channel blocker -conotoxin GVIA has been used to identify N-type Ca_V channels in -cells (Table 2). The mouse islet -cell does not appear to possess N-type Ca_V channels, as application of -conotoxin GVIA did not affect the mouse -cell Ca_V currents (96). Evidence for the presence of N-type Ca_V channels in the rat islet -cell was obtained by measuring [Ca²⁺]_i (80). This study revealed that arachidonic acid induced Ca²⁺ influx into purified rat pancreatic -cells (80). The L-type Ca_V channel blocker nifedipine only partially blocked the effect. Interestingly, -conotoxin GIVA, an N-type Ca_V channel blocker, decreased arachidonic acid-induced Ca²⁺ influx by a magnitude similar to that of nifedipine. This indicates that rat -cell N-type Ca_V channels mediate Ca²⁺ influx induced by arachidonic acid (80). Electrophysiological and pharmacological evidence does not support N-type Ca_V channels being situated in human -cells (19). Some groups have detected N-type Ca_V currents in HIT-15T, RINm5F, and INS-1 cells (89, 91, 96). Contradictory to this, other groups reported that whole cell Ca_V currents in HIT-15T, RINm5F, and INS-1 cells were insensitive to -conotoxin GVIA (64, 89, 96). The role of N-type Ca_V channels in insulin exocytosis is controversial. The N-type Ca_V channel blocker -conotoxin GVIA indeed gave a measurable inhibition of the second phase of glucose-induced insulin secretion from rat islets but had no effects on the first phase. The effect on the second phase of glucose-induced insulin secretion was attributed to toxic effects of the high concentration of -conotoxin GVIA used (53).

-Cell P/Q-type Ca_V currents have been recorded in various types of -cells, including mouse, rat, and human islet -cells as well as INS-1 and RINm5F cell lines (Table 2). Recently, the presence of P/Q-type Ca_V channels has been verified in the mouse islet -cell. A mixture of the L-type Ca_V blocker isradipine and R-type Ca_V blocker SNX-482 reduced Ca_V currents recorded from the mouse islet -cell by 80%. A cocktail of isradipine, SNX-482, and -agatoxin IVA almost fully blocked mouse -cell Ca_V currents (94). The role of P/Q-type Ca_V channels in stimulus-secretion coupling of mouse -cells remains to be examined. The involvement of P/Q-type Ca_V channels in insulin secretion from rat -cells has been demonstrated by electrophysiological and pharmacological means (58). The P/Q-type Ca_V channel blocker -agatoxin IVA partially blocks HVA Ca²⁺ currents in the rat -cell and inhibits the DHP-resistant component of glucose-induced insulin secretion by 30% (58). Previous work showed that a portion of Ca_V currents and Ca²⁺-dependent insulin secretion in human islet cells remained in the presence of both the L-type Ca_V channel blocker nifedipine and the N-type Ca_V channel blocker -conotoxin GVIA (19). Recently, 25% of human -cell Ca_V currents have been verified as P/Q-type Ca_V currents by application of -agatoxin IVA. The effect of -agatoxin IVA on insulin release from human -cell is significant, but less than that of the L-type Ca_V channel blocker (96).

It was difficult to evaluate whether the R-type Ca_V channel was present in -cells and involved in Ca²⁺-dependent insulin secretion. These problems were solved by the application of mice lacking the Ca_V2.3 (_1E) subunit and selective peptide antagonist of the R-type Ca_V channel (71, 76). Pharmacological manipulation has dissected R-type Ca_V currents from whole cell Ca_V currents in mouse islet -cells and INS-1 cells (Table 2). The Ca_V2.3 subunit-selective peptide antagonist SNX-482 inhibits 60% of isradipine-resistant Ca_V currents in mouse islet -cells (94). Evidence indicates that Ca²⁺ influx through R-type Ca_V channels is coupled to insulin exocytosis (28, 76, 105, 106). Ca_V2.3-deficient mice exhibited disturbances in glucose tolerance and insulin secretion as well as hyperglycemia (76). An appreciable proportion of the increase in mouse -cell capacitance, reflecting insulin exocytosis in response to depolarizations, can be blocked by SNX-482. Glucose- and KCl-induced insulin secretion from INS-1 cells was inhibited by SNX-482 in a dose-dependent manner (105).

Although the mouse -cell does not carry T-type Ca_V channels, they have been identified in human and rat islet -cells as well as in RINm5F and INS-1 cells (Table 2). Interestingly, the occurrence of T-type Ca_V channels has been observed in nonobese diabetic (NOD) mouse -cells (108). The -cell T-type Ca_V channel is likely to be a player in stimulus-secretion coupling (13, 52, 68). The T-type Ca_V channel blocker NiCl₂ has been shown to inhibit insulin secretion from INS-1 cells in a dose-dependent manner (13). However, it is not known about the role of the T-type Ca_V channel in insulin secretion from rat and human islet -cells. It would be attractive to evaluate the possible contribution of the T-type Ca_V channel to stimulus-secretion coupling in these islet -cells.

	-CELL CAV CHANNELS AND -CELL DEATH

TOP ABSTRACT PHYSIOLOGICAL TYPES OF CaV... MOLECULAR STRUCTURE OF Cav... {beta}-CELL CaV CHANNELS AND... {beta}-CELL CaV CHANNELS AND... CUSTOMARY MECHANISMS OF {beta}... REGULATION OF {beta}-CELL L-TYPE... REGULATION OF {beta}-CELL L-TYPE... REGULATION OF {beta}-CELL L-TYPE... CONCLUSIONS GRANTS REFERENCES

 
The -cell Ca_V channel also plays an important role in the maintenance of -cell viability. It coordinates with non-voltage-gated Ca²⁺ channels, Ca²⁺ buffering systems, and Ca²⁺ pumps to effectively control dynamics and homeostasis of [Ca²⁺]_i, a life-and-death signal (10, 11, 48, 74). The sophisticated interplay between Ca_V channels and non-voltage-gated Ca²⁺ channels and Ca²⁺ buffering systems as well as Ca²⁺ pumps in the cell can form an infinity of functional combinations spatially and/or temporally. Some of them play an important role in cell growth, proliferation, and differentiation. Others trigger necrosis and apoptosis. Therefore, it is easy to understand why [Ca²⁺]_i can be either a life or a death signal (11). Generally speaking, Ca²⁺ influx through the properly opened Ca_V channels provides cells with a life signal. On the contrary, hyperactivation of Ca_V channels may result in too high a [Ca²⁺]_i, this divalent cation then serving as a death signal (74). For example, the enhancement of -cell L-type Ca_V activity by type 1 diabetic serum causes typical apoptosis (48). Moreover, the long-term L-type Ca_V channel opening induced by glucose and tolbutamide results in pancreatic -cell apoptosis (24). Cytokines trigger pancreatic -cell death through activation of T-type Ca_V channels (109). The loss of -cells occurs in both type 1 and type 2 diabetes. Type 1 diabetes is characterized by the absolute loss of pancreatic -cells; type 2 diabetes is defined by not only the progressive loss of -cell function but also increased -cell apoptosis (62). It is likely that hyperactivation of -cell Ca_V channels is involved in the loss of -cells in both type 1 and 2 diabetes (49).

	CUSTOMARY MECHANISMS OF -CELL CAV CHANNEL REGULATION

TOP ABSTRACT PHYSIOLOGICAL TYPES OF CaV... MOLECULAR STRUCTURE OF Cav... {beta}-CELL CaV CHANNELS AND... {beta}-CELL CaV CHANNELS AND... CUSTOMARY MECHANISMS OF {beta}... REGULATION OF {beta}-CELL L-TYPE... REGULATION OF {beta}-CELL L-TYPE... REGULATION OF {beta}-CELL L-TYPE... CONCLUSIONS GRANTS REFERENCES

 
Unlike receptors, there are no endogenous selective activators or inhibitors to control individual types of Ca_V channels. Physiologically, only the degree of membrane depolarization determines the opening of LVA or HVA Ca²⁺ channels. There must be some other distinct mechanisms to fine tune individual Ca_V channel functions. The striking structural differences among Ca²⁺-conducting pore subunits lay a foundation for distinct regulations of Ca_V channels (15).

Phosphorylation, the most prevalent reversible, covalent modification, is a highly effective means of regulating the activities of target proteins. Regulation of L-type Ca_V channels by protein phosphorylation is an important example. A variety of protein kinases and phosphatases are present in the pancreatic -cell (Fig. 2) (22, 70, 97). Activation of cAMP-dependent protein kinase (PKA) markedly increases Ca²⁺-dependent insulin secretion from permeabilized rat pancreactic islets (102). However, activation of PKA leads to only a marginal increase in L-type Ca_V currents in mouse pancreatic -cells, which accounts for a minor proportion of the total increase in insulin exocytosis by PKA (1, 2). A positive impact on insulin exocytosis by protein kinase C (PKC) activation has been found in rat pancreatic -cells (102). Interestingly, regulation of L-type Ca_V channels by PKC-mediated phosphorylation is quite different. Acute application of a PKC activator does not affect -cell Ca_V channel activity (4). However, -cell Ca²⁺ influx through -cell Ca_V channels dramatically decreases after deprivation of PKC (4). This means that PKC plays a tonic role in maintaining a proper function of -cell Ca_V channels and stimulus-secretion coupling (4). cGMP-dependent protein kinase (PKG) is present in the pancreatic -cell (118). The effects of PKG activators 8-bromo-cGMP and dibutyryl-cGMP on -cell Ca_V channels have been evaluated by measuring [Ca²⁺]_i or patch clamp techniques; however, the results are not consistent. Although [Ca²⁺]_i measurements in combination with application of L-type Ca_V channel blockers indicate that cGMP increased Ca²⁺ influx through L-type Ca_V channels in rat pancreatic -cells, direct recordings of Ca_V channel currents show that cGMP does not alter Ca_V channel activity in mouse -cells (43, 118). It should be noted that cGMP analogs can produce a direct effect on Ca_V channels bypassing PKG (20). Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) is expressed in pancreatic -cells and is involved in the regulation of insulin secretion (22). It is well known that binding of Ca²⁺/calmodulin to the L-type Ca_V channel is responsible for Ca²⁺-dependent inactivation of this channel (98). However, it is unknown whether CaM kinase II per se modulates -cell Ca_v channel activity. Tyrosine kinase signaling plays an important role in regulation of -cell proliferation, survival, and differentiation (112). It is still unclear whether tyrosine kinases affect -cell Ca_V channels by phosphorylating them, although Ca²⁺ influx through -cell Ca_V channels has been shown to partially contribute to the increase in [Ca²⁺]_i produced by stimulation of insulin receptors (84). The marginal or no changes in Ca_V channel activity following activation of the aforementioned protein kinases in primary -cells indicate that there is a striking difference in Ca_V channel regulation by protein phosphorylation between primary -cells and other excitable cells, such as neurons and muscles. It is likely that Ca_V channels in primary -cells are highly phosphorylated under basal conditions. Therefore, it is difficult to further phosphorylate these channels following stimulation (1, 2, 4).

View larger version (54K): [in this window] [in a new window]   Fig. 2. Schematic representation of customary mechanisms of CaV channel regulation by protein phosphorylation, G protein, and Ca2+/calmodulin as well as stimulus-secretion coupling in the pancreatic -cell. AC, adenylyl cyclase; Ca2+/CaM, Ca2+/calmudulin; CAC, citric acid cycle; DAG, diacylglycerol; G, GTP-binding protein; InsP3, inositol 1,4,5-triphosphate; P, phosphoryl group; PIP2, phosphatidylinositol 4,5-bisphosphate; PKA, protein kinase A; PKC, protein kinase C; PLC: phospholipase C; PPase, protein phosphatase.

Ca²⁺ not only flows through Ca_V channels to produce currents but also functions as the feedback regulator of these channels. The Ca²⁺-dependent inactivation of L-type Ca_V channels is thought to offer an important physiological feedback mechanism protecting against Ca²⁺ overload resulting from activation of these channels during action potentials (98). The Ca²⁺-dependent inactivation of -cell L-type Ca_V channels was first described in the mouse pancreatic -cell, where the majority of voltage-activated Ca²⁺ currents are mediated by L-type Ca_V channels (79). Later on, this phenomenon was also found in -cells from other species (52). Ca²⁺-dependent inactivation of -cell L-type Ca_V channels results in a Ca²⁺ current decay during depolarization. The most marked decay of -cell Ca_V currents occurs at the potential evoking the largest current. The -cell Ca_V current decay disappears when the charge carrier Ca²⁺ is replaced with Ba²⁺. Originally, the binding of Ca²⁺ or some mediators activated by Ca²⁺ to the Ca_V channel was proposed to be responsible for the Ca²⁺-dependent inactivation (52, 79). Now the COOH-terminal IQ domain of the Ca_V1.2 subunit has been demonstrated to bind Ca²⁺-activated calmodulin. This binding initiates Ca_V conformational changes, which cause Ca²⁺-dependent inactivation (98).

Inhibitory coupling between G proteins and Ca_V channels is one of the classical pathways of Ca_V channel regulation. This regulation is voltage dependent and membrane delimited. N-type and P/Q-type Ca_V channels in particular are modulated by direct interaction with G proteins. Ca_V channel regulation by the direct membrane-delimited interaction with G protein is characterized by a positive shift in the voltage dependence and a slowing of channel activation (21). The G-subunit was thought to be responsible for this action on the channel (21, 35). However, later the G-subunits were demonstrated to play the key role in regulation of Ca_V channels (21). It is clear that G-subunits bind to the I-II linker of N-type and P/Q-type Ca_V channels. The binding site in the I-II linker has been mapped. The QQIER sequence is essential for G binding (21). Additionally, NH₂ and COOH termini of the Ca_{V 1}-subunit are also involved in G-binding (21). It has been suggested that activation of G protein-coupled receptors significantly inhibits insulin secretion via inhibition of -cell Ca_V channel activity (27, 39, 40, 50). Indeed, P/Q-type Ca_V channels are present in pancreatic -cells (19, 36, 58, 63, 99). However, the L-type Ca_V channel in insulin-secreting cell lines is the major mediator of the inhibition of insulin secretion by the activation of G protein-coupled receptors, including ₂-adrenergic, galanin, and somatostatin receptors (27, 39, 40, 50). Conversely, the stimulation of these G protein-coupled receptors in mouse islet -cells does not influence voltage-activated Ca²⁺ influx (82). Moreover, the low conductance G protein-dependent K⁺ channel is drastically activated by the stimulation of the mouse -cell ₂-adrenergic receptors (85). Indeed, emerging evidence indicates that Ca_V1.2 channels are negatively regulated by a direct membrane-delimited interaction with G proteins. A recent study shows that the G-subunits directly bind to cytosolic NH₂ and COOH termini of the Ca_V1.2 subunit and significantly inhibit L-type Ca_V channel activity. It should be noted that in vitro binding assays and coexpression in Xenopus oocytes were employed in this study (44). Interestingly, it has been demonstrated that the activation of µ-opioid receptors dramatically increases Ca_V1.3 channel activity (93). Collectively, the molecular mechanisms whereby the activation of G protein-coupled receptors regulate L-type Ca_V channel activity and insulin secretion remain to be established.

	REGULATION OF -CELL L-TYPE CAV CHANNELS BY EXOCYTOTIC PROTEINS

TOP ABSTRACT PHYSIOLOGICAL TYPES OF CaV... MOLECULAR STRUCTURE OF Cav... {beta}-CELL CaV CHANNELS AND... {beta}-CELL CaV CHANNELS AND... CUSTOMARY MECHANISMS OF {beta}... REGULATION OF {beta}-CELL L-TYPE... REGULATION OF {beta}-CELL L-TYPE... REGULATION OF {beta}-CELL L-TYPE... CONCLUSIONS GRANTS REFERENCES

 
In the early 1990s, antibodies against syntaxin or synaptotagmin were reported to coimmunoprecipitate -conotoxin-binding proteins (8, 57). This was indicative of possible regulation of Ca_V channels by exocytotic proteins. Considerable efforts have been made to investigate the physical association and functional interaction of Ca_V channels with exocytotic proteins. The molecular mechanisms of the regulation of N- and P/Q-type Ca_V channels by exocytotic proteins have been manifested by combining the techniques of electrophysiology, protein chemistry, and molecular biology (12, 15). Although these mechanisms are not involved in the regulation of neuronal L-type Ca_V channels, they do exert direct control of -cell L-type Ca_V channels and represent an additional mechanism whereby the -cell Ca_V channel can be regulated. Experimental evidence has demonstrated that the L-type Ca_V channel has a similar association with the exocytotic machinery as the neuronal N- and P/Q-type Ca_V channel (47, 51, 113, 116).

Deconvolution analysis of fluorescence images revealed that the expressed Ca_V1.3 subunit-enhanced green fluorescent protein (EGFP) and enhanced blue fluorescent protein-syntaxin 1 colocalized in the -cell plasma membrane (116). Furthermore, subcellular fractionation showed that the endogenous Ca_V1.3 subunit and syntaxin 1A also colocalized in the -cell plasma membrane. This led to the hypothesis that syntaxin 1A might interact with the Ca_V1.3 subunit in the pancreatic -cell, and this was subsequently found to be the case (116). Interestingly, the polyclonal antibody against the intracellular domain of syntaxin 1A efficiently coimmunoprecipitated the Ca_V1.3 subunit from the -cell plasma membrane fractions. These results strongly suggest that syntaxin 1A forms a complex with the Ca_V1.3 subunit of the L-type Ca_V channel. The physical association of the Ca_V1.3 subunit with syntaxin 1A exhibits clear functional consequences. On the one hand, -cell L-type Ca_V channel activity drastically runs down following anti-syntaxin 1A antibody interference with the formation of a syntaxin 1A-Ca_V1.3 subunit complex. On the other hand, the dissociation of syntaxin 1A from the Ca_V1.3 subunit dramatically perturbs insulin exocytosis independently of the rundown of L-type Ca_V channel activity. This indicates that the syntaxin 1A-Ca_V1.3 subunit complex plays an important role in maintaining both L-type Ca_V channel activity and syntaxin 1A function (116).

The -cell Ca_V1.2 channel is also modulated by exocytotic proteins (113). Pull-down experiments with His₆-fused Ca_V1.2 subunit peptides show that syntaxin 1A, synaptosome-associated protein of 25 kDa (SNAP-25), and synaptotagmin physically associate with the Ca_V1.2 channel at the II-III loop of the Ca_V1.2 subunit (L_C753–893) (113). The coexpressed syntaxin 1A slightly alters the inactivation and activation rate and significantly decreases the amplitude of Ca_V1.2 currents recorded in Xenopus oocytes injected with Ca_V1.2/_2A/₂. The inhibitory effects are partially reversed by the coexpression of synaptotagmin. The interruption of the physical association of the Ca_V1.2 channel with exocytotic proteins by the intracellular application of Ca_V1.2_753–893 peptide almost completely blocks depolarization-evoked exocytosis without significant influence on Ca²⁺ influx (113). Similar effects were observed in insulin-secreting cell lines overexpressing syntaxin 1A and 3. Overexpression of syntaxin 1A and 3 dramatically inhibits L-type Ca_V channel activity and Ca²⁺-dependent insulin secretion in insulin-secreting cell lines (51).

Modulation of L-type Ca_V channel activity by distinct domains within SNAP-25 has been characterized in -cells (47). L-type Ca_V currents in mouse -cells are significantly decreased by intracellular application of SNAP-25_(1–206). The coapplication of Ca_V1.2_753–893 peptide occludes the reduction in L-type Ca_V currents. HIT cells overexpressing or injected with wild-type SNAP-25 show smaller L-type Ca_V currents than control cells. This inhibition is also prevented by the Ca_V1.2_753–893 peptide. Interestingly, expression of SNAP-25_(1–197) increases L-type Ca_V currents, and these effects are blocked by the Ca_V1.2_753–893 peptide. In contrast, intracellular application of SNAP-25_(198–206) into untransfected cells significantly reduces L-type Ca_V currents, these inhibitory effects dominating over the stimulatory effects of SNAP-25_(1–197) overexpression. The results clearly reveal that the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein SNAP-25 possesses distinct inhibitory and stimulatory domains that act on the L-type Ca_V channel (47).

Collectively, the -cell L-type Ca_V channel physically associates with the exocytotic machinery. This physical association may not only serve as a fine-tuning mechanism of -cell L-type Ca_V channel function but also as an anchoring machinery to optimally organize this channel at the site of insulin exocytosis (Fig. 3).

View larger version (31K): [in this window] [in a new window]   Fig. 3. Newly characterized mechanisms of -cell CaV channel regulation in physiology and pathophysiology. Pathways of -cell CaV channel regulation by exocytotic proteins, inositol hexakisphosphate (InsP6), and type 1 diabetic (T1D) serum are illustrated.

	REGULATION OF -CELL L-TYPE CAV CHANNELS BY INOSITOL HEXAKISPHOSPHATE

TOP ABSTRACT PHYSIOLOGICAL TYPES OF CaV... MOLECULAR STRUCTURE OF Cav... {beta}-CELL CaV CHANNELS AND... {beta}-CELL CaV CHANNELS AND... CUSTOMARY MECHANISMS OF {beta}... REGULATION OF {beta}-CELL L-TYPE... REGULATION OF {beta}-CELL L-TYPE... REGULATION OF {beta}-CELL L-TYPE... CONCLUSIONS GRANTS REFERENCES

The aforementioned results attracted us to examine the possible role of InsP₆ in the regulation of -cell Ca_V channels. As we have learned more recently, InsP₆ significantly inhibits the activity of purified catalytic subunits of serine/threonine protein phosphatase types 1, 2A, and 3 as well as corresponding holoenzymes in insulin-secreting cell extracts (55). In addition to the plasma membrane receptor-mediated pathway, glucose stimulation also results in a rapid InsP₆ rise in insulin-secreting cells (55). Furthermore, intracellular application of InsP₆ dramatically potentiates L-type Ca_V channel activity in insulin-secreting cell lines (55). These results lead to the conclusion that, under physiological conditions, -cell L-type Ca_V channels are activated not only by glucose metabolism-mediated depolarization but also by glucose-induced elevation of intracellular InsP₆, which inhibits protein phosphatases and as well activates other mechanisms (see below) leading to an increase in -cell L-type Ca_V channel activity (Fig. 3) (55).

The above-mentioned study raises two questions. 1) Does InsP₆ selectively modulate L-type Ca_V channels? 2) Are there other mechanisms involved in the modulation of L-type Ca_V channels by InsP₆? To tackle these questions, we chose hippocampal neurons because they are equipped with all known physiological types of Ca_V channels, including L-, P/Q-, N-, R-, and T-types, and should also contain other possible InsP₆-mediated signaling pathways (14, 33, 34, 101). We observed that intracellular application of InsP₆ selectively enhances L-type Ca_V currents, although multiple types of Ca_V channels exist in the hippocampal neuron. Interestingly, we found that InsP₆ significantly increases adenylyl cyclase (AC) activity in hippocampal membrane preparations without influencing cAMP phosphodiesterase (PDE). Physiological consequences of the InsP₆ effect on AC were examined in both biochemical and electrophysiological experiments. In the presence of InsP₆, more cAMP is produced by AC in the hippocampal membrane preparation, resulting in a more effective activation of PKA in the hippocampal cytosol, compared with in the absence of InsP₆. Furthermore, the effect of 8-(4-chlorophenylthio)-cAMP, a membrane-permeable cAMP analog, on L-type Ca_V channel activity is counteracted by pretreatment with InsP₆ (117).

The combination of our findings in -cells and in hippocampal neurons leads to the novel view that InsP₆ acts as a general intracellular signaling molecule to fine tune L-type Ca_V channel activity via both inhibition of protein phosphatases and stimulation of the AC-PKA cascade in native excitable cells (Fig. 3).

	REGULATION OF -CELL L-TYPE CAV CHANNELS BY TYPE 1 DIABETIC SERUM

TOP ABSTRACT PHYSIOLOGICAL TYPES OF CaV... MOLECULAR STRUCTURE OF Cav... {beta}-CELL CaV CHANNELS AND... {beta}-CELL CaV CHANNELS AND... CUSTOMARY MECHANISMS OF {beta}... REGULATION OF {beta}-CELL L-TYPE... REGULATION OF {beta}-CELL L-TYPE... REGULATION OF {beta}-CELL L-TYPE... CONCLUSIONS GRANTS REFERENCES

 
Under certain pathophysiological conditions, an increased Ca²⁺ influx through the hyperactivated Ca_V channels can overload cells with Ca²⁺. The Ca²⁺ overload results in both the disintegration of cells (necrosis), through the activity of Ca²⁺-sensitive proteases, and the activation of the apoptotic cell death program (10, 11, 74). It is clear that a lethal Ca²⁺ influx occurs in pancreatic -cells when L-type Ca_V channels are hyperactivated by exposure to type 1 diabetic serum (Fig. 3) (48). Our initial study revealed that -cells exposed to type 1 diabetic serum show an increase in L-type Ca²⁺ currents at both the whole cell and single channel levels. Indeed, an abnormal Ca²⁺ entry via L-type Ca_V channels causes a Ca²⁺ overload in -cells exposed to type 1 diabetic serum, as manifested by measurements of [Ca²⁺]_i. The Ca²⁺ overload, in turn, leads to -cell apoptosis. The apoptotic effect of type 1 diabetic serum is blocked by L-type Ca_V channel blockers (48).

Although we are still far from understanding the mechanisms whereby type 1 diabetic serum enhances -cell Ca_V channel activity and causes -cell apoptosis, evidence indicates that multiple factors are involved. Fas-specific antibodies in type 1 diabetic serum elevate [Ca²⁺]_i in neuroblastoma cells and cause their apoptosis (78). The rat dorsal root ganglion neurons incubated with the serum from the Bio Bred/Worchester diabetic rat (type 1 diabetic model) display enhancement of both HVA and LVA Ca²⁺ channel activity, associated with impaired regulation of the inhibitory G protein-Ca_V channel complex (31, 83). Recently, our group (16) found that incubation with type 1 diabetic serum promotes T-type Ca_V channel expression in a particular type of neurons with triangular soma in cerebellar granule cell cultures. More recently, we (49) demonstrated that type 1 diabetic serum contains significantly elevated concentrations of apolipoprotein C-III. This serum factor increases L-type Ca_V channel activity, thereby overloading -cells with Ca²⁺, resulting in Ca²⁺-dependent -cell apoptosis. Anti-apolipoprotein C-III antibody effectively abolishes both type 1 diabetic serum- and apolipoprotein C-III-induced increases in [Ca²⁺]_i and apoptosis. It is thus possible to postulate that the elevated concentration of apolipoprotein C-III in the blood of type 1 diabetic patients likely aggravates the disease development on top of the autoimmune attack. The mechanisms of -cell destruction in type 1 diabetes are not fully understood, although T-lymphocyte-mediated -cell death has been considered as a major one (65). Apparently, the effect of type 1 diabetic serum on -cells involves both multiple factors in the serum and numerous targets on the -cells. At the moment, we are searching for additional factors in type 1 diabetic serum, which hyperactivate -cell Ca_V channels, and additional targets on -cells, which mediate -cell apoptosis.