Regulation of high-voltage-activated Ca²⁺ channel function, trafficking, and membrane stability by auxiliary subunits

Abstract

Voltage-gated Ca²⁺ (Ca_V) channels mediate Ca²⁺ ions influx into cells in response to depolarization of the plasma membrane. They are responsible for initiation of excitation-contraction and excitation-secretion coupling, and the Ca²⁺ that enters cells through this pathway is also important in the regulation of protein phosphorylation, gene transcription, and many other intracellular events. Initial electrophysiological studies divided Ca_V channels into low-voltage-activated (LVA) and high-voltage-activated (HVA) channels. The HVA Ca_V channels were further subdivided into L, N, P/Q, and R-types which are oligomeric protein complexes composed of an ion-conducting Ca_Vα₁ subunit and auxiliary Ca_Vα₂δ, Ca_Vβ, and Ca_Vγ subunits. The functional consequences of the auxiliary subunits include altered functional and pharmacological properties of the channels as well as increased current densities. The latter observation suggests an important role of the auxiliary subunits in membrane trafficking of the Ca_Vα₁ subunit. This includes the mechanisms by which Ca_V channels are targeted to the plasma membrane and to appropriate regions within a given cell. Likewise, the auxiliary subunits seem to participate in the mechanisms that remove Ca_V channels from the plasma membrane for recycling and/or degradation. Diverse studies have provided important clues to the molecular mechanisms involved in the regulation of Ca_V channels by the auxiliary subunits, and the roles that these proteins could possibly play in channel targeting and membrane Stabilization.

INTRODUCTION

Voltage-gated Ca²⁺ (Ca_V) channels are a family of transmembrane proteins widely distributed in excitable cells and also found in many nonexcitable cells. These channels open when the plasma membrane becomes depolarized mediating Ca²⁺ entry in response to action potentials and subthreshold depolarizing signals. Ca²⁺ entering the cell through Ca_V channels serves as the second messenger of electrical signaling, initiating a variety of cellular events including neurotransmitter and hormone release, muscle contraction, enzyme activation, and gene expression, among many others. Signal transduction in different cell types involves diverse molecular subtypes of Ca_V channels, which mediate currents with distinct physiological and pharmacological properties.¹

Ca_V channels have been grouped into two distinct classes depending on their voltage sensitivity (Table 1). Channels that are activated at relatively hyperpolarized voltage ranges (less than or equal to −40 mV) have been named low-voltage-activated (LVA) or T-type, whereas channels that are activated at potentials more positive to −40 mV have been termed high-voltage-activated (HVA). HVA Ca_V channels can be further subdivided into L-, N-, P-, Q-, and R-types (Table 1), by virtue of their distinct functional and pharmacological profiles.¹

TABLE 1.

Ca_Vα₁ subunits can be divided into three classes according to amino acid sequence identity, as shown in the dendrogram. The Ca_V1 and Ca_V2 classes are termed High-Voltage-Activated (HVA) channels. The Ca_V3α₁ subunits form the Low-Voltage-Activated (LVA) channels. The original names, molecular nomenclature, and type of currents are given in purple, yellow, and pink, respectively.

Members of the HVA class are heteromultimers of a pore-forming Ca_Vα₁ subunit that coassembles into a functional channel complex with auxiliary Ca_Vβ, Ca_Vα₂δ, and in some cases with Ca_Vγ subunits (Figure 1(a)). In contrast, members of the LVA channel Ca_V class are thought to be Ca_Vα₁ subunit monomers.² The Ca_Vα₁ subunit defines the channel subtype, whereas the auxiliary subunits regulate the Ca_Vα₁ subunit function and plasma membrane expression.^1,3 Molecular studies have shown that the Ca_Vα₁ subunit is comprised of four homologous repeats each containing six transmembrane helices (S1–S6) and a pore lining P-loop domain (Figure 1(b)). These homologous membrane repeats are connected via cytoplasmic loops and flanked by intracellular N-and C-termini.¹

FIGURE 1.

Subunit composition of high-voltage-activated Ca²⁺ channels and structural domains of the auxiliary subunits. (a) Channel complex is composed of the pore-forming Ca_Vα₁ and the auxiliary Ca_Vα₂δ, Ca_Vβ, and Ca_Vγ subunits. The Ca_Vα₂δ and Ca_Vγ subunits contain transmembrane domains, whereas Ca_Vβs are intracellular. (b) The Ca_Vα₁ subunits consist of four transmembrane domains (I–IV) and the linker joining I and II encompasses the α-interaction domain (AID). (c)The Ca_Vβ subunits are formed by three conserved domains: PSD95/Dlg1/ZO-1 (PDZ), Scr homology 3 (SH3), and guanylate kinase (GK). The Ca_Vβ subunit interaction domain (BID) is one of the regions involved in the interaction of the protein with the Ca_Vα₁ subunit. (d) The Ca_Vα₂δ subunits consist of α₂ (blue), which is an extracellular subunit, disulphide-bonded to the δ subunit (orange), which is membrane-associated. The approximate positions of the vWA domain and the two bacterial chemosensory domains (Cache; C) are also indicated. The sites of interaction between the Ca_Vα₁ subunit and the Ca_Vα₂δ subunit are unknown.

Ten different Ca_Vα₁ subunits grouped into three subfamilies are expressed in mammals¹ (Table 1). The Ca_V1 subfamily encodes L-type Ca²⁺ channels and includes four members (Ca_V1.1–Ca_V 1.4). The Ca_V2 subfamily consist of three members (Ca_V2.1–Ca_V2.3) that encode the neuronal P/Q-type, N-type, and R-type channels, respectively. The P- and Q-type channels appear to arise from alternate splicing of Ca_V2.1 and/or coassembly with distinct Ca_Vβ subunits.^4,5 Last, the Ca_V3 subfamily includes the LVA (T-type) channels with three members (Ca_V3.1–Ca_V3.3).

EFFECTS OF THE AUXILIARY SUBUNITS ON HVA Ca_V CHANNEL FUNCTION, TRAFFICKING, AND MEMBRANE STABILITY

Ca_V channel trafficking and regulation mechanisms involve many different aspects including intrinsic channel protein structural determinants, interactions with regulatory elements such as second messengers and/or with extrinsic proteins to the channel complex. However, as we shall discuss later, associations with auxiliary subunits Ca_Vβ and Ca_Vα₂δ are thought to play crucial roles in fine-tuning channel gating, regulating channel modulation by other proteins and signaling molecules, as well as in forward trafficking and removal of Ca_V1 and Ca_V2 channels from the plasma membrane.^6,7

THE Ca_Vβ SUBUNITS

The Ca_Vβ subunits are cytoplasmic proteins that bind to the proximal region of the intracellular loop between domains I and II of the Ca_V1α₁ and Ca_V2α₁ subunits termed the α-interaction domain (AID)⁸ (Figure 1(b)). There are four subfamilies of Ca_Vβ subunits (Ca_Vβ₁–Ca_Vβ₄), each with splice variants, encoded by four distinct genes.⁹ All Ca_Vβ subunits contain conserved Src homology 3 (SH3) and a guanylate kinase-like (GK) domains^9,10 (Figure 1(c)), placing them into the membrane-associated guanylate kinase (MAGUK) protein family, scaffold proteins that function at neuronal synapses, tight junctions, immunological synapses, and neutrophil membranes.¹¹ The general structural features of guanylate kinases are preserved in the GK domain of the Ca_Vβ subunit (Cavβ-GK) though many key amino acid residues are absent rendering it catalytically inactive.¹² Instead, this domain has evolved into a protein–protein interaction module, binding tightly to the Ca_Vα₁ subunit through the AID.^13–15 Likewise, a large portion of the Ca_Vβ-GK domain remains free to interact with other proteins, such as RGK GTPases^16–18 and the largeconductance Ca²⁺-dependent K⁺ channels.¹⁹ The SH3 and Cavβ-GK conserved domains are flanked by regions of variable sequence and length between isoforms and splice variants of each isoform.^9,12,20

When coexpressed in heterologous systems with Ca_V1α₁ or Ca_V2α₁, Ca_Vβ auxiliary subunits significantly enhance Ca²⁺ currents and affect the voltage dependence and kinetics of current activation and inactivation.^21–27 Furthermore, Ca_Vβ subunit presence may be important for Ca_V1 and Ca_V2 channel modulation by different protein kinases,^28–32 G proteins,^33–35 Ras-related monomeric small GTP-binding proteins^36–39 as well as by proteins of the vesicle release machinery including Rab3-interacting molecule 1.^40–43 Results from Ca_Vβ subunit knockout mice have confirmed the physiological relevance of these auxiliary subunits. Ca_Vβ₁ and Ca_Vβ₂ subunit knockouts are lethal,^44,45 while Ca_Vβ₃ and Ca_Vβ₄ knockouts result in severe malfunction of the central nervous system.^46,47

Likewise, diverse studies have suggested that the Ca_Vβ subunits may participate in physiological events independent of their association with Ca_V channels. It has been suggested that a truncated splice variant of Ca_Vβ₄ lacking the GK and the C-terminal domains interacts directly with the heterochromatin protein 1 (HP1), attenuating its function as gene silencing factor.⁴⁸ More recently, Zhang et al.⁴⁹ confirmed that full-length Ca_Vβ subunits may function as transcription regulators. In particular, Ca_Vβ₃ can interact and suppress the transcriptional activity of Pax6(S), a critical factor for the development of the eye and the nervous system. In addition, upon neuronal differentiation, Ca_Vβ₄ can interact with B56δ, a nuclear regulatory subunit of the phosphatase 2A (PP2A), and the heterochromatin protein, HP1γ. This complex relocates to the nucleus, where it regulates the dephosphorylation of histones, a key mechanism in transcriptional regulation, and associates to the tyrosine hydroxylase (TH) promoter through the nuclear transcription factor thyroid hormone receptor α. This signaling cascade evidences the role of Ca_Vβ₄ as a repressor recruiting platform to control neuronal gene expression.^50,51

Given their essential role in the functional expression and modulation of HVA Ca_V channels, it is not surprising that Ca_Vβ auxiliary subunit mutations have been implicated in disease. Cavβ knockout animals and spontaneous occurring mutants have severe phenotypes that are in some cases lethal.^9,12,52 For instance, the Ca_Vβ_1a subunit isoform is selectively expressed in skeletal muscle where it partners with Ca_V 1.1α₁ subunit and is necessary for excitation-contraction coupling. Consequently, the Cavβ_1a subunit knockout mice, similar to Ca_V1.1α₁ knockouts, are motionless and die after birth from asphyxiation.⁴⁴ Similarly, Ca_Vβ_2b expression is predominant in the heart and knockouts of this subunit die prenatally due to lack of cardiac contractions.⁴⁵ Ca_Vβ₃ subunit knockouts show altered perception of inflammatory pain as a result of reduced N-type Ca_V channel expression in dorsal root ganglia⁴⁷ and have compromised sympathetic control, likely due to reduced N- and L-type channel activity.⁵³ Last, lethargic (lh) mice are naturally occurring Ca_Vβ₄ subunit knockouts. The lethargic phenotype includes ataxia, seizures, absence epilepsy, and paroxysmal dyskinesia,^46,52 associated with reduced excitatory neurotransmission in thalamic neurons.⁵⁴

In humans, two point mutations in the Ca_Vβ_2b subunit have been implicated in cardiovascular diseases. The first mutation contributes to a type of sudden death syndrome characterized by a short QT interval and an elevated ST-segment,⁵⁵ and the other is linked to the Brugada syndrome.⁵⁶ Likewise, mutations in the CACNB4 gene have been linked to epilepsy and episodic ataxia.⁵⁷

As mentioned earlier, Ca_V1α₁ and Ca_V2α₁ channel subunits show poor surface expression by themselves, however, upon coexpression of the auxiliary subunits currents are significantly increased reflecting either enhanced channel open probability or cell surface expression.^58,59 The increase in surface expression can be observed both in native and recombinant channels with any of the Ca_Vβs and seems to be dependent on binding to the AID, as point mutations that disrupt the AID/Ca_Vβ interaction reduce or abolish Ca_Vβ-mediated current stimulation.⁸ Interestingly, in heterologous systems the GK domain can recapitulate the function of the full-length Ca_Vβ subunits increasing channel surface expression.^60,61

An initial explanation of how the Ca_Vβ subunits enhance channel surface expression was that they antagonize an endoplasmic reticulum (ER) retention signal located in the I-II linker that severely restricts the plasma membrane incorporation of the Ca_Vα₁ subunit⁵⁸ (Figure 2(a)). More recently, this idea has been enriched based on data obtained after transferring the intracellular linkers of the Ca_V1.2α₁ subunit into a T-type channel (Ca_V3.1). The results of these experiments suggest that the I-II linker of Ca_V1.2α₁ has an ER export signal of 9 amino acids downstream the AID.⁶² On the other hand, all other intracellular linkers, including the N- and C-termini, were found to contain ER retention signals. Consequently, it was proposed that the intracellular regions in the Ca_Vα₁ subunit form a complex that yields a prevailing ER retention signal, and when the Ca_Vβ subunit binds to the I-II linker, it triggers a switch in the channel complex such that the ER export signal becomes dominant, enhancing Ca_Vα₁ surface expression (Figure 2(b)). In this process, the Ca_Vα₁ C-terminus may play also an important role for Cavβ-dependent channel upregulation.^12,62,63

FIGURE 2.

Two distinct models for Ca_Vβ subunit-induced increase in Ca_V channel expression at the plasma membrane. (a) In the absence of Ca_Vβ, the Ca_Vα₁ subunit remains trapped within the endoplasmic reticulum (ER) by binding via the I-II loop onto an ER retention protein (ERRP) of unknown identity. Expression and subsequent binding of the Ca_Vβ subunit to the I-II loop relieves the trafficking clamp imposed by the ERRP and allows Ca_V channel complexes to be targeted to the cell surface. (b) In the absence of Ca_Vβ, an ER export signal (blue) present on the Ca_Vα₁ subunit I-II loop is functionally overcome by ER retention signals (pink) present in different regions of the protein, leading to channels being retained in the ER. Upon Ca_Vβ binding to the Ca_Vα₁ subunit, a C-terminus-dependent conformational change of the intracellular domains occurs that diminishes the strength of ER retention signals leading to channel transport to the plasma membrane.

There are also studies that point to the importance of intramolecular domains in the physiology of the Ca_Vβ subunits. In particular, it has been reported that Ca_Vβ₃ contains two PEST-like sequences, potential signals for rapid protein degradation,⁶⁴ sensitive to the Ca²⁺-dependent protease calpain. Ca_Vβ₃ mutants lacking the PEST sequences induce increased Ca_V2.2 current densities,⁶⁴ suggesting that Ca_Vβ subunit proteolytic cleavage may be important for Ca²⁺ channel surface expression.

Likewise, the role of Ca_Vβs as critical determinants of channel cell surface expression has been also evidenced by protein–protein interaction assays. Using a yeast two-hybrid screening approach, Béguin et al.³⁶ initially uncovered a link between regulation of Ca_V channel trafficking and signaling by small guanosine triphosphatases (GTPases). Using the Ca_Vβ₃ subunit as bait to screen a cDNA library these authors identified the Ras-like GTPase kir/Gem as an interacting partner. Interestingly, the binding of kir/Gem to Ca_Vβ₃ inhibited their assembly with the Ca_Vα₁ subunit, such that kir/Gem cotransfection drastically inhibited Ca_V channel functional expression.³⁶

In line with this, using an extracellular epitope to probe for surface expression of the channels, subsequent studies showed a drastic reduction of fluorescent signal when channels and their subunits were coexpressed with the small GTPase RGK.^37,38 This led to the initial conclusion that the RGK proteins disrupt the Ca_Vα₁/Ca_Vβ subunit association, thus retaining newly synthesized channels in the ER and reducing the number of functional channels in the plasma membrane. However, it has been shown more recently that the reduction in surface expression of Ca_V 1.2 channels by small GTPases (e.g. Rem), seems to occur by enhancing the dynamin-dependent endocytosis pathway rather than by a sequestration of the Ca_Vβ subunits by the small GTPase.¹⁶ Interestingly, restoring Ca_V1.2 surface expression by coexpressing a dominant negative dynamin mutant was not sufficient to restore current densities, suggesting that Rem has at least one other mode of action to inhibit currents, most likely an effect on channel open probability.^65,66

Recent work from Colecraft’s group offers new insight into the complex mode of RGK-mediated Ca_V channel inhibition. According to their proposal which is based on a customized mechanism at both the channel and GTPase level, distinct RGKs differentially use Ca_Vβ-binding-dependent and Ca_Vα₁-binding-dependent mechanisms to inhibit Ca_V1 and Ca_V2 channels.¹⁷ In this scenario, Rem may inhibit Ca_V 1.2 channel using both Ca_Vβ-binding-dependent and independent mechanisms and binding to Ca_Vβ is required for Rem-mediated decrease in Ca_V1.2 channel surface density and open probability. On the other hand, Rem associates directly with Ca_V1.2α₁ to initiate β-binding-independent inhibition while inhibits Ca_V2.2 channels using a solely Ca_Vβ-binding-dependent mechanism.¹⁷

The Ca_Vβ subunits have been also implicated in trafficking of Ca_V channels to specific subcellular regions. Vendel et al.⁶⁷ found that Ca_Vβ_4a, one of the two alternative splicing variants of the N-terminal A domain of the Ca_Vβ₄ auxiliary subunit,⁶⁸ may exert functions other than modulation of channel gating and trafficking by interacting with the microtubule-associated protein 1A (MAP1A) as well as synaptotagmin I, an important protein for presynaptic vesicle release. These interactions suggest that the Ca_Vβ_4a subunit may act as a scaffolding element to facilitate coupling of Ca²⁺ signaling with neurotransmitter release. Since the Cavβ_4a-synaptotagmin I interaction is disrupted by Ca²⁺,⁶⁷ it has been speculated that at basal Ca²⁺ levels Ca_Vβ_4a may interact both with the Ca_V2.1α₁ subunit and the vesicle protein to organize the machinery necessary for vesicle release, and that Ca²⁺ entry via Ca_V channels disrupts this interaction, thus releasing the vesicle to allow fusion with the plasma membrane.⁴³

More recently, it has been found that different members of the RIM family, putative effectors of Rab3, may functionally link Ca_V channels to proteins in the active zone at the presynapsis which are related to the machinery for exocytosis⁶⁹ (Figure 3). Moreover, it has been reported that several proteins in the active zone including RIM1 modulate neuronal Ca_V2.1 channels through interactions with the Ca_Vβ subunits. These interactions not only modify the inactivation properties of the channels and cause a sustained Ca²⁺ influx, but also anchor the neurotransmitter-containing vesicles to the channels in the vicinity.^40,41 Similar results have been reported for recombinant L-type channels (of the Ca_V1.2 and Ca_V1.3 class) in insulin-secreting cells.⁴²

FIGURE 3.

Functional coupling between Ca_V channels and secretory vesicles via the Ca_Vβ subunit. (a) RIMs anchor synaptic vesicles next to channels through its interaction with zone-specific proteins (Rab3) and the Ca_Vβ subunit. After depolarization, RIMs regulate the time course of channel inactivation resulting in a sustained Ca²⁺ influx. This molecular organization favors hormone and neurotransmitter release.

THE Ca_Vα₂δ SUBUNITS

As in the case of Ca_Vβs, the ability of the Ca_Vα₂δ auxiliary subunits to promote membrane expression of HVA Ca_V channels is well established.^6,70 Four subtypes of Ca_Vα₂δ proteins encoded by four separate genes with several known splice variants have been described.^71,72 In general, the Cacna2d genes (coding the Ca_Vα₂δ subunits) are translated as precursor proteins that are proteolytically cleaved into a transmembrane δ region and an extracellular α₂ domain, that remain linked by a disulphide bond.^3,6,70 The extracellular α₂ region is heavily glycosylated and contains several functional domains, including a 178 amino acid von Willebrand factor-A (vWFA) domain similar to extracellular matrix-binding regions of integrins, a 5 amino acid metal ion-dependent adhesion site (MIDAS) motif and a poorly understood 92 amino acid Cache domain⁶ (Figure 1(d)). The transmembrane domain of δ and some extracellular regions of α₂ are thought to associate with the Ca_Vα₁ subunit of HVA channels.⁷³

Although the functional relevance of the Ca_Vα₂δ subunits is not fully understood, there is compelling evidence indicating a role in trafficking and also in the modulation of channel biophysical properties. Heterologous expression of Ca_Vα₂δ with various Ca_Vα₁ and Ca_Vβ subunits results in increased current densities, as well as altered current kinetics and current–voltage relationships.^{23,25,74–79} The increase in current density is generally explained by improved targeting of Ca_V channels to the plasma membrane^74,78 and significant reduction in the rate of entry of surface resident channels into degradation pathways.⁸⁰ Additionally, the Ca_Vα₂δ subunits have been involved in cell surface organization of Ca_V channels. It has been shown that Ca_Vα₂δ-1 (and likely the other Ca_Vα₂δ subunits) not only partitions into lipid rafts itself but also mediates raft-partitioning of neuronal Ca_V2.2 channel complexes.^81–83

Recently, a mechanism has been proposed for Ca_V channel localization to lipid rafts which challenges the conventional structural model of the Ca_Vα₂δ subunits. This suggests that Ca_Vα₂δ associates with the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor in the δ domain.⁸⁴ In order to examine the role of membrane anchoring of the Ca_Vα₂δ-1 subunit on its biochemical and functional properties, Dolphin and her colleagues generated a Ca_Vα₂δ-1 truncated at the putative GPI-anchor ω-site, which removes the C-terminal hydrophobic domain (α₂δ-1 ΔC-term⁸⁵). Unexpectedly, the α₂δ-1 ΔC-term protein was able to produce a significant increase of Ca²⁺ currents in cells expressing Ca_V2.1/Ca_Vβ₁ channels. Likewise, these authors did not observe any effect upon external application of secreted α₂δ-1Δ C-term, which suggested that an intracellular interaction with other subunits may be needed for the complete functionality of the Ca_Vα₂δ-1 subunit in the channel complex. Last, a large proportion of α₂δ-1 Δ C-term was secreted into the medium, but despite the lack of a C-terminal membrane anchor, the α₂δ-1Δ C-term proteins remained partially associated with the plasma membrane. It has been speculated that this interaction may occur via a noncovalent linkage formed during the maturation and trafficking of the protein.⁸⁵ Other studies, have also shown that the raft localization of Ca_Vα₂δ is preserved after replacement of the GPI anchoring motif,⁸³ which is consistent with the idea that the Ca_Vα₂δ subunits may reside in lipid rafts via protein–protein and/or specialized lipid–protein interactions.

As mentioned earlier, during biosynthesis the Ca_Vα₂δ subunits are generated as precursor proteins which undergo post-translational cleavage, oxidation, and glycosylation to yield mature proteins comprised of disulphide-linked α₂ and δ glycopolypeptides.^86–88 Although the precise functional role of the post-translational modifications of the Ca_Vα₂δ subunits has not yet been fully defined, it has been suggested that they could play a role in channel trafficking. Initial experiments by Gurnett et al.⁷⁵ showed that N-linked glycosylation play a critical role for surface expression of Ca_V2.2 channels since the Ca²⁺ current stimulation normally observed after Ca_Vα₂δ coexpression was virtually abolished by deglycosylation. Moreover, by combining electrophysiology with site-directed mutagenesis two N-glycosylation sites were found in the α₂ domain of the protein that are required for the auxiliary subunit-induced current stimulation. Given that the mutation of these sites prevented current stimulation without altering its kinetic properties, a regulation on the number of functional channels at the plasma membrane was suggested.⁸⁶

In addition, alterations in the proteolytic cleavage that typically separates the extracellular α₂ from the transmembrane δ domain⁸⁹ may interfere with proper assembly and trafficking of the Ca_V2.2 channel complex. After mutation of the amino acids in the proteolytic site, Ca_Vα₂δ becomes insensitive to proteolytic cleavage and loses its ability to increase currents. These changes are not accompanied with alterations in the voltage dependence or kinetics of the channels, suggesting a reduction in the number of channels targeted to the plasma membrane.⁸⁷

Disulfide bond formation seems to be also important for channel cell surface expression. Recent studies have shown that a pair of conserved cysteine residues at positions 404 and 1047, located in the vWFA region of α₂ and the extracellular domain of δ, respectively, form a single intermolecular disulfide bridge required for normal α₂δ−1 subunit function.⁸⁸ In these studies, it was shown that whereas there was a significant increase in current density through Ca_V2.2α₁/β₃ channels upon cotransfection with wild-type Ca_Vα₂δ−1, Cys404Ser, and Cys1047Ser mutations had little influence in current density, implying that formation of an intra-subunit disulfide bond between these residues is essential for Ca_Vα₂δ-1 functional enhancement of currents, and perhaps for Ca_V channel trafficking to the plasma membrane.

Likewise, some insights into the role of the Ca_Vα₂δ auxiliary subunits in vivo come from a series of spontaneously occurring mutations in mice. These include two mutations in Ca_Vα₂δ-2 (ducky and entla) that result in similar phenotypes characterized by ataxia, paroxysmal dyskinesia, and spike wave seizures.^52,90,91 In humans, a mutation in Ca_Vα₂δ-4 that introduces a premature stop codon that truncates one-third of the corresponding open reading frame presumably underlies a channelopathy leading to cone-rod dysfunction in the visual system.⁹² More recently, a novel gene defect in CACNA2D2 encoding the Ca_Vα₂δ-2 subunit has been associated with early infantile epileptic encephalopathy a disease that usually manifest as severely impaired cognitive and motor development.⁹³ Last, mutations in the CACNA2D1 gene might be associated with some forms of cardiac dysfunction, including the Brugada and long QT syndromes.^94,95

Interestingly, it has been reported that experimental peripheral nerve injury results in increased levels of Caα₂δ-1 mRNA in damaged dorsal root ganglion (DRG) sensory neurons, as well as a corresponding increase in protein levels in the neuronal cell bodies and at their presynaptic terminals in the spinal cord.^96–98 Furthermore, mice overexpressing the Ca_Vα₂δ-1 subunit show a neuropathic phenotype of hyperalgesia and tactile allodynia in the absence of nerve injury,⁹⁹ suggesting that Ca_Vα₂δ-1 is instrumental to the excitability of DRG neurons and the expression of neuropathic pain.

Evidence supporting the role of the Ca_Vα₂δ subunits in the promotion of surface expression of channels comes, in part, from the fact that Ca_Vα₂δ is the principal target for a group of small molecules with analgesic effects called gabapentinoids.¹⁰⁰ Although the mechanisms by which these drugs alleviate pain is not well understood, they seem to act intracellularly after chronic but not acute treatment.^79,101–105 Therefore, Vega-Hernandez and Felix¹⁰² initially formulated the hypothesis that gabapentinoids could be impairing the ability of Ca_Vα₂δ to enhance the number of channels in the plasma membrane, via an effect on trafficking. Subsequent studies showed that indeed GBP acting chronically by displacing an endogenous ligand that is normally a positive modulator of Ca_Vα₂δ, could be interfering with the function of the vWFA domain impairing Ca_V channel trafficking to the membrane^78,104 (Figure 4).

FIGURE 4.

Hypothetical mechanisms of action of gabapentin (GBP). The effect of GBP may be to displace an endogenous ligand (L-leucine) and impair the ability of the auxiliary Ca_Vα₂δ subunit to increase the number of functional channels at the plasma membrane. GBP may be entering the cells using the system-L transporter protein LAT4 and might be exerting its effect on intracellular Ca_Vα₂δ subunits during assembly and trafficking of the Ca_V channel complex to the cell surface.

In addition, gabapentin has been shown to inhibit Rab11-dependent recycling of N-type Ca_V channels from the plasma membrane.¹⁰⁶ However, not only the trafficking and recycling of recombinant Ca_V channels appears to be impeded by gabapentin but also the enhancement of native N-type channel at the cell surface associated with chronic pain.⁹⁸ It is worth mentioning that endosomal trafficking may represent an important mechanism for maintaining proper plasma membrane expression of distinct native Ca_V channels,^107,108 though the role of Ca_Vα₂δ in this process, if any, is yet to be determined.

Last, although investigations regarding the role of the auxiliary subunits on subcellular distributions of Ca_V channels have primarily focused on the Ca_Vβ subunits in neurons,^40,41 initial studies using L-type channels have revealed that the Ca_Vα₂δ subunits may also play a role in this process. Hence, it has been reported that the RalA GTPase regulates Ca_V channel activity, and acts as a chaperone for hormone secretory granules to L- and R-type channels on the plasma membrane, where these granules become tethered, in preparation for the formation of excitosome complexes with vesicle and plasma membrane SNARE proteins. Notably, this interaction occurs through a physical association between RalA and Ca_Vα₂δ-1,¹⁰⁹ suggesting that the RalA/Ca_Vα₂δ-1 complex likely engage and traffic secretory granules to the plasma membrane-bound Ca_Vα₁ subunits to be able to then conduct Ca²⁺ through functional channels at precise sites where hormone release occurs.

ROLE OF THE AUXILIARY SUBUNITS ON HVA Ca_V CHANNEL REGULATED DEGRADATION

Recent studies have proposed that the Ca_Vβ auxiliary subunits increase channel surface expression by preventing Ca_Vα₁ ubiquitination and proteasomal degradation.^63,110 Indeed, Ca_V1.2 channels are tonically ubiquitinated and the degree of ubiquitination seems to be increased in the absence of the Ca_Vβ subunits by an ER associated ubiquitin ligase called RFP2. Hence, in the absence of Ca_Vβ, an interaction occurs between the channel and proteins related to the ER Associated Protein Degradation (ERAD) system. The end result of this is channel retrotranslocation and proteasomal degradation in the cytosol. On the other hand, in the absence of the Ca_Vβ subunits, a proteasome inhibitor can rescue Ca_Vα₁ surface expression.¹¹⁰

These results suggest that the Cavβ subunits may be required to help Ca_Vα₁ (particularly of the Ca_V1.2 and Ca_V2.2 class) escape the ubiquitin proteasome system (UPS) degradation pathway.^63,110 Consistent with this, reduced levels of bulk Ca_V2.2 and Ub-Ca_V2.2 proteins are observed in the absence of Ca_Vβ subunits.¹¹¹ In this regard, elements in the proximal C-terminus of a Ca_V2.2α₁ alternative splice variant (e37b) predispose cloned and native channels to downregulation by the UPS.¹¹¹ Therefore, the question arises as to whether sequences unique to e37b may destabilize the Ca_V2.2/Ca_Vβ subunit interaction leading to increased ubiquitination and degradation by the UPS.

In a similar manner, it has been suggested that the Ca_Vα₂δ-1 subunit may play a role in stabilizing Ca_V channel functional expression once delivered to the plasma membrane. By examining the binding, internalization, and degradation kinetics of recombinant N-type (Ca_V2.2α₁δ/Ca_Vβ_1b) channels in the presence or the absence of Ca_Vα₂/δ-1, Bernstein and Jones⁸⁰ proposed that the Ca_Vα₂δ subunits may act, at least in part, reducing the entry of surface resident Ca_V channels into degradative pathways. Computer modeling of trafficking data showed that Ca_Vα₂δ stabilizes N-type channels after their delivery to the cell surface through a significant reduction in the rate constants for internalization and degradation.⁸⁰ These kinetic changes help to explain the enhanced N-type channel expression levels and suggest that, in conjunction with forward trafficking effects (see the preceding section), the Ca_Vα₂δ subunits play a significant role in defining HVA Ca_V channel functional expression.

Last, it is well established that skeletal muscle L-type Ca²⁺ channels contain a Ca_Vγ₁ subunit which includes four transmembrane helices.^1,3 It was subsequently shown that neurons express at least four homologues of Ca_Vγ₁,¹¹² most notably Ca_Vγ₂ also known as stargazin,^113,114 which in addition to regulate Ca_V channel function may also play a role in mediating AMPA receptor trafficking to the synapsis.¹¹⁵ In general, the γ subunits appear to be inhibitory and their physiological relevance is emphasized by the epileptic phenotype in the stargazer mouse, a spontaneous mutant that lacks the Ca_Vγ₂ subunit.^{52,112,113,115}

Although it has been shown that Ca_Vγ₁, Ca_Vγ₂, and Ca_Vγ₇ drastically reduce Ca²⁺ current density in heterologous expression systems,^116–119 it is worth noting that these effects may not be associated with altered channel trafficking but most likely occur as a result of changes in channel expression. Interestingly, the effect of the Ca_Vγ subunits resemble the inhibition of the Ca_V2.2 currents induced by the coexpression of truncated Ca_V2.2 constructs,¹²⁰ which occurs after the activation of PERK, a component of the unfolded protein response (UPR).^118,121 When UPR is initiated, an immediate consequence is the inhibition of protein biosynthesis through phosphorylation of the translation initiation factor eIF2α.¹²²

PERSPECTIVES

Molecular and cellular biological studies have begun to explore the intriguing question of the spatial and temporal control of Ca_V channel density in the plasma membrane. These studies will surely be followed with more mechanistic analyses of the trafficking, targeting, recycling, and degradation, as well as their interactions with the cytoskeleton and regulation by cellular activity. Future studies must consider also other important variables such as distinct cell phenotypes, developmental, and cell cycle stages, and the dynamic arrangement of microdomains within a given cell. Therefore, the molecular characterization of the possible roles of Ca_V channel auxiliary subunits is only one of the crucial early steps towards understanding how different cells regulate membrane expression and organization of Ca_V channels for functional purposes. Undoubtedly, the current shortage of knowledge will be the motivation for intensive work in this field in the near future.