The β Subunit of Voltage-Gated Ca²⁺ Channels

Abstract

Calcium regulates a wide spectrum of physiological processes such as heartbeat, muscle contraction, neuronal communication, hormone release, cell division, and gene transcription. Major entry-ways for Ca²⁺ in excitable cells are high-voltage activated (HVA) Ca²⁺channels. These are plasma membrane proteins composed of several subunits, including α₁, α₂δ, β, and γ. Although the principal α₁ subunit (Ca_vα₁) contains the channel pore, gating machinery and most drug binding sites, the cytosolic auxiliary β subunit (Ca_vβ) plays an essential role in regulating the surface expression and gating properties of HVA Ca²⁺ channels. Ca_vβ is also crucial for the modulation of HVA Ca²⁺ channels by G proteins, kinases, and the Ras-related RGK GTPases. New proteins have emerged in recent years that modulate HVA Ca²⁺ channels by binding to Ca_vβ. There are also indications that Ca_vβ may carry out Ca²⁺ channel-independent functions, including directly regulating gene transcription. All four subtypes of Ca_vβ, encoded by different genes, have a modular organization, consisting of three variable regions, a conserved guanylate kinase (GK) domain, and a conserved Src-homology 3 (SH3) domain, placing them into the membrane-associated guanylate kinase (MAGUK) protein family. Crystal structures of Ca_vβs reveal how they interact with Ca_vα₁, open new research avenues, and prompt new inquiries. In this article, we review the structure and various biological functions of Ca_vβ, with both a historical perspective as well as an emphasis on recent advances.

I. INTRODUCTION

Calcium is arguably one of life’s most important elements. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) is kept at very low levels (~100 nM) under resting conditions, but it rises sharply (to tens or hundreds of µM) upon stimulation. This allows Ca²⁺ to play a crucial role in numerous biological processes, including neurotransmitter and hormone release, muscle excitation-contraction coupling, cell division, tumorigenesis, differentiation, migration, and cell death. In addition, Ca²⁺ influx across the plasma membrane causes changes in cellular excitability. Mechanisms that rigorously control intracellular Ca²⁺ levels are therefore essential for eukaryotic cell function. [Ca²⁺]_i is maintained at low levels by Ca²⁺-ATPases through active extrusion of cytosolic Ca²⁺ to the extracellular milieu or into intracellular organelles. On the other hand, Ca²⁺ entry into cells is mediated primarily by passive flow through voltage-, ligand-, temperature-, and mechanical stretch-gated ion channels.

The principal Ca²⁺ entryways of nerve, muscle, and some endocrine cells are voltage-gated Ca²⁺ channels (VGCCs). They were discovered in 1953 with the unexpected observation that crab muscle action potentials (APs) persist in the absence of external Na⁺, unlike squid nerve APs (145). Muscle APs were then found to increase with increasing extracellular Ca²⁺ concentration ([Ca²⁺]_o), consistent with a Ca²⁺ conductance (200). Similar currents were later found in nerve, endocrine, and other tissues in diverse organisms (12, 221,225, 253, 291, 304). Based on the membrane voltage required for activation, VGCCs were subsequently classified into high-voltage activated (HVA) and low-voltage activated (LVA) channels (65, 66,146, 293). Later studies further classified Ca²⁺ currents into L-, N-, P/Q-, R-, and T-type currents, which exhibit distinct biophysical and pharmacological properties (127, 137,292, 335, 359, 408, 444, 446, 500).

Molecular characterization of VGCCs began with the purification and cloning of the skeletal muscle Ca²⁺ channel (also called dihydropyridine receptor or DHPR) (107, 430, 434). The purified channel complex is composed of five subunits, termed α₁ (175 kDa), α₂ (143 kDa), β (54 kDa), δ (24–27 kDa), and γ (30 kDa). α₂ and δ are linked posttranslationally by disulfide bonds into a single subunit referred to as α₂δ (430). Subsequent research showed that L-, N-, P/Q- and R-type channels are made up of α₁, α₂δ, β, and, in some tissues, γ subunits (Fig. 1A). T-type channels, on the other hand, appear to require only an α₁ subunit (351, 352).

Fig. 1.

Molecular organization of voltage-gated Ca²⁺ channels. A: subunit composition of high-voltage activated (HVA) Ca²⁺ channels. B: schematic representation of the predicted transmembrane topology of Ca_vα₁, with the location of the α-interaction domain (AID) marked. C: Ca²⁺ channel current types and the corresponding α₁ subunits of the channels that produce them. D: list of all cloned auxiliary HVA Ca²⁺ channel subunits. E: amino acid sequence alignment of the AID from the indicated Ca_vα₁. Residues involved in interactions with Ca_vβ are marked in red, with the most critical residues underlined. Residue numbers are indicated on both sides of the sequence.

The α₁ subunit (Ca_vα₁) is the principal component of VGCCs and is responsible for their unique biophysical and pharmacological properties. However, proper trafficking and functioning of L-, N-, P/Q- and R-type channels require the auxiliary subunits. In particular, the β subunit (Ca_vβ) plays a crucial role in trafficking the channels to the plasma membrane, fine-tuning channel gating, and regulating channel modulation by other proteins and signaling molecules. Crystal structures of the core region of three distinct Ca_vβs have opened up new avenues for investigating the molecular basis of Ca_vβ’s actions. There is also emerging evidence that Ca_vβ may possess functions unrelated to VGCCs. This review focuses on the molecular biology, structure, function, and channelopathy of Ca_vβ, beginning with a brief overview of all VGCC subunits. Summaries of classical and recent work on VGCC electrophysiology, pharmacology, biochemistry, molecular biology, modulation, cell biology, and pathophysiology can be found in numerous excellent reviews (20, 22,72–74, 108, 126, 138, 189, 216, 220, 234, 237, 246, 318, 351, 389, 423, 440, 445, 495).

A. The α₁ Subunit

Ca_vα₁ is the principal subunit of VGCCs. It is a 190- to 250-kDa protein containing four homologous repeats (I–IV) connected through cytoplasmic loops (Fig. 1B). Each repeat has six predicted transmembrane segments (S1–S6) and a reentrant pore-forming loop (P-loop) between S5 and S6. The four P-loops form the ion-selectivity filter, where four highly conserved negatively charged amino acids (glutamate or aspartate), one from each P-loop, form a signature locus that is essential for selecting and conducting Ca²⁺ (256, 266,389, 482). Similar to K⁺ channels (128, 243, 290), the S6 segments form the inner pore (505), and the S4 segments’ positively charged amino acids form part of the voltage sensor. The voltage-dependent movement of this sensor results in channel opening and closing. Furthermore, the majority of drug and toxin binding sites are located on Ca_vα₁ (72). Thus Ca_vα₁ possesses all the key features that define a VGCC, including pharmacological and biophysical properties such as gating, ion selectivity, and permeation.

Mammalian Ca_vα₁ are encoded by 10 distinct genes. Based on amino acid sequence similarity, Ca_vα₁ are divided into three subfamilies: Ca_v1, Ca_v2, and Ca_v3 (reviewed in Refs. 10, 72, 141, 486). The Ca_v1 subfamily includes channels that conduct L-type Ca²⁺ currents; the Ca_v2 subfamily includes channels that conduct N-, P/Q-, and R-type Ca²⁺ currents; and the Ca_v3 subfamily includes channels that conduct T-type Ca²⁺ currents (Fig. 1C).

B. The α₂δ Subunit

The Ca_v1 and Ca_v2 subfamilies contain an auxiliary α₂δ subunit (reviewed in Ref. 112). To date, there are four known α₂δ subunits, each encoded by a unique gene and all possessing splice variants (Fig. 1D). Each α₂δ protein is encoded by a single messenger RNA and is posttranslationally cleaved and then linked by disulfide bonds (259, 367). The δ peptide, originally presumed to be transmembrane but recently shown to be attached to the membrane through a glycosylphosphatidylinositol linker (113), anchors the larger extracellular α₂ peptide in place (Fig. 1A). α₂δ subunits can modify channel biophysical properties (63, 406, 459), but their main role is to increase Ca²⁺ channel current (63, 111,174, 259, 260, 322, 406, 459) by promoting trafficking of Ca_vα₁ to the plasma membrane and/or by increasing its retention there (32, 64,194, 385). More recently, it was reported that α₂δ functioned as a thrombospondin receptor to regulate excitatory synpatogenesis, independently from its regulation of VGCC activity (140, 267).

In two different mouse strains, naturally occurring mutations that lead to the loss of the full-length α₂δ₂ protein cause the ducky phenotype. This is characterized by shortened life spans, absence epilepsy, spike wave seizures, cerebellar ataxia, and decreased Purkinje cell dendritic arborization and firing rates (112, 260). α₂δ₂ knockouts also have abnormalities in the cardiovascular, immune, respiratory, and nervous systems. Irregularities in the cardiovascular system are also found in α₂δ₁ knockouts (169). α₂δ₃-null Drosophila are not viable, and the mutants have significantly impaired synaptic transmission (123, 267). Upregulation of α₂δ₁, on the other hand, is associated with neuropathic pain (283, 284). Importantly, α₂δ₁ is the main target of the antiepilepsy and antineuropathic pain drugs gabapentin and pregabalin, respectively (150, 169, 254).

C. The γ Subunit

There are eight different γ subunit genes, all yielding proteins with four transmembrane segments and intracellular amino (NH₂) and carboxy (COOH) termini. γ₁ was the first cloned γ subunit (182, 238, 430) and was copurified with muscle VGCCs, consistent with its primary role as a VGCC subunit. γ₁ knockout mice are viable, morphologically indistinguishable from wild-type (WT) mice, but have larger Ca²⁺ currents with altered inactivation kinetics (168). γ₂, γ₃, and γ₄ also associate with VGCCs (250, 399). γ_1–4 subunits have been shown to produce varying effects on VGCC activity, depending on the partnered Ca_vα₁ and Ca_vβ (134, 168,2017, 250, 258, 277, 379, 406, 467). The most consistent effect is a small reduction of current, caused mainly by a hyperpolarizing shift of the voltage dependence of inactivation and/or a positive shift of the voltage dependence of activation. However, unlike α₂δ subunits, whose primary role is regulating VGCCs, γ subunits have more diverse biological functions. Since the discovery that mutations in γ₂ underlie the stargazer mouse phenotype (277), which includes absence epilepsy and defects in the cerebellum and inner ear, it has become clear that γ₂ and three other closely related γ subunits (γ₃, γ₄, and γ₈) regulate the trafficking, localization, and biophysical properties of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (41, 82,249, 343, 442). They are therefore referred to as transmembrane AMPA receptor regulatory proteins (TARPs). Indeed, acting as TARPs seems to be the primary role of γ₂, γ₃, γ₄, γ₈, and probably γ₇ (252). While the function of γ₅ remains unknown, γ₆ is suggested to inhibit Ca_v3.1 channels (288), and γ₇ is involved in the turnover of the mRNA of Ca_v2.2 and other proteins (149, 323). For recent reviews on γ subunits, see References 41, 82, 249, 320, 343, 350, 382.

D. The β Subunit

Purified Ca_v1 and Ca_v2 channels contain a tightly bound cytosolic Ca_vβ protein. There are four subfamilies of Ca_vβs (β₁– (β₄), each with splice variants, encoded by four distinct genes. All four Ca_vβs can dramatically enhance Ca²⁺ channel currents when they are coexpressed in heterologous expression systems along with a Ca_v1 or Ca_v2 (α₁ subunit (268, 319,322, 361, 405, 450, 467, 470). Ca_vβs also change the voltage dependence and kinetics of activation and inactivation (247, 268,322, 332, 406, 412, 418, 450, 495); however, they do not affect ion permeation (183, 405,458; but see Ref. 390). Furthermore, Ca_vβ either regulates or is indispensable for the modulation of Ca_v1 and Ca_v2 channels by protein kinases, G proteins, and small RGK (Rem, Rem2, Rad, Gem/Kir) proteins. Not surprisingly, Ca_vβ knockouts are either nonviable (in the case of (β₁ and (β₂) or result in a severe pathophysiology (in the case of (β₃ and (β₄).

The rest of this review is devoted to Ca_vβ.

II. CLONING OF Ca_vβ

Molecular studies on Ca_vβ can be traced back to the first purification and identification of the components of the skeletal muscle DHPR (107). With the use of a combination of chromatography, sucrose gradient sedimentation, and labeling with a high-affinity DHPR-specific ligand, three noncovalently attached subunits were purified: the largest 160-kDa subunit was named (α, a 53-kDa subunit was named (β, and a 32-kDa subunit was named (γ (107). Subsequent purification studies of skeletal and neuronal Ca²⁺ channel complexes showed the presence of similar protein bands (4, 59,114, 162, 278, 322, 381, 430, 434) and established that the DHPR actually consisted of five subunits, including (α₁ (175 kDa), (α₂ (143 kDa), (β (54 kDa), (δ (24–27 kDa), and (γ (30 kDa) (430, 434).

Cloning of the first Ca_vβ was accomplished by Ruth et al. (381) using a classical approach based on peptide sequences derived from a purified skeletal muscle (β subunit. This (β subunit is now referred to as (β_1a. This cloning paved the way for the identification of other (β subunits, their genes, and splice variants. Using a labeled skeletal muscle (β_1a cDNA, Pragnell et al. (362) screened a rat brain cDNA library and cloned a new (β subunit, which later turned out to be a splice variant of (β₁ named (β_1b (360) (see sect. IV). Perez-Reyes et al. (353) also screened a rat brain cDNA library with (β_1a and, using low-stringency hybridization, uncovered another new (β subunit, which was encoded by a different gene and named (β₂ (now named (β_2a). Screening a cardiac cDNA library, Hullin et al. (230) found (β_2a and two other (β₂ splice variants ((β_2b and (β_2c); in addition, they isolated the cDNA for (β₃. Meanwhile, using degenerate primers corresponding to the conserved domains of (β₁ and (β₂ to perform reverse-transcription PCR, Castellano et al. (67, 68) cloned (β₃ and (β₄ from a rat brain cDNA library.

The cloning of Ca_vβs subsequently led to the mapping of the four Ca_vβ genes (55, 94,143, 347, 438) to chromosomes 17, 10, 12, and 2 for (β₁, (β₂, (β₃, and (β₄, respectively, and to the discovery of many other splice variants (see sect. IV).

III. STRUCTURE OF Ca_vβ

Prior to the determination of the crystal structure of Ca_vβ, it was already well recognized, based on amino acid sequence alignment, biochemical and functional studies, and molecular modeling, that Ca_vβ has a modular structure consisting of five distinct regions (40, 93,119, 203, 342, 361). The first, third, and fifth regions are variable in length and amino acid sequence, whereas the second and fourth regions are highly conserved and are homologous to the Src homology 3 (SH3) and guanylate kinase (GK) domains, respectively. The SH3 domain is a common protein interaction module present in diverse groups of proteins (reviewed in Ref. 307). The GK domain, originally found in guanylate kinase from baker’s yeast (416), is also engaged in protein-protein interactions (136, 170, 431). The middle three regions of Ca_vβ constitute the so-called Ca_vβ core, which is able to reconstitute many key functions of Ca_vβ (83, 84,119, 176, 206, 313, 342, 502). In addition, early studies determined that Ca_vβ binds with high affinity to Ca_vα₁. This high-affinity site is located in the cytoplasmic loop connecting the first two homologous repeats (i.e., the I–II loop) of Ca_vα₁ and was named the (α-interaction domain or AID (121, 361, 472) (Fig. 1E).

In 2004, three groups simultaneously and independently reported the crystal structure of the core of (β_2a, (β₃ and (β₄, either alone or in complex with the AID (84, 341, 447). The structures show that the Ca_vβ core indeed contains an SH3 domain and a GK domain, which are connected by a so-called HOOK region (Fig. 2A).

Fig. 2.

Ca_vβ crystal structure. A: crystal structure of the β₃ core in complex with the AID (PDB accession code 1VYT). This structure reveals the following regions: the NH₂ terminus (light blue, residues 38–59), an SH3 domain (gold, residues 60–120 and 170–175), a HOOK region (purple, residues 121–169), and a GK domain (green, residues 176–360). Residues 137–166 were disordered and are not included. Residues 226–244 (forming the α₄ helix of the GK domain) were disordered in this molecule but were well-resolved in another one in the same asymmetric unit. Residues 422–446 of Ca_v1.2 containing the entire AID are colored in orange. B: same structure as in A but with the BID (β₃ residues K163-T193) highlighted in dark blue. The BID spans parts of the SH3-HOOK-GK motif but is not directly involved in binding the AID. C: close-up of the interface between β₃ and AID. Some residues involved in the interactions are shown. [Adapted from Chen et al. (84)].

The existence of an SH3-HOOK-GK module places Ca_vβ in a family of proteins called the membrane-associated guanylate kinases (MAGUKs). MAGUKs, which include proteins such as PSD95, SAP97, CASK, Shank, and Homer, function as scaffold molecules that play a key role in organizing multiprotein complexes at functionally specialized regions such as synapses and other cellular junctions (136, 170, 431). MAGUKs contain an SH3-HOOK-GK module; in addition, they also contain one or more PDZ domains in the NH₂ terminus, which serve protein-protein interaction and oligomerization functions. Ca_vβ is only partially related to MAGUKs structurally, however, because it does not contain a well-defined PDZ domain. Not surprisingly, the functions of Ca_vβ are markedly different from those of MAGUKs.

A. The GK Domain

Guanylate kinases are members of the nucleotide monophosphate kinase family that exists in organisms ranging from bacteria to humans. They catalyze the reversible phosphoryl transfer from ATP to GMP to produce ADP and GDP. Crystal structures of yeast guanylate kinases show that these enzymes have a compact structure with well-defined domains and folds and a catalytic site harboring the GMP- and ATP-binding pockets (43, 415, 416). The general structural features of yeast guanylate kinases are preserved in the Ca_vβ GK domain (Ca_vβ-GK), but large structural variations exist in the catalytic site, and many key catalytic residues are absent in Ca_vβ-GK (84, 341, 447). Thus Ca_vβ-GK is catalytically inactive. Similarly, the GK domain of MAGUKs does not possess catalytic activity, as indicated by the structural changes in the catalytic site and the lack of critical catalytic residues (285, 312, 437). Instead, the GK domains in these proteins have evolved into a protein interaction module. The Ca_vβ structures show that Ca_vβ-GK binds tightly to the AID in Ca_vα₁ (84, 341, 447) (Fig. 2A), an interaction that will be further discussed in detail.

B. The SH3 Domain and the HOOK Region

Classical SH3 domains have a well-conserved and compact fold consisting of five sequential β-strands (βstrand 1–5) assembled into two orthogonally packed sheets (271). They mediate specific protein-protein interactions by binding to PxxP-containing motifs in target proteins, through a surface formed by a cluster of highly conserved hydrophobic residues. The Ca_vβ SH3 domain (Ca_vβ-SH3) has a similar fold as canonical SH3 domains do, but its last two β sheets are noncontinuous, separated by the HOOK region (84, 341, 447) (Fig. 2A). This split configuration is also shared by the SH3 domain of PSD-95, a MAGUK (312, 437). Ca_vβ-SH3 contains a well-preserved PxxP motif-binding site and therefore has the potential to bind PxxP motif-containing proteins. However, in the crystal structures, this binding site is partly shielded by the HOOK region and a long loop connecting two of the four continuous β sheets. Thus access to this site requires movement of these two regions. Such conformational changes are conceivable when Ca_vβ is bound to full-length Ca_vα₁ and/or when it interacts with other partners, but are yet to be demonstrated. In contrast, the PxxP motif-binding site of the SH3 domain of PSD-95 is unobstructed (312, 437), consistent with the observation that the SH3 domain of MAGUKs can associate directly with PxxP motif-containing proteins (177, 306).

The HOOK region is variable in length and amino acid sequence among the Ca_vβ subfamilies (Fig. 3). In the crystal structures, a large portion of the HOOK is unresolved due to poor electron density, indicating that it has a high degree of flexibility (84, 341, 447). As will be discussed below, the HOOK region plays an important role in regulating channel inactivation.

Fig. 3.

Amino acid sequence alignment of Ca_vβ subtypes. The four included subtypes are β_1b (GenBank accession number, NP-000714), β_2a (M80545), β₃ (M88751), and β_4a (L02315). Light blue indicates the NH₂ terminus, gold the SH3 domain, purple the HOOK region, green the GK domain, and gray the COOH terminus. Secondary structure elements are indicated in the top line as arrows for β sheets and solid lines for α helices (based on the crystal structure of β₃). Residues involved in interactions with the AID are marked in red.

C. The NH₂ Terminus

The NH₂ and COOH termini of Ca_vβ (abbreviated as Ca_vβ-NT and Ca_vβ-CT) are highly variable in length and amino acid composition (Fig. 3). There is yet no structure available for Ca_vβ-CT. However, an NMR structure of the NH₂ terminus of β₄ was solved recently, revealing a fold consisting of two α-helices and two antiparallel β sheets (451). This structure also shows that, unlike previously thought (203), Ca_vβ-NT does not have a PDZ fold, which consists of five β sheets (380). Incidentally, one of the two α-helices in the NMR structure is equivalent to the very first α-helix in the Ca_vβ core structures. Superposition of this helix in the two structures reveals that the NH₂ terminus is oriented away from the core (Fig. 4).

Fig. 4.

Structural model of a partial Ca_vα₁/Ca_vβ complex on the plasma membrane. A side view and an inside-to-outside view are presented. The partial structure of Ca_vα₁ includes only the S5, P-loop, and S6 segments and is based on a Ca_vα₁ homology model developed in Stary et al. (411). IS5 is colored orange, and IS6 is red. The IS6-AID linker from Ca_v1.2 is modeled as an α-helix and is joined with IS6 at its NH₂ terminus and the AID at its COOH terminus. The structure of Ca_vβ is based on the crystal structure of the β₄ core region (84) and the NMR structure of the β₄ NH₂ terminus (451); there is no Ca_vβ COOH terminus. Since the structure of the β₄-AID complex is not available, we docked the AID to β₄ based on the crystal structure of the β₃ core-AID complex (84). The regions of Ca_vβ are color coded as in Figures 2 and 3 (NH₂ terminus in light blue, SH3 in gold, HOOK in purple, and GK in green).

D. The SH3-GK Intramolecular Interaction

The crystal structures of the Ca_vβ core show that the SH3 and GK domains interact intramolecularly (84, 341, 447). The affinity of this interaction is unknown, but the interaction is strong enough such that hemi-Ca_vβ fragments containing the NT-SH3_{βstrand 1–4}-HOOK module and the SH3_{βstrand 5}-GK-CT module can associate biochemically in vitro and reconstitute the functionality of full-length Ca_vβs when they are coexpressed in cells (298, 313,342, 431, 432, 447). In fact, one of the β_2a structures was obtained from cocrystals of two β_2a hemifragments truncated at the HOOK region (447).

The last β sheet of Ca_vβ-SH3 (SH3_{βstrand 5}), which is separated from the rest of the SH3 domain by the HOOK region, is critical for the strong intramolecular SH3-GK interaction (83, 298,313, 342, 431, 432). This β sheet is directly connected to the GK domain, and it interacts extensively with both the GK domain and the rest of the SH3 domain (83). As a result, SH3_βstrand5 glues the NT-SH3_{βstrand1–4}-HOOK module and the SH3_βstrand5-GK-CT module together and strengthens the otherwise weak interactions at the SH3-GK interface (83).

As in MAGUKs, the SH3-GK intramolecular interaction is important for the function of Ca_vβ (83, 298,313, 431, 432). Weakening this interaction by mutating the SH3-GK interface or by inserting flexible linkers between the SH3 domain and the GK domain severely compromises the gating effects of Ca_vβ (83). Thus mutations, modifications, or protein-protein interactions that alter the SH3-GK intramolecular interaction may produce significant functional consequences.

E. The AID-Ca_vβ Interaction

Which regions anchor Ca_vβ to Ca_vα₁? By screening an epitope library of 20,000 Ca_v1.1 fragments, Pragnell et al. (361) identified a region in the I–II loop that binds β_1b. This region, known as the AID, is comprised of 18 residues, with a conserved consensus motif (QQxExxLxGYxxWIxxxE) in all Ca_v1 and Ca_v2 α₁ subunits (Fig. 1E). The AID binds to all four Ca_vβs (121). The affinity of the AID-Ca_vβ interaction ranges from 2 to 54 nM, depending on the AID/Ca_vβ or Ca_vα₁/Ca_vβ pair and the method of affinity measurement (30, 56,62, 120, 121, 179, 342, 371, 395, 448). Single mutations of several conserved residues in the AID, including Y10, W13, and I14, greatly weaken the AID-Ca_vβ interaction, as indicated by in vitro binding experiments and by the reduction or abolishment of Ca_vβ-induced stimulation of Ca²⁺ channel current in heterologous expression systems (33, 34,56, 120, 181, 185, 206, 218, 276, 361, 448). Thus the role of the AID as the principal interacting domain with Ca_vβ is firmly established.

Which region(s) of Ca_vβ interact with the AID? In an influential study, De Waard et al. (119) described a 31-amino acid segment of Ca_vβ, referred to as the β-interacting domain or BID, as the main binding site for the AID. The BID was able to slightly enhance Ca²⁺ channel current and modulate gating (119), and several BID point mutations were able to weaken the Ca_vβ/Ca_vα₁ interaction and reduce BID-stimulated Ca²⁺ channel currents (119, 120).

For the next decade, it had been generally accepted that Ca_vβ interacted with Ca_vα₁ primarily through the BID. Surprisingly, however, the crystal structures of two different AID-Ca_vβ core complexes reveal that the AID does not bind the BID (84, 341, 447). Indeed, the AID and the BID do not come into direct contact (Fig. 2B). Instead, the AID binds to a hydrophobic groove in the GK domain termed the AID-binding pocket (ABP; Fig. 2C) (84, 447, 448). The AID occupies only a tiny fraction of the Ca_vβ surface area, raising the possibility that other domains of Ca_vβ are involved in interactions with other regions of Ca_vα₁ or with other proteins. As will be discussed later, both are indeed the case.

The AID-GK domain interactions are extensive and predominantly hydrophobic (Fig. 2C). These interactions account for the 2–54 nM affinity of the AID-Ca_vβ binding. Functional studies show that mutating two or more key residues in the ABP severely weakens or completely abolishes the AID-Ca_vβ interaction (206, 502).

The binding of the AID with Ca_vβ does not significantly change the Ca_vβ structure, except for some small and localized changes near the ABP. Importantly, however, the AID undergoes a dramatic change in secondary structure when it is engulfed by the ABP. When alone, the AID exists as a random coil in solution, as determined by circular dichroism spectrum measurements (341). When bound to Ca_vβ, the AID forms a continuous α-helix, as shown in the crystal structures. Together with the observation that the 22-amino acid linker between the AID and the first S6 segment of Ca_vα₁ (i.e., IS6) also forms an α-helix (9), a picture emerges that the entire region encompassing IS6 and the AID adopts a continuous α-helical structure in the presence of Ca_vβ (Fig. 4). This structural hallmark is crucial for the regulation of Ca²⁺ channel gating by Ca_vβ, as will be discussed later.

Since the publication of the Ca_vβ structures, some investigators have been continuing to perform or interpret experiments based on the notion that the BID interacts with Ca_vα₁ (85, 281,302, 388, 441, 506), so before leaving this section, we briefly revisit the BID. The crystal structures show that the BID spans three different regions of Ca_vβ (SH3, HOOK and GK) and that most of it is completely buried (Fig. 2B). Thus the BID does not directly interact with Ca_vα₁; rather, it is crucial for maintaining the SH3-GK intramolecular interaction and the structural integrity of Ca_vβ. Of the four residues in the BID whose mutations weakened the Ca_vβ/Ca_vα₁ interaction, three were proline and one was tyrosine (119, 120). Mutating these residues most likely alters the folding and/or structure of Ca_vβ, which explains its inability to bind Ca_vα₁.

But how could the BID enhance Ca²⁺ channel current (119)? While the mechanism of this action remains unclear, it reminds us of an experiment of our own in which a random 43-amino acid peptide (which has no sequence similarity in GenBank) was coexpressed with Cav2.1 and α₂δ in Xenopus oocytes. This random peptide significantly increased Ca²⁺ channel currents (compared with no Ca_vβ), to ~50% of β₃-induced current. This obvious nonspecific effect, reported in 2004 (84), suggests that the BID-induced current increase may also be a nonspecific effect. Given these structural and functional information, it is prudent to exercise caution when interpreting experimental data concerning the BID.

IV. Ca_vβ SPLICE VARIANTS AND THEIR TISSUE DISTRIBUTION

Mammalian Ca_vβs are encoded by four distinct genes, Cacnb 1–4. They all have 14 exons except Cacnb3, which has 13, and each Ca_vβ has 2 or more splice variants. Figures 5 and 6 show most of the human Ca_vβ splice variants found thus far. The five distinct domains and regions of Ca_vβ are mapped onto their corresponding exons and protein sequences. Alternative splicing occurs in those exons that encode the variable domains or regions, namely, the NH₂ and COOH termini and the HOOK region. The four different Cacnb genes utilize different alternative splicing sites. Cacnb1 and Cacnb2, which produce β₁ and β₂, respectively, exhibit alternative splicing in exon 7, giving rise to divergent HOOK regions. Cacnb1 is also alternatively spliced in the COOH terminus, with exon 14 either included or excluded. On the other hand, Cacnb2 is alternatively spliced extensively in the NH2 terminus, yielding highly diversified NH₂ termini. Cacnb4 has no alternative splicing in the HOOK region but has NH₂- and possibly COOH-terminal alternative splicing. Cacnb3 has no alternatively spliced exons, but like all other Cacnb genes, produces a truncated isoform.

Fig. 5.

Human Ca_vβ splice variants. Fourteen Ca_vβ exons (13 for β₃) are color-coded based on the regions they give rise to: the NH₂ terminus (light blue), the SH3 domain (gold), the HOOK (purple), the GK domain (green), and the COOH terminus (gray). Exons are numbered, and some exons have additional letters to indicate alternatively spliced variants. The thick full and dashed lines at the very top indicate highly or somewhat conserved exons, respectively. Of the weakly conserved regions, similar exons are placed in the same column (e.g., β₁ exon 2 is homologous to β₂ exon 2A). Exons 13 and 14 of β₁ were originally designated as 13a and 13b, respectively (222). The names of splice variants are, from left to right columns, those used in this article, those proposed by Foell et al. (166), and those proposed by Yang and Berggren (486). β_2a is the only splice variant that can be palmitoylated (wave). The jagged edge (e.g., exon 6 of β_1d) indicates missing amino acids resulting from exon skipping and/or frame-shifts. Striped exons (e.g., exon 8 of β_1d) are translated with a frame shift; hence, their amino acid sequence is unrelated to the “conventional” sequence produced by that exon. **Direct submission by M. E. Williams, 1997. ***AK316045; direct submission by T. Isogai and J. Yamamoto, 2008.

Fig. 6.

Amino acid sequence alignment of Ca_vβ splice variants. The 5 Ca_vβ regions, their corresponding exons, and the exon boundaries are marked. Color coding follows the same scheme as in previous figures, with the NH₂ terminus in light blue, the SH3 domain in gold, the HOOK in purple, the GK domain in green, and the COOH terminus in gray. Exon numbers are indicated in the color bar, and some exons have additional letters to indicate alternatively spliced variants. Arrows and bold amino acids mark exon boundaries. A single bold residue indicates that exon splicing occurs within its codon, whereas two bold residues indicate that splicing occurs between their codons. Shaded in black are missense sequences resulting from a frame-shift. # Indicates a premature stop codon. The GenBank accession number of each sequence is indicated at the end of the sequence, except for two sequences where the original reference is given. All sequences are from human except Cβ_4c, which is a chicken isoform. In regions where alternative splicing occurs (e.g., the NH₂ terminus of β₂), the amino acid sequence is aligned with its parent exon; thus the alignment in these regions does not necessarily indicate amino acid sequence similarity.

Table 1 shows the tissue distribution of some Ca_vβ splice variants. As expected, Ca_vβs are abundantly expressed in excitable tissues such as the brain, heart, and muscles. While some splice variants (e.g., β_1b and β_2b) are widely expressed, others (e.g., β_1a, β_2d, and β_2e) have a more restricted expression. The expression of some splice variants is developmentally regulated. For example, β_1b and β₄ expression increases with development, whereas β_2c, β_2d, and β_2e expression decreases with development. It is important to note that, in most cases, protein but not mRNA expression was listed. Immunolocalization experiments should be interpreted cautiously since an antibody may recognize several splice variants, for example, an antibody against the β₂ COOH terminus will recognize all β₂ splice variants.

TABLE 1.

Tissue distribution of Ca_Vβ

CaVβ	Tissue Distribution	Reference Nos.
β1	Expressed in brain (cerebral cortex, habenula, hippocampus, and olfactory bulb), heart, skeletal muscle, spleen, and T cells, but not in kidney, liver, or stomach.	360, 419, 435
β1a	Expressed only in skeletal muscle (but see Ref. 89). Exclusive partner of CaV1.1 and irreplaceable for excitation-contraction coupling.	360, 381, 395, 472
β1b	Expressed in brain (cerebellum and cerebral cortex), nerve endings at the NMJ, and pancreas. Expression is detected at P0 in rat brains and increases from P7 to adulthood by ~3-fold.	311, 344, 353, 355, 360, 395, 456, 472
β1c	Expressed in brain and spleen, but not in kidney, liver, muscle, or stomach.	360
β1d	Expressed in heart.	92
β2	Expressed in brain (hippocampus–becoming the most abundant isoform there, cerebellum, pontine nucleus, susbtantia nigra, central grey, habenula, pineal gland, thalamic nuclei, cerebrum), heart, lung, nerve endings at the NMJ, T cells, and osteoblasts, but not in kidney, liver, pancreas, or spleen. Brain expression is constant during development, but see hippocampus data (391).	121, 294, 311, 344, 353, 355, 391, 398, 419, 435, 456
β2a	Expressed in brain, heart, and aorta; its heart and brain levels seem lower than other β subunits and isoforms.	215, 229, 230
β2b	Expressed in brain, heart, and aorta. It is the most abundant CaVβ in human heart.	89, 215, 230
β2c	Expressed in brain and heart, where it is the second most abundant CaVβ. Its expression declines in adults.	89, 215, 230
β2d,e	Expressed in heart. β2e expression is robust only in young animals.	89, 215
β3	Expressed mostly in brain (cerebellum, cerebral cortex, habenula, hippocampus, olfactory bulb, and striatum), but also in heart, aorta, kidney, lung, skeletal muscle, smooth muscle, spleen, thalamus, T cells, and trachea, but not in liver, pancreas, or testis. Expression remains constant in the brain and heart during development. It is the most predominant partner of CaV2.2 (N-type) channels in the brain, and it pairs with ~40% of brain L-type channels.	58, 68, 89, 230, 294, 310, 329, 344, 355, 395, 419, 435, 449, 456, 472, 473
β3trunc	Expressed in brain, heart, and aorta.	230
β4	Expressed in brain (cerebellum–the most abundant Cavβ there, brain stem, cerebral cortex, dentate gyrus, habenula, hippocampus, olfactory bulb, striatum, thalamus, and hypothalamus), kidney, nerve endings at the NMJ, ovary, skeletal muscle, spinal cord, T cells, and testis, but not detected in heart (except in young animals, Ref. 89), liver, lung, spleen, or thymus. The expression increases in rat brain by 10-fold from P0 to adult. It is the most prevalent partner of CaV2.1 (P/Q-type) channels in brain, and, like β3, it pairs with ~40% of brain L-type channels.	55, 67, 294, 311, 355, 391, 395, 419, 435, 449, 456, 472
β4a	Expressed in spinal cord and cerebellum.	208, 209, 452
β4b	Expressed in spinal cord and forebrain.	209

NMJ, neuromuscular junction.

Since the association between Ca_vβ and Ca_vα₁ is promiscuous (i.e., any full-length Ca_vβ can associate with any Ca_v1 or Ca_v2 α₁ subunit), alternative splicing greatly increases the molecular diversity and functionality of HVA Ca²⁺ channels. Furthermore, some splice variants may take on functions other than regulating HVA Ca²⁺ channels (see sect. XII). Thus a major future challenge (and a fruitful area of research) is to determine how alternative splicing is regulated in various tissues and at different developmental stages.

V. Ca_vβ REGULATES THE SURFACE EXPRESSION OF HIGH-VOLTAGE ACTIVATED Ca²⁺ CHANNELS

The α₁ subunit of Ca_v1 and Ca_v2 channels cannot reach the membrane by itself; it shows no surface expression and produces very small or no currents when expressed without auxiliary subunits. Coexpression of Ca_vβ with Ca_vα₁ increases currents by orders of magnitude, depending on factors such as the expression system, DNA or RNA concentration, VGCC turnover rate, inhibitory factors present, the α₁/β combination, etc. (reviewed in Refs. 10, 40, 125, 237, 495). The current increase reflects enhanced channel expression on the plasma membrane and also an increase in channel open probability. In this section we discuss the evidence and mechanisms of increased channel surface expression.

A. Ca_vβ Is Required for Normal Channel Expression

It has been well established that Ca_vβ can function as a chaperone to dramatically increase the surface expression of Ca_v1 and Ca_v2 channels. This is observed in various heterologous expression systems with all four subfamilies of Ca_vβ and all Ca_v1 and Ca_v2 subunits (14, 50,88, 93, 191, 245, 247, 248, 276, 322, 353, 458, 481, 495). The increased surface expression can be detected by Ca_vα₁ epitope tag staining, surface biotinylation, gating charge measurements, or increased Ca²⁺ channel current. An important point to mention is that Xenopus oocytes, a widely used expression system for studies of VGCCs, have two endogenous β subunits that share 98% homology with β₃ (436). These endogenous subunits are expressed at sufficient levels to transport a small number of exogenously expressed Ca_vα₁ to the plasma membrane and hence lead to small Ca²⁺ channel currents in the absence of an exogenous Ca_vβ. Antisense oligonucleotides against endogenous β₃ are able to suppress these currents (62, 436). Little or no endogenous Ca_vβ was detected in widely used mammalian cell lines such as HEK 293 cells, COS cells, and CHO cells (276, 315). Nevertheless, expression of Ca_vα₁ alone in these cells can produce measureable, albeit miniscule, Ca²⁺ channel currents (245, 247,248, 303, 407, 418, 436), suggesting that either a very small fraction of Ca_vα₁ can be trafficked to the plasma membrane in the absence of Ca_vβ or these cells contain low levels of endogenous Ca_vβs.

Ca_vβ also enhances Ca²⁺ channel surface expression in vivo. For example, β₁ and β₂ knockout mice have severely reduced Ca²⁺ currents in muscle and heart (see sect. XIII). Knockdown of Ca_vβ also decreases endogenous Ca²⁺ currents in neuronal cells (35, 279). Conversely, overexpression of Ca_vβ using adenoviruses increases Ca²⁺ channel current density in native cardiac cells, suggesting that Ca²⁺ channel surface expression may be limited by the availability of Ca_vβ (336, 465).

Binding of Ca_vβ to the AID of Ca_v1 and Ca_v2 is essential for its chaperone effect. Point mutations in the AID that weaken or abolish the AID-Ca_vβ interaction severely reduce or abolish Ca_vβ-stimulated Ca²⁺ channel current (33, 34,56, 120, 181, 185, 206, 218, 276, 336, 361, 448). Deleting the AID altogether, not surprisingly, abolishes Ca_vβ-induced current enhancement (185, 298). Likewise, mutations in the ABP that weaken or abolish the AID-Ca_vβ interaction also reduce or abolish Ca_vβ-stimulated Ca²⁺ channel expression and current (206, 502). Recent studies show that the GK domain itself can largely recapitulate the chaperone function of full-length Ca_vβs, greatly increasing Ca²⁺ channel surface expression and current in Xenopus oocytes and mammalian cells (129, 206).

How does Ca_vβ enhance Ca²⁺ channel surface expression? One hypothesis is that Ca_vβ shields or disrupts one or more ER retention signals on the I–II loop of Ca_vα₁ (39), and several lines of evidence support this hypothesis. The I–II loop of Cav1.2 and Ca_v2 can trap α₁ subunits in the ER (except Ca_v1.1), but the I–II loop of Cav3.1 (a T channel) fails to do so. Also, tagging a Shaker K⁺ channel with the I–II loop of Ca_v1.2 or Ca_v2.1 decreases its expression by approximately sevenfold, while coexpression of Ca_vβ prevents this downregulation (39). Moreover, deleting the I–II loop from Ca_v1.2 (Δ389–423) increases its surface expression in the absence of Ca_vβ (39).

However, some results are inconsistent with this hypothesis. 1) The I–II loop of Ca_v1.1 does not cause ER retention of a CD8 peptide (99). 2) CD4 fusion constructs of the I–II loop of Ca_v1.2 and Ca_v2.2 are trafficked efficiently to the plasma membrane, rather than being retained in the ER (5). 3) Transplanting the I–II loop of Ca_v2.2 into Ca_v3.1 causes Ca_vβ-independent current upregulation instead of downregulation (9).

An alternative possibility is that additional trafficking signals exist in the NH₂ and COOH termini of Ca_vα₁ (99, 163,175, 262, 466). However, the NH₂ and COOH termini of Ca_v1 and Ca_v2 are not conserved, and yet, the chaperone function of Ca_vβ is universal, suggesting that any ER retention signals in the NH₂ and COOH termini may only be modulatory.

Recently, a new study suggested that Ca_vβ increases Ca_vα₁ expression on the plasma membrane by preventing its ubiquitination and proteasomal degradation (5). Thus Ca_vβ may simply be required to help Ca_vα₁ escape the degradation pathway.

B. Membrane Association and Subcellular Targeting of Ca_vβ

Ca_vβs are expected to have a cytosolic localization based on analyses of their amino acid sequence (353, 381). This is true for the majority of Ca_vβ splice variants when they are expressed alone, without a Ca_vα₁ (with a few exceptions discussed below; Refs. 176, 181). However, some Ca_vβs, most notably β_2a, can be localized to the plasma membrane on their own. β_2a is linked to the plasma membrane through palmitoyl groups that are covalently attached to two cysteines (Cys 3, 4) in the NH₂ terminus (86, 87). When palmitoylation is abolished, in a double Cys→Ser mutant, membrane localization disappears (87). Importantly, β_2a palmitoylation can be dynamically regulated in vivo, adding a layer of physiological control (232, 464). However, palmitoylation alone may not be sufficient for membrane localization because implanting the β_2a NH₂ terminus into other Ca_vβs does not yield membrane localization (87, but see Ref. 369). Thus β_2a probably possesses additional determinants that help target it to the plasma membrane. Another β₂ subunit, β_2e, is not palmitoylated but is found at the plasma membrane (433). The underlying mechanism is yet unknown. Finally, β_1b is localized to the plasma membrane in COS-7 cells (44, 50), but this is not observed in tsA201 cells (87) or primary cardiomyocytes (93). The reason for the discrepancy is unclear, but in COS-7 cells, the membrane localization is attributed to a COOH-terminal acidic motif (WEEEEDYEEE) whose deletion diminishes membrane localization. When this motif is fused to β₃, which is normally cytosolic, it migrates to the plasma membrane (44, 50). As will be discussed in section VI, membrane localization of Ca_vβ coincides with many functional effects, especially slowed inactivation.

In the presence of Ca_vα₁, all Ca_vβs localize to the plasma membrane through their association with Ca_vα₁; however, they may be targeted to different subcellular locations depending on which Ca_vα₁ they associate with. For example, β₃ and β₄, which predominantly associate with presynaptic Ca_v2 channels, can be found in axons, whereas β₁ is scarce in this compartment (336); instead, β₁ is found in postsynaptic compartments (soma and dendrites). In skeletal muscle, β_1a is targeted to the triads through its association with Ca_v1.1 (333). When exogenously expressed in epithelial cells, β_1b is localized on the apical membrane with Ca_v2.1 but on the basolateral membrane with Ca_v1.2 (44). Conversely, Ca_vβ may affect the subcellular localization of Ca_vα₁. For example, β_1a helps arrange L-type Ca²⁺ channels as tetrads in the t tubules of skeletal muscles (see sect. XIA; Refs. 164, 191, 394, 507), and β₄ is implicated in the synaptic localization of P/Q-type channels in cultured hippocampal neurons (474). Furthermore, through interactions with different proteins, Ca_vβ helps attach Ca²⁺ channels to synaptic vesicles (257), the cytoskeleton (223), or the surface of sarcoplasmic reticulum (333). These examples illustrate the role of Ca_vβ as a scaffold protein.

VI. Ca_vβ REGULATION OF Ca²⁺ CHANNEL GATING

Once the Ca²⁺ channel complex reaches the plasma membrane, Ca_vβ powerfully modulates its gating. The main features of gating modulation are the enhancement of voltage-dependent activation (VDA) and voltage-dependent inactivation (VDI). β_2a is unique in that it inhibits VDI. This section describes these Ca_vβ effects and their mechanisms.

A. Ca_vβ Enhances Voltage-Dependent Activation

All Ca_vβs shift the voltage dependence of activation to more hyperpolarized voltages (by ~10–15 mV, Table 2 and Fig. 7). This was shown for both Ca_v1 and Ca_v2 channels in various expression systems (61, 116,229, 245, 268, 322, 406, 412, 414, 443, 495). The shift can also be observed in vivo in some knockout mice (191, 325, 468), while in some other cases, it is probably obscured by the compensatory effects of other Ca_vβ genes (31, 330, 331). In addition, the speed of activation is increased in general (268, 412), but it could appear slower depending on the stimulus voltage (353) and the particular α₁/β pair (184, 245,443, 459).

TABLE 2.

Effect of Ca_Vβ on HVA Ca²⁺ channel gating properties

CaVβ	Kinetics of Activation	Voltage Dependence of Activation	Kinetics of Inactivation	Voltage Dependence of Inactivation
β1	Accelerates activation by 2- to 100-fold; acceleration is weaker for CaV2 compared with Cav1 channels (40, 119, 206, 245, 268, 406, 412, 443, 450, 467).	Hyperpolarizing shift of −10 to −15 mV (40, 44, 92, 206, 245, 417, 450, 467).	Accelerates inactivation by 2- to 10-fold (40, 206, 218, 276, 340, 406, 417, 443, 450). α2δ may be required in some instances.	Hyperpolarizing shift of −5 to −30 mV (40, 44, 119, 206, 218, 245, 276, 314, 340, 406, 443). α2δ or γ may promote larger shifts.
β2a	Accelerates activation by 2- to 4-fold; acceleration is less evident with CaV2 channels (40, 119, 206, 230, 245, 353).	Hyperpolarizing shift of −5 to −20 mV (40, 206, 230, 245, 276, 340, 353, 417).	Slows inactivation by ~10-fold (except for the unaffected CaV1.4 channels) (40, 58, 206, 218, 265, 276, 340, 369).	Depolarizing shift of 10 to 40 mV with CaV2 channels; much less or no effect on L-type channels (40, 119, 132, 206, 218, 245, 276, 314, 340, 369).
Other β2	Accelerate activation kinetics ~2- to 4-fold (110, 215, 230, 433)	Hyperpolarizing shift of −7 to −20 mV (40, 110, 215, 230, 340, 433).	Inactivation is accelerated except with β2e (58, 215, 340).	Hyperpolarizing shift of approximately −10 mV except β2e (215, 340, 433).
β3	Accelerates activation by 0 to 3.5-fold; acceleration is less evident with CaV2 channels (68, 119, 206, 230, 245).	Hyperpolarizing shift of −6 to −15 mV (44, 68, 206, 245, 276, 386, 417, 477).	Accelerates inactivation by 2- to 7-fold (68, 206, 265, 329).	Hyperpolarizing shift of −5 to −30 mV; α2δ may lessen the shift (44, 68, 119, 206, 245, 276, 386).
β4	Accelerates activation by ~2- to 3-fold; acceleration is less evident with CaV2 channels (67, 119, 206, 208, 209, 245).	Hyperpolarizing shift of −5 to −25 mV (67, 159, 179, 206, 208, 245, 386, 452). For differences between splice variants, see Refs. 208, 209.	Accelerates inactivation by 2- to 4-fold (67, 206, 208), but see (417).	Hyperpolarizing shift of −5 to −30 mV; less evident for L-type channels (67, 119, 179, 206, 245, 386). For some differences between splice variants, see Refs. 208, 209.

Reference numbers are given in parentheses.

Fig. 7.

Modulation of Ca²⁺ channel gating by Ca_vβ. A: voltage dependence of activation of P/Q-type Ca²⁺ channels containing β_1b, β_2a, β₃, or β₄ or no β (β⁻). In this and all other panels, currents were recorded in cell-attached macropatches from oocytes expressing Ca_v2.1 and α₂δ, without or with the indicated β subunit. B: voltage dependence of inactivation. C: representative current traces evoked by a depolarization to ~30 mV, showing the kinetics of voltage-dependent inactivation. Currents are shown only from the first 2.5 s of a 25-s pulse. D and E: comparison of V_1/2 and t_1/2 of voltage-dependent inactivation of P/Q-type Ca²⁺ channels containing no β (β⁻) or the indicated β module: the GK domain, β core (SH3-HOOK-GK), or full-length (FL) β. V_1/2 is the membrane voltage at the midpoint of voltage-dependent inactivation, and t1/2 is the time for the current to inactivate to 50% of the peak value in C. Note the logarithmic scale of the y-axis in E. [All data from He et al. (206).]

These effects are also visible at the single-channel level. Thus channels without a Ca_vβ tend to open less frequently, open for a shorter duration, and require more positive activation voltages. Ca_vβ coexpression greatly increases channel open probability (P_o) and shortens the latency to first opening (93, 125,215, 229, 295, 457). Notably, β_2a produces the most dramatic increase in P_o (76, 93, 132).

Normal VDA is largely reconstituted by the core region of Ca_vβ (206). Deleting the entire Ca_vβ COOH terminus has no effect on VDA, at least for Ca_v2.1 channels expressed in Xenopus oocytes (206). The NH₂ terminus, however, appears to have a small role in modulating VDA. For example, β_4b, which has a longer NH₂ terminus compared with β_4a, induces a larger hyperpolarizing shift in the activation of some Ca_vα₁ (208).

B. Ca_vβ Promotes Voltage-Dependent Inactivation, Except β_2a

VDI reduces the amount of Ca²⁺ entering the cell following depolarization and decreases the number of channels responsive to subsequent depolarizations. Ca_vβ is a key modulator of VDI, as first demonstrated in 1991 (268, 406, 450) and subsequently confirmed for various α₁/β combinations in different expression systems (116, 137,245, 340, 348, 414, 417, 458). Several aspects of VDI are affected by Ca_vβ. 1) β₁, most β₂ splice variants, β₃, and β₄ shift the voltage dependence of inactivation to more hyperpolarized voltages (by ~10–20 mV; Table 2 and Fig. 7), whereby weaker depolarizations are able to inactivate the channels. β_2a, however, causes a shift to more depolarized voltages (by ~10 mV) (40, 93,119, 132, 206, 218, 245, 276, 314, 340, 369). 2) Ca_vβs (except β_2a) promote the process of “closed state” inactivation exhibited by Ca_v2 channels when they rapidly transition between closed and open states, such as during a train of action potentials (β3 > β1b = β₄ >> β2a; Refs. 348, 491). Similarly, a large hyperpolarization of steady-state inactivation (approximately ~40 mV) is observed when β₃ is overexpressed with N- and R-type channels, dramatically increasing the population of inactivated channels at resting conditions (491). 3) β₁, most β₂ splice variants, β₃, and β₄ speed up the inactivation kinetics, whereas β_2a and β_2e slow down inactivation (Table 2 and Fig. 7).

The unique effects of β_2a on VDI are largely abolished when palmitoylation of β_2a is disrupted by mutating its two NH₂-terminal cysteine residues to serine (β_2a C3,4S) (365, 369). WT β_2a-like properties can be restored when a transmembrane segment of an unrelated membrane protein is fused to this mutant, suggesting that membrane anchorage rather than palmitoylation per se is critical for β_2a’s unique functions (369). Supporting this idea, the nonpalmitoylated but membrane-attached β_2e has properties similar to β_2a (433).

Multiple domains and regions of Ca_vβ are involved in the regulation of VDI. The GK domain alone, when expressed together with Ca_v2.1 and α₂δ in Xenopus oocytes, has been shown to speed up VDI and hyperpolarize the voltage dependence of VDI (206). The GK domain of all four subfamilies of Ca_vβ produces the same effects (Fig. 7, D and E; Ref. 206), as expected from its high degree of amino acid conservation. Similarly, the GK domain of β_2a greatly accelerates VDI and hyperpolarizes the voltage dependence of VDI of Ca_v2.2 channels expressed in oocytes and tsA-201 cells (129, 372). On the other hand, it has been reported that refolded and purified proteins of β_2a and β_1b GK domains slow down VDI and depolarize the voltage dependence of Ca_v2.3 channels expressed in oocytes (187). The discrepancy between these studies may result from the use of different Ca_vα₁ or from RNA versus protein injection, but it should be noted that the refolded and purified GK domains appear to be dimerized proteins (187), and it is unknown whether and how dimerization changes the function of the GK domain.

The HOOK plays an important role in regulating VDI, as first suggested by chimeric studies between different Ca_vβs (364, 420). Two recent studies based on structurally defined Ca_vβ domains provide more definitive evidence. 1) Swapping the HOOK between the core regions (SH3-HOOK-GK) of β_1b and β_2a, which have opposite effects on VDI, also swaps their effects on VDI (206). 2) Deleting the HOOK in either β_2a core or full-length β_2a results in increased VDI (372). These studies, in conjunction with those discussed earlier, indicate that both membrane attachment through palmitoylation and a long HOOK region contribute to the unique effects of β_2a on VDI.

The role of the NH₂ terminus of Ca_vβ in regulating VDI has long been established. Deleting or shortening the NH₂ terminus, or swapping the NH₂ terminus of different Ca_vβs markedly alters VDI (236, 340,364, 420). β₂ or β₄ splice variants differing in the NH₂ terminus exhibit markedly different VDI (208, 209,215, 433). As discussed above, the palmitoylation site of β_2a is in the NH₂ terminus.

Surprisingly, the COOH terminus of Ca_vβ seems to play a very limited or no role in regulating VDI, even though it is highly variable among the four Ca_vβ subfamilies. Thus, although a very small change in the inactivation kinetics of Ca_v2.1 channels is observed when the COOH terminus of β₄ is deleted (460), exchanging the COOH terminus between β₃ and β₄ or deleting the entire COOH terminus of any of the four Ca_vβs has little effect on VDI of Cav2.2 channels or Ca_v2.1 channels (206, 420). It remains to be determined whether the COOH terminus exerts a more prominent effect on VDI under other conditions and for certain combinations of Ca_vα₁ and Ca_vβ. Intriguingly, a β₄ COOH-terminal truncation mutant missing the last 38 amino acids, which causes slightly faster inactivation of Ca_v2.1 channels at moderate depolarizations, was identified in a juvenile myoclonic epilepsy patient (142). Whether the very subtle change in Ca²⁺ channel inactivation underlies the disease is unclear.

C. A Unified Model for Ca_vβ Regulation of Ca²⁺ Channel Gating

How does Ca_vβ regulate VDA and VDI of Ca_v1 and Ca_v2 channels? Before addressing this question, we first briefly discuss the pore structure, the location of the activation gate, and the mechanism of VDI of VGCCs.

The external pore, including the ion selectivity filter, of VGCCs is formed by the pore loop between the S5 and S6 transmembrane segments of each of the four homologous repeats of Ca_vα₁; point mutations in this region, especially of the four conserved glutamate or aspartate residues, drastically alter ion selectivity, permeation, and pore blockage (256, 266,389, 482). The inner pore is formed by all four S6 segments of Ca_vα₁, as demonstrated by the substituted cystine accessibility method (505). Cystine accessibility studies also indicate that the activation gate is located at the cytoplasmic end of the S6 segments (476). The S6 segments, together with the I–II loop and the NH2 and COOH termini of Ca_vα₁, are involved in controlling or regulating VDI (for review, see Refs. 212, 422). Although the precise molecular mechanism of VDI is unknown, a prevalent model is that the I–II loop of Ca_vα₁ functions as a “hinged lid” to physically occlude the pore by binding to the cytoplasmic ends of the S6 segments (421, 422), reminiscent of VDI of voltage-gated Na⁺ channels (71). Which amino acids form the inactivation gate and its receptor site remain unknown. An alternative model is that VDI is produced by a constriction of the pore (151). Either way, the S6 segments constitute a converging point through which both VDA and VDI are controlled and regulated.

The biochemical, functional, and structural studies presented above support a unified model for Ca_vβ regulation of VDA and VDI of VGCCs (9, 125,151, 206, 298, 341, 448, 455, 461). This model has two central components.

First, the high-affinity AID-GK domain interaction and a rigid IS6-AID linker are essential for Ca_vβ regulation of VGCC gating. As mentioned in section IIIE, in the presence of Ca_vβ, through the AID-GK domain interaction, the entire region encompassing the IS6 segment and the end of the AID becomes a continuous α-helix (9, 151, 341). Via this rigid structure, Ca_vβ gains a lever with which to regulate both activation and inactivation (Fig. 4). Thus Ca_vβ binding adds mass and tension to IS6 and the I–II loop, which most likely affects the energetics of voltage-dependent movement of both IS6 and the inactivation gate, thereby directly changing the voltage dependence and kinetics of activation and inactivation. This explains why the GK domain alone is capable of affecting both activation and inactivation (129, 206, 372). Equally important, the AID-GK domain interaction anchors Ca_vβ to Ca_vα₁, thereby enabling interactions between Ca_vβ and other parts of Ca_vα₁ that are of intrinsic low affinity but are important for Ca_vβ’s gating effects (see below). Supporting an essential role of the AID-GK domain interaction, many studies show that Ca_vβ regulation of gating is abolished by mutations in the AID (33, 34,56, 120, 181, 185, 206, 218, 276, 361, 448) or in the ABP (206, 502). However, one difficulty in interpreting these and similar experiments is that those mutations dramatically reduce or abolish Ca_vβ-stimulated Ca²⁺ channel surface expression, leaving minuscule currents to be scrutinized. This problem is circumvented in several recent studies where the rigid α-helical structure of the IS6-AID linker was disrupted by substituting linker residues with glycines, or inserting multiple glycines in the linker, while leaving the AID-GK domain interaction intact. These substitutions or insertions do not affect Ca_vβ-enhanced Ca²⁺ channel surface expression, but they severely compromise or eliminate the ability of Ca_vβ to regulate Ca_v1 and Ca_v2 channel activation and inactivation (151, 455, 502). These results underscore the essential role of a rigid IS6-AID linker in Ca_vβ regulation of VGCC gating.

An additional factor that is important for Ca_vβ regulation of gating is the orientation of Ca_vβ relative to Ca_vα₁ (455, 502). Inserting five alanine residues in the IS6-AID linker, which is expected to maintain the α-helical structure of the linker but induce a 180° rotation of Ca_vβ with respect to Ca_vα₁, markedly diminishes Ca_vβ regulation of activation, while insertion of seven alanines, which produces two full turns, has no significant detrimental effect (502). Similarly, deleting one or three residues in the IS6-AID linker totally abolishes Ca_vβ regulation of both activation and inactivation (455). These studies are consistent with the notion that additional contacts between Ca_vβ and Ca_vα₁ besides the AID-GK domain interaction are critical for Ca_vβ regulation of VGCC gating.

Second, intrinsically low-affinity interactions between Ca_vβ and Ca_vα₁ are crucial for Ca_vβ regulation of VGCC gating (especially VDI), and these interactions confer each Ca_vβ its distinct modulatory effect and α₁/β pair-specific gating properties. Besides the AID-GK domain interaction, other direct contacts between Ca_vβ and Ca_vα₁ have been observed in vitro. For example, the Ca_vβ SH3 domain interacts with the I–II loop, but at a region different from the AID (298), and a COOH-terminal region conserved only in β₂ binds to a COOH-terminal region of Ca_v1.2 where calmodulin (CaM) also binds (270). The same Cav1.2 COOH-terminal region also binds to a β_2a construct containing the N-SH3_{βstrand1–4}-HOOK module (501). Other regions of Ca_vα₁, including the NH₂ and COOH termini and the III–IV loop, have also been shown to interact directly with Ca_vβ (366, 436,460, 461). It remains to be determined which regions of Ca_vβ they associate with, but the Ca_vβ NH₂ terminus and HOOK are prime candidates since they are critically involved in regulating VDI. These additional α₁/β interactions have intrinsic low affinity, and on their own, do not produce significant gating effects. However, the strength of these interactions increases dramatically when Ca_vβ is anchored to Ca_vα₁ by the AID-GK domain interaction. These notions are supported by the aforementioned mutagenesis/insertion studies in the AID, the ABP, and the IS6-AID linker. Further supporting these ideas, Chen et al. (83) reported that, without changing the AID-GK domain interaction, splitting β_2a into two connected modules (N-SH3-HOOK and GK-C) through the insertion of increasingly longer flexible linkers between the SH3 and GK domains leads to a gradual diminishment of the effect of the N-SH3-HOOK module on VDI (83). This result indicates that keeping the N-SH3-HOOK module near Ca_vα₁ is essential for its modulatory effect. A future challenge is to develop ways to precisely map the interface of intrinsically low-affinity Ca_vα₁/Ca_vβ interactions, which might be too weak to be identified biochemically and might require more than one Ca_vα₁ region.

How low-affinity α₁/β interactions regulate gating is unclear. These interactions could pull on Ca_vβ and thereby modulate the movement of IS6 and the presumed inactivation gate in the I–II loop. They may also interfere with intramolecular interactions between the I–II loop and other parts of Ca_vα₁, such as the NH₂ and COOH termini and the III–IV loop, where point mutations and deletions cause marked changes of VDI (for review, see Refs. 212, 422). These intramolecular interactions, as well as the low-affinity α₁/β interactions, are α₁ or α₁/β pair specific (2, 99,178, 262, 369, 386, 404, 436, 460, 501). Thus, to fully appreciate the physiological importance of Ca_vβ regulation of VGCC gating, it is crucial to examine the pairing of Ca_vα₁ and Ca_vβ in different tissues and cell types, in different subcellular locations, and at different developmental stages.

A final point that should be mentioned here is that many proteins that interact directly with Ca_vβ have been shown to regulate VGCC gating, such as RGK proteins (see sect. IX), Best1 (493), and RIM1 (257) (see sect. XI).

D. Can Ca_vβ Produce AID-Independent Gating Effects?

Several reports, which at first seemed to contradict the model presented above, are in fact in accord with the model upon closer examination. It has been shown that β_2a is able to modulate VDA and VDI of Ca²⁺ channels formed by a mutant Ca_v2.1 subunit (Ca_v2.1_ΔAID) whose AID is deleted (298). This result led the authors to conclude that essential Ca_vβ modulatory properties are AID independent. This result, however, has an alternative explanation: β_2a can be anchored to the plasma membrane through palmitoylation, and this membrane tethering might mimic, at least partially, the anchoring role of the AID-GK domain interaction, bringing β_2a near Cav2.1_ΔAID subunits and promoting the functionally important low-affinity α₁/β interactions alluded to above. Indeed, this result lends strong support to the second part of the model discussed above, i.e., there are low-affinity interactions between Ca_vβ and Ca_vα₁ that are crucial for Ca_vβ regulation of VGCC gating.

Several studies reported that a 41-amino acid β₂ COOH-terminal fragment and some Ca_vβ splice variants, including β_2f, β_2Δg, β_1d, and chicken β_4c, all of which lack most or the entire GK domain (and hence cannot bind the AID), are all able to enhance Ca²⁺ channel currents and/or regulate their gating (92, 204, 270). However, these effects are much weaker than those produced by full-length Ca_vβs. Moreover, the specificity of these effects is called into question by the clear nonspecific effects of two short peptides that do not exist in nature: a 35-amino acid peptide containing the BID and a 43-amino acid peptide with a random sequence, both of which are able to stimulate Ca²⁺ channel expression and weakly modulate gating (84, 119, 120). Nevertheless, given that β_2f and β_2Δg are found in native cells (204, 270), they could affect Ca²⁺ channel gating through low-affinity α₁/β interactions if they are expressed at very high levels. At present, the physiological role of β_2f and β_2Δg remains unknown.

E. Ca_vβ Regulation of Ca²⁺-Dependent Inactivation and Facilitation

HVA Ca²⁺ channels are strongly regulated by another type of inactivation that depends on Ca²⁺ influx, namely, Ca²⁺-dependent inactivation (CDI) (for reviews, see Refs. 53, 78, 201, 358), which serves as a negative-feedback mechanism. CDI is mediated by the ubiquitous Ca²⁺-sensing protein CaM, which is constitutively bound to the Ca_vα₁ COOH terminus (494, 509). The exact molecular mechanism of CDI is unclear, as is the relationship between CDI and VDI, but a recent study shows that two of the elements critical for VDI, Ca_vβ and a rigid IS6-AID linker, are also essential for CDI (151). Glycine (but not alanine) substitutions that disrupt the α-helix of the IS6-AID linker dramatically slow CDI. The absence of Ca_vβ binding to Ca_vα₁, ensured by mutating the AID, produces similar results (151). Thus CDI and VDI appear to share a common mechanism by which conformational changes caused by CaM-Ca_vα₁ interactions or Ca_vβ-Ca_vα₁ interactions are transmitted to the pore through the rigid IS6-AID linker.

HVA Ca²⁺ channels also undergo Ca²⁺-dependent facilitation (CDF), which occurs during repetitive channel activation, such as during a train of action potentials (for reviews, see Refs. 53, 201, 358). This process, which is dependent on CaM binding to the Ca_vα₁ COOH terminus, also requires Ca_vβ binding to the AID and an intact IS6-AID α-helix (151). Interestingly, CDF is readily observed with β_2a but not with β_1b or β₄ (80, 272). The main reason for this difference is probably that channels with β_2a inactivate much slower; slow inactivation not only allows the unmasking of CDF but also further stimulates CDF by permitting a larger Ca²⁺ influx.

F. Ca_vβ Regulation of Voltage-Dependent Facilitation

L-type Ca²⁺ channels exhibit voltage-dependent facilitation (VDF) (47). VDF is manifested as a gradual increase in L-type current during high-frequency action potentials, and it partly explains activity-dependent enhancement of L-type currents in skeletal muscle, brain, and heart. VDF can be differentiated from CDF by using Ba²⁺ as the charge carrier; it is accompanied by an increase in high P_o gating (357) and may be dependent on phosphorylation (273). Like CDF, VDF depends on the presence of Ca_vβ (47, 75; but see Ref. 274); it is supported by β₁ and β₃ but not β_2a (47, 75,365; but see Ref. 109). Some of the discrepancies in the literature may result from the following reasons. 1) Differences in L-type channel splice variants and the α₂δ subunits used affect the results. For example, α₂δ₁ and α₂δ₃ seem to mask VDF by increasing inactivation (109). 2) The β_2a-containing channels already have a high P_o, so VDF is harder to observe in these channels. 3) Nonpalmitoylated β_2a mutants can restore VDF (365), suggesting that different levels of palmitoylation may contribute some variations in the results.

The GK domain alone appears to be necessary and sufficient to confer VDF; deleting other domains, including the SH3 domain, separately or in combination, spares VDF (77). Hence, it is possible that VDF, just like CDF, CDI, VDI, and VDA, relies on the rigid IS6-AID linker and Ca_vβ to affect gating. It would be of interest to investigate whether glycine substitution or insertion in the IS6-AID linker also affects VDF.

VII. STOICHIOMETRY AND REVERSIBILITY OF THE Ca_vα₁-Ca_vβ INTERACTION

How many β subunits need to bind to each Ca_vα₁ to bring about the aforementioned trafficking and gating effects? Is the Ca_vα₁-Ca_vβ interaction reversible? This section discusses these two important issues.

A. Ca_vα₁ and Ca_vβ Are Paired With a 1:1 Stoichiometry

Early biochemical studies suggest that skeletal and neuronal VGCCs contain a single Ca_vα₁ and a single Ca_vβ (430, 473). This remains the prevalent view today, but it comes after a brief competition with the idea of a 1 Ca_vα₁:2 or more Ca_vβ stoichiometry (62, 436).

As mentioned in section VA, Xenopus oocytes express two endogenous β₃-like subunits, called β_3xo (436). When Ca_vα₁ cRNA is injected into Xenopus oocytes alone, a small fraction of the Ca_vα₁ is transported to the plasma membrane by β_3xo (436). Coinjection of a mammalian Ca_vβ or either of the two Xenopus β subunits greatly increases Ca²⁺ channel current and changes its gating properties. These results led to the proposal that the “Ca_vα₁-alone” channels in fact contained a β_3xo and that one or more exogenous Ca_vβ bind the Ca_vα₁/β_3xo complex to form a higher order complex with modulated gating (436). Subsequently, by varying the concentration of coexpressed β₃, it was found that β₃ produced the trafficking effect with a sevenfold higher apparent affinity than it did gating modulation (17 vs. 120 nM) (62). This result was initially explained by one of two hypotheses: either two Ca_vβs bind a single Ca_vα₁ or the mature Ca_vα₁ on the plasma membrane has a lower affinity for Ca_vβ than the nascent Ca_vα₁ does (62).

Subsequent extensive studies indicate that Ca_vβ associates with Ca_vα₁ in a 1:1 stoichiometry and that this stoichiometry is determined by the AID-GK domain inter- action. 1) Channels coexpressed with a mixture of β_2a and β₃ form two biophysically distinct channel populations, rather than a single population of “mixed”-channel type (245). 2) Colecraft and colleagues (110) covalently linked a single β_2b to the COOH terminus of Ca_v1.2 (creating Ca_v1.2-β_2b) and found that the channels formed by Cav1.2-β_2b exhibited the same gating properties as channels formed by the coexpression of Ca_v1.2 and β_2b did. Moreover, coexpression of β_2a and Cav1.2-β_2b did not further change channel gating. 3) The crystal structures of the AID-Ca_vβ core complexes clearly show that each Ca_vβ binds a single AID (84, 341, 447). 4) Mutations of key residues in the AID or the ABP abolish both Ca_vβ-mediated Ca²⁺ channel surface expression and gating modulation (33, 34,56, 120, 181, 185, 206, 218, 276, 361, 448, 502).

B. The Ca_vα₁-Ca_vβ Interaction Is Reversible

The affinity of the AID-Ca_vβ interaction measured in vitro is very high, with a K_d ranging from 2 to 54 nM (30, 56,62, 120, 121, 179, 342, 371, 395, 448). The affinity of Ca_vα₁-Ca_vβ interactions in cells is less certain but seems to be lower (218), probably partly due to competition for Ca_vα₁ and Ca_vβ binding by other proteins. The lower affinity likely permits a more dynamic Ca_vα₁-Ca_vβ interaction. Indeed, several studies support the notion that the Ca_vα₁-Ca_vβ interaction is reversible in intact cells. 1) Injection of β₃ protein into oocytes expressing L-type Ca_vα₁ alone quickly alters Ca²⁺ channel gating properties, suggesting that some channels on the plasma membrane are devoid of Ca_vβ (480). 2) A synthetic AID peptide can significantly reduce the P_o of channels formed by L-type Ca_vα₁ and β_2a in HEK 293 cells when it is applied to the cytoplasmic side of inside-out membrane patches, but it has no effect on channels containing no β_2a, suggesting that the AID peptide can compete off bound β_2a (224). 3) Injection of β_2a protein into oocytes expressing Cav2.3 and β_1b results in a dramatic inhibition and slowing down of inactivation, consistent with β_2a replacing previously bound β_1b and overtaking the channel (218).

That Xenopus oocytes have endogenous Ca_vβs and that the Ca_vα₁-Ca_vβ interaction has a 1:1 stoichiometry and is reversible provide a straightforward explanation for why Ca²⁺ currents can be recorded in oocytes expressing Ca_vα₁ alone, and why the gating properties of these currents can be modulated by exogenous Ca_vβ: the endogenous β_3XO subunits are expressed at high enough levels to interact with a small fraction of the nascent Ca_vα₁ in the ER and transport them to the plasma membrane; there, β_3XO eventually dissociates from Ca_vα₁, leaving most of the channels devoid of a β subunit because the cytoplasmic concentration of β_3XO is too low to rebind these β-less channels. The β-less channels, however, can associate with exogenously overexpressed Ca_vβ to form a stable Ca_vα₁/Ca_vβ complex, as long as the cytoplasmic concentration of Ca_vβ is a few fold higher than the K_d of the Ca_vα₁-Ca_vβ interaction. It is likely that this is also the scenario in mammalian expression systems.

A dynamic and reversible Ca_vα₁-Ca_vβ association might play an important role in regulating Ca²⁺ channel activity, especially during development when changes in the expression level of different Ca_vβ isoforms occur (311, 435, 449). It has been shown that the Ca_vβ component of N-type Ca²⁺ channels changes during postnatal development, from β_1b > β₃ >> β₂ at P2 to β₃ > β_1b = β₄ at P14 and adult age (449). This study further shows that although no N-type channels associate with β₄ at P2, 14 and 25% of N-type channels contain β₄ at P14 and adult age, respectively.

VIII. ROLE OF Ca_vβ IN G PROTEIN INHIBITION OF Ca_V2 CHANNELS

VGCCs are susceptible to negative-feedback inhibition by hormones and neurotransmitters through the activation of G protein-coupled receptors (GPCRs). An extensively studied form of inhibition is the G protein-mediated, membrane-delimited, and voltage-dependent inhibition of members of the Ca_v2 channel family (i.e., N-, PQ-, and R-type channels). It is believed that this inhibition contributes to presynaptic inhibition and short-term synaptic plasticity (36, 52, 126, 219, 440, 471). This inhibition is mediated by the direct binding of G protein G_βγ subunits to the channel (213, 233), and it demonstrates three hallmarks: 1) it shifts channel activation to more depolarized potentials (23); 2) it is accompanied by a slowing of channel activation (23), resulting from latent G_βγ unbinding from the channel (139, 244, 349); and 3) it can be reversed by a strong conditioning depolarizing prepulse, which accelerates G_βγ dissociation from the channel in a phenomenon known as prepulse facilitation, or PPF (23, 139, 251). Below we discuss the role of Ca_vβ in the G_βγ-mediated, voltage-dependent inhibition. For in-depth reviews on other aspects of this inhibition, see References 117, 126, 138, 424, 440, and 497.

A. Ca_vβ Is Required for Voltage-Dependent G_βγ Inhibition

It has long been observed that some effects of G_βγ on VGCCs, such as the slowing of activation and the depolarizing shift of the voltage dependence of activation, are opposite to those of Ca_vβ, raising the possibility that G_βγ and Ca_vβ compete with each other (48, 60). Supporting this idea, early studies found that knockdown of endogenous Ca_vβ in neurons increased GPCR-induced inhibition of Ca²⁺ currents (60), and coexpression of Ca_vβ with Ca_vα₁ in oocytes decreased G protein-mediated inhibition (48, 366). However, later studies showed that in COS-7 cells G protein inhibition of N-type Ca²⁺ channels was markedly enhanced by coexpressed Ca_vβs (315), and that in tsA-201 cells, a mutant Ca_v2.2 that contained a point mutation in the AID (W391A) and was unable to associate with Ca_vβ could no longer display voltage-dependent G protein inhibition (276). The latter studies indicate that Ca_vβ is essential for voltage-dependent G protein inhibition of N-type Ca²⁺ channels.

The discrepancy among these studies could arise from many factors. In particular, in the early studies (48, 60, 366), G protein inhibition was examined at a single voltage, which could complicate the interpretation because G_βγ and Ca_vβ both shift the voltage dependence of channel activation, but in opposite directions. Another factor could be the difficulty of 1) characterizing inhibition of tiny Ca²⁺ channel currents typically recorded in the absence of coexpressed Ca_vβ, and 2) excluding the contribution of endogenous Ca_vβs. To overcome these difficulties, a mutant β_2a subunit (named β_2a_Mut2) was created by mutating two key AID-binding residues (M245 and L249) to alanine (502). When coexpressed with Ca_v2.1 in Xenopus oocytes, β_2a_Mut2 is still capable of promoting channel trafficking, but owing to its reduced affinity for the AID, it can be washed off from the surface of Ca²⁺ channels in excised membrane patches (502). With the use of this approach, large populations of Ca²⁺ channels devoid of Ca_vβ can be generated on the plasma membrane. Such β-less channels are still inhibited by purified G_βγ protein applied to the cytoplasmic side of the channels; however, all the hallmarks of voltage-dependent inhibition are absent (502). This finding strongly supports the notion that Ca_vβ is indispensible for voltage-dependent G_βγ inhibition.

Although β-less channels do not display voltage-dependent G protein inhibition, they can still be inhibited by G proteins in a voltage-independent manner. For example, the mutant Ca_v2.2 harboring the W391A mutation is still susceptible to voltage-independent G protein inhibition (276). Likewise, the β-less Ca_v2.1 channels (produced by washing off the bound β_2a_Mut2 in inside-out membrane patches) are also still inhibited by G_βγ, but without any voltage-dependent features (502). These findings indicate that G_βγ can bind Ca_vα₁ in the absence of Ca_vβ. Thus the essential role of Ca_vβ is to enable voltage-dependent dissociation of G_βγ from the inhibited channels, a process that gives rise to the voltage dependence of G_βγ inhibition (45).

B. G_βγ Does Not Displace Ca_vβ

Another important question is whether Ca_vβ and G_βγ coexist on Ca_vα₁ during voltage-dependent inhibition. The apparent opposing actions of G_βγ and Ca_vβ prompted the hypothesis that G_βγ displaces Ca_vβ from Ca_vα₁ (48, 60, 366, 375). This conclusion was also reached in a study showing that Förster resonance energy transfer (FRET) signals between Ca_vβ and Ca_vα₁ change during G_βγ inhibition (387). However, functional antagonism does not necessarily indicate direct competition, and, while FRET signal changes are indicative of protein conformational changes, they are inadequate in demonstrating protein dissociation (3, 231, 490).

On the contrary, several lines of evidence indicate that Ca_vβ remains associated with Ca_vα₁ during G_βγ modulation. 1) Different subfamilies of Ca_vβ have different effects on the magnitude and properties of voltage-dependent G_βγ inhibition, with β_2a being the least effective in promoting this inhibition (61, 129, 147, 316, 376). 2) Ca_vβ increases the rate of G_βγ dissociation (as determined by the time constant of PPF) from the inhibited channels, but the efficacy of the four Ca_vβs is different (with a rank order of β₃ > β₄ > β_1b > β_2a; Refs. 61, 147). These observations (1 and 2) are most easily explained if Ca_vα₁, Ca_vβ, and G_βγ form a tripartite complex during G_βγ modulation. 3) Since Ca_vβ critically affects VDA and VDI, these properties are expected to change if Ca_vβ were dislodged from the channel. However, the voltage dependence and kinetics of VDI remain unchanged before, during, and after G_βγ modulation (23, 316, 502). Similarly, the voltage dependence of activation is unchanged before and after G_βγ modulation (502). These results support the notion that Ca_vβ is not dislodged by G_βγ from the inhibited channels.

C. The Rigid IS6-AID α-Helix Is Necessary for Voltage-Dependent G_βγ Inhibition

The antagonistic effects of Ca_vβ and G_βγ on channel activation suggest that their actions are related structurally and mechanistically. As mentioned in section VIC, disruption of the α-helical structure of the IS6-AID linker (by inserting 3–7 glycine residues in the linker or by substituting 3 linker residues with glycine) abolishes Ca_vβ modulation of VGCC gating (151, 455, 502). The same maneuver also completely eliminates voltage-dependent G_βγ inhibition but spares voltage-independent G_βγ inhibition (502), indicating that a rigid IS6-AID helix is not necessary for G_βγ binding to Ca_vα₁ but is essential for voltage-dependent dissociation of G_βγ. Strikingly, both voltage-dependent and -independent G_βγ inhibition are abolished when five alanine residues are inserted into the IS6-AID linker of Ca_v2.1, which is likely to maintain the β-helical structure of the linker but produce a ~180° rotation of Ca_vβ with respect to Ca_vα₁ (502). It is possible that G_βγ can no longer bind to this mutant Ca_v2.1, but further investigation is necessary to confirm this speculation.

Consistent with the requirement of a rigid IS6-AID linker, two recent studies show that the GK domain alone is sufficient to support voltage-dependent G protein inhibition in both N- and P/Q-type channels (129, 502). On the other hand, the observation that different isoforms of Ca_vβ differentially modulate voltage-dependent G protein inhibition indicates that other Ca_vβ domains and regions can fine-tune this process. Indeed, the HOOK region has been suggested to play a role in enhancing the voltage-dependent dissociation of G_βγ (129).

On another note, the differential effect of different Ca_vβs on G_βγ-mediated inhibition can be further exposed by the expression of other proteins. For example, RGS2, which is a member of the regulators of G protein signaling that catalyze GTP hydrolysis and terminate G protein signaling (498), can unmask differences in G-protein modulation of P/Q-type channels containing different types of Ca_vβ (300).

D. Model for the Voltage Dependence of G_βγ Inhibition

Before presenting a model for voltage-dependent G_βγ inhibition, we first consider the molecular components involved in this process. 1) Several distinct regions in Ca_vα₁, all of which bind G_βγ in vitro, play a role in voltage-dependent G_βγ inhibition, including the NH₂ terminus (3, 345), the I–II loop (118, 346, 439, 496), and to a lesser extent, the COOH terminus (3, 189, 231, 282, 366, 499). The NH₂ terminus of Ca_vα₁ binds directly to the I–II loop, and together they form a G_βγ-gated inhibitory module (3). 2) The I–II loop has two G_βγ binding sites: one extends from the COOH-terminal end of IS6 to the NH₂-terminal end of the AID and contains a signature G_βγ-interacting QxxER motif (QQIER in Ca_v2.1), and the other is located further downstream of the AID (118, 439, 496). The downstream site, termed the G protein interaction domain or GID, is likely to be an anchoring site for G_βγ. It has a ~20 nM affinity for G_βγ (118, 496), and when applied as a 21-amino acid peptide, it can prevent PPF. The upstream site containing the QxxER motif has a ~60 nM affinity for G_βγ, and its mutations attenuate G_βγ modulation (214). However, this site may serve only as a secondary G_βγ-binding site in the holo-channel for three reasons. 1) It is partially buried by Ca_vβ, as shown by the AID-Ca_vβ core crystal structures (84, 341, 447). Hence, G_βγ binding to the upstream site is significantly weaker in the presence of Ca_vβ than in the absence of Ca_vβ (502). 2) The QxxER motif is unlikely to become completely available, since Ca_vβ does not vacate from the G_βγ-bound channels (61, 231, 315, 502), as discussed above. When seven alanine residues are inserted in the upstream site, which is expected to prevent G_βγ binding to this site, voltage-dependent G_βγ inhibition remains intact (502). 3) As discussed in section IIIE, Ca_vβ binding to the AID results in the formation of an α-helix extending continuously from IS6 to the AID. The integrity of this α-helix is critical for voltage-dependent G_βγ inhibition, as mentioned above.

Figure 8 depicts an allosteric model proposed recently for the origin of the voltage dependence of G_βγ inhibition of Ca_v2 channels (502). This model links voltage-dependent dissociation of G_βγ to the voltage-dependent movement of IS6 and to the obligatory role of Ca_vβ. The pocket where G_βγ binds in the holo-channel to produce the voltage-dependent inhibition is still unknown, but it is postulated to be downstream of the COOH-terminal end of the AID and is formed collectively by portions in the NH₂ terminus, the I–II loop, and the COOH terminus (and possibly yet unknown regions). Under the resting condition and in the presence of G_βγ, the channel is inhibited (Fig. 8A, left). Upon depolarization, the S6 segments of Ca_vα₁ move, and owing to the continuous rigid α-helical structure of the IS6-AID linker, this movement is transmitted to and beyond the AID, resulting in a movement of the distal I–II loop and, consequently, a conformational change of the G_βγ-binding pocket. Such a chain of events ultimately leads to the disassembly of the NH₂ terminus-I–II loop inhibitory module and the dissociation of G_βγ from the channel (Fig. 8A, right), which account for the slowing of the activation kinetics and prepulse facilitation. In the absence of Ca_vβ, G_βγ can still bind to the holo-channel but cannot be discharged by the depolarizing potential, because the G_βγ-binding pocket is uncoupled from IS6 as a result of the unwinding of the AID into a random coil (Fig. 8B). Such uncoupling can also be produced by glycine insertions in the IS6-AID linker (Fig. 8C). In summary, this model postulates that the voltage dependence of G_βγ inhibition of Ca_v2 channels arises from the voltage-dependent movement of IS6 and that Ca_vβ and a rigid IS6-AID linker play a pivotal role in translating this movement to G_βγ dissociation.

Fig. 8.

Model for the voltage dependence of G_βγ inhibition. The G_βγ-binding pocket in the holo-channel is postulated to be formed by a region of the I–II loop distal to the AID, the NH₂ terminus, and the COOH terminus of Ca_vα₁. A: WT channel: depolarization moves IS6; this movement is propagated through the rigid IS6-AID α-helix, consequently altering the conformation of the G_βγ-binding pocket and resulting in G_βγ dissociation. B: β-less channel: the AID relaxes into a random coil in the absence of Ca_vβ, uncoupling IS6 from the G_βγ-binding pocket. G_βγ can bind and inhibit the channel but does not dissociate in a voltage-dependent way. C: channel containing Ca_vβ but with a flexible IS6-AID linker: insertion of 3–7 glycine residues in the IS6-AID linker disrupts the α-helix, uncoupling IS6 from the G_βγ-binding pocket and abolishing voltage-dependent dissociation of G_βγ. [Adapted from Zhang et al. (502)].

IX. ROLE OF Ca_vβ IN RGK INHIBITION OF HIGH-VOLTAGE ACTIVATED Ca²⁺ CHANNELS

The RGK (Rad, Rem, Rem2, Gem/Kir) family of Ras-related monomeric small GTP-binding proteins has emerged as potent inhibitors of HVA Ca²⁺ channels (27, 155). There are four members in this family: Rad (Ras associated with diabetes; Ref. 370), Rem (or Ges = human ortholog; Ref. 153), Rem2 (157), and Gem/Kir (91, 296). They share a conserved Ras-like core but differ from other Ras members in that their GTP/GDP-binding domains have nonconserved mutations that alter or abolish the GTP/GDP cycle (410, 489). They also contain extended NH₂ and COOH termini. The COOH terminus has a motif that can anchor them to the membrane (reviewed in Refs. 81, 104, 211, 296) and is critical for their function (81, 104, 105, 488). RGK proteins have two known functions: shaping cytoskeletal dynamics and inhibiting HVA Ca²⁺ channels (24, 27, 104, 255, 324). These two functions can be differentially regulated; for example, RGK modification of cytoskeletal reorganization, but not inhibition of HVA Ca²⁺ channels, is attenuated by dephosphorylation of certain RGK residues (152, 463). The physiological importance of RGK inhibition of HVA Ca²⁺ channels is illustrated by recent in vivo studies that manipulate endogenous Rad levels with consequences for the heart (79, 462, 478). For example, dominant negative suppression of endogenous Rad in the heart increases L-type Ca²⁺ channel currents and action potential duration in cardiac cells and causes longer QT intervals and arrhythmias (478). Here, we only discuss RGK inhibition of HVA Ca²⁺ channels and the role of Ca_vβ in this process. For more comprehensive reviews of RGK proteins and their functions, see References 104 and 255.

A. Ca_vβ Is Essential for RGK Inhibition of HVA Ca²⁺ Channels

All members of the RGK family are able to inhibit, in a voltage-independent manner, HVA Ca²⁺ channels when expressed in various heterologous expression systems (15, 24–27, 81, 101, 102, 154–156, 165, 281, 397, 478, 487, 488). This inhibition depends on Ca_vβ, as RGK proteins do not affect Ca²⁺ channel currents recorded in cells expressing only Ca_vα₁ (27, 155, 397 but see Ref. 106). Consistent with this notion, RGK proteins do not affect the activity of T-type Ca²⁺ channels, which do not associate with Ca_vβ nor require Ca_vβ for their activity (81, 155). Furthermore, RGK proteins interact directly with Ca_vβ, both in vitro and in cells (24–28, 101, 102, 154–156, 165, 281, 487), and this interaction seems to be promiscuous whereby any RGK protein can interact with any full-length Ca_vβ. A structural model of Gem-β₃ interaction has been recently developed (28) based on systematic mutagenesis analysis and homology modeling based on the crystal structure of the AID-β₃ core complex (84) and a crystal structure of GDP-bound Gem (PDB 2G3Y). This model shows that Gem binds to the β₃ GK domain at a site distinct from the AID-binding pocket and that residues D194, D270, and D272 in β₃ and R196, V223, and H225 in Gem are critical for this interaction. Supporting this model, mutating these residues individually or in combination severely weakens or abolishes in vitro binding of Gem and β₃ (28, 144).

Three mechanisms of RGK inhibition have been reported. The first mechanism, reported in the first study on RGK regulation of HVA Ca²⁺ channels (27) and advanced in subsequent studies mostly from the same groups (24–26, 28, 388, 478), is that all RGK proteins disrupt the trafficking of HVA Ca²⁺ channels to the plasma membrane and hence reduce the number of surface Ca²⁺ channels, as determined primarily by imaging the subcellular localization of epitope-tagged Ca_vα₁. It was hypothesized that RGK proteins compete with Ca_vα₁ by sequestering Ca_vβ in the cytoplasm and/or the nucleus, thus leaving Ca_vα₁ trapped in the ER. Several observations are consistent with this hypothesis. 1) Nuclear targeting of Rem and Rad causes nuclear sequestration of Ca_vβ (24). 2) In addition to Ca_vβ, all RGK proteins also interact with CaM and 14 –3-3 (24 –26, 158, 297). Abolishing CaM and 14 –3-3 binding or CaM binding alone results in nuclear accumulation of RGK proteins (24 –26), suggesting that these interactions regulate the subcellular localization of RGK proteins. Moreover, Rem2 and Gem (but not Rad and Rem) mutants deficient in CaM binding are unable to inhibit HVA Ca²⁺ channel currents (24 –27, 463).

On the other hand, other findings are inconsistent with the aforementioned mechanism (i.e., sequestration of Ca_vβ and disruption of channel surface expression). 1) Ca_vβ-Ca_vα₁ binding through the AID-GK domain interaction is much stronger than RGK-Ca_vβ binding (154), making it unlikely for RGK proteins to compete off Ca_vβ from Ca_vα₁. 2) Several studies show that Ca_vα₁, Ca_vβ, and RGK proteins form a trimeric complex in vitro and in cells (28, 101, 144, 154, 487). 3) Work from several groups show that RGK proteins can inhibit HVA Ca²⁺ channels without affecting their surface expression, with the latter determined by surface binding of a radioactive toxin (81), surface biotinylation (144, 154, 156), or gating charge measurement (487). These observations suggest that cytoplasmic and/or nuclear sequestration of Ca_vβ by RGK proteins occurs via a mechanism other than competition with the AID and may, if at all, only partially account for the observed RGK inhibition of HVA Ca²⁺ channels.

The second mechanism, closely related to the first one, is that RGK proteins decrease the number of surface Ca²⁺ channels by increasing their internalization. A recent study shows that Rem enhances dynamin-mediated endocytosis of L-type channels expressed in HEK 293 cells (488).

The third mechanism of RKG inhibition is the suppression of the activity of channels already on the plasma membrane. Such direct inhibition has been observed for different RGK proteins and different HVA Ca²⁺ channels in a variety of expression systems. For example, Rem2 inhibits endogenous surface N-type channels in native neurons (81), Rem inhibits surface L-type channels expressed in pancreatic β-cells (154, 156) and HEK 293 cells (488), and rapid translocation of a recombinant Rem derivative acutely inhibits L- and N-type channels expressed in tsA201 cells (487). Recently, Gem inhibition of P/Q-type channels was reconstituted in inside-out membrane patches by direct application of a purified Gem protein domain (144). Furthermore, it was found that this acute inhibition was completely abolished when Ca_vβ was washed away from the patch (using the strategy mentioned in sect. VIIIA), leaving P/Q channels β-less, but it was fully restored after application of a purified Ca_vβ protein, demonstrating that Ca_vβ is indispensable for Gem inhibition of surface P/Q channels. Finally, in agreement with a recent study (281), it was found that the Ca_vβ GK domain alone was sufficient to support Gem inhibition of P/Q channels expressed in Xenopus oocytes (144).

The absolute requirement of Ca_vβ for RGK-mediated inhibition of HVA Ca²⁺ channels is reminiscent of such a requirement for voltage-dependent G_βγ inhibition of N- and P/Q-type channels. The latter process further requires a rigid α-helical structure of the IS6-AID linker. It was shown, however, that disrupting the IS6-AID α-helix by glycine insertions did not affect Gem inhibition of P/Q channels (144). This result suggests that Gem inhibition uses a fundamentally different mechanism from voltage-dependent G_βγ inhibition and may not involve IS6-transmitted conformational changes in the pore.

The three mechanisms of RGK inhibition discussed above operate on different time scales and are not mutually exclusive. For example, in the case of Rem inhibition of L-type channels expressed in HEK 293 cells, both reduction of surface channel density and direct inhibition of surface channels occur (488). Which mechanism dominates or is utilized in native cells remains to be determined, but it likely depends on the type and expression level of RGK proteins as well as Ca²⁺ channel subunits.

B. A New Paradigm for RGK Inhibition of HVA Ca²⁺ Channels

As mentioned above, all members of the RGK family can interact with all four subfamilies of Ca_vβ. The RGK-Ca_vβ interaction has been widely presumed to be essential for RGK inhibition of HVA Ca²⁺ channels (15, 24–27, 81, 101, 102, 104, 154–156, 165, 206, 281, 397, 478). However, it was recently reported that while Ca_vβ is required for Gem inhibition of surface P/Q-type channels, the interaction between Ca_vβ and Gem is not (144). In this study, residues D194, D270, and D272 in β₃ and R196, V223, and H225 in Gem were simultaneously mutated to alanine; these residues are predicted and shown to be critical for the Gem-β₃ interaction (28, 144). This combination of mutations completely abolished binding of Gem and β₃, and yet, the mutant Gem (named Gem_Mut3) was still fully capable of inhibiting P/Q channels containing either the mutant or WT β₃ (144). Mean-while, it was found that Ca_v2.1 could be coimmunoprecipitated by WT Gem or Gem_Mut3, either in the presence or absence of β₃, suggesting that Gem directly interacts with Ca_v2.1. Chimeric studies with PQ- and the RGK-insensitive T-type channels indicate that the IIS1–IIS3 region of Ca_vα₁ is essential for gem inhibition (144).

Based on these results and those discussed above, we propose a “Ca_vβ-priming” model for Gem inhibition of P/Q-type Ca²⁺ channels on the plasma membrane (Fig. 9; Ref 144). (This model may be applicable to Rem and Rem2 since they also inhibit surface Ca²⁺ channels.) A distinct feature of this model is that the interaction between Gem and Ca_vβ is not necessary for Gem’s inhibitory effect, but a direct association between Gem and Ca_v2.1 is essential. In this model, Gem interacts directly with Ca_v2.1 through an anchoring site, with or without Ca_vβ being present. In the presence of Ca_vβ and Gem, Ca_v2.1 forms a multimeric complex with both proteins on the plasma membrane (Fig. 9A). Binding of Ca_vβ to Ca_v2.1 produces a conformational change, resulting in the formation of an inhibitory site in Ca_v2.1 where Gem binds to produce inhibition (Fig. 9A). When Ca_vβ dissociates or is washed off from surface Ca_v2.1, the inhibitory site disappears, rendering Gem unable to inhibit Ca_v2.1, even though it can remain attached to Ca_v2.1 via the anchoring site (Fig. 9B). When Ca_vβ and Gem mutants that cannot bind to each other are used (Fig. 9C), inhibition can nevertheless proceed since the ability of Ca_vβ and Gem to bind Ca_v2.1 is not compromised. Thus the essential role of Ca_vβ is to convert Ca_v2.1 into a state permissive for Gem inhibition.

Fig. 9.

The “Ca_vβ-priming” model of Gem inhibition of surface HVA Ca²⁺ channels. Gem associates directly with Ca_vα₁ via an anchoring site in Ca_vα₁ (indicated by the purple patch). A: WT channel: binding of Ca_vβ to Ca_vα₁ induces an inhibitory site in Ca_vα₁ (indicated by the red patch), where Gem binds to induce inhibition. B: β-less channel: Gem can still associate with Ca_vα₁ via the anchoring site, but it does not inhibit the channel because Ca_vα₁ lacks the Ca_vβ-induced inhibitory site. C: WT channel with mutually noninteracting Ca_vβ and Gem: disrupting the interaction between Ca_vβ/Gem with mutations in the Ca_vβ/Gem interface does not affect Gem inhibition, since the interactions between Ca_vβ and Ca_vα₁ and between Gem and Ca_vα₁ remain intact. [Modified from Fan et al. (144).]

At present, this model remains speculative, and many questions remain unanswered. For example, where is the anchoring site for Gem on Ca_v2.1? Where is the inhibitory site? How does Ca_vβ binding to Ca_v2.1 create the inhibitory site? How does Gem binding to the inhibitory site lead to channel inhibition? Does the Gem-Ca_vβ interaction play any role at all? With regard to the last question, we speculate that in native cells, with physiological levels of Gem protein, the Gem-Ca_vβ interaction may increase the effective concentration of Gem near surface Ca²⁺ channels and thereby facilitate Gem inhibition.

X. ROLE OF Ca_vβ IN PHOSPHO- AND LIPID REGULATION OF HIGH-VOLTAGE ACTIVATED Ca²⁺ CHANNELS

Phosphorylation allows dynamic regulation of protein functions, including those of HVA Ca²⁺ channels. During the “fight-or-flight” response, for example, β-adrenergic stimulation leads to PKA-dependent upregulation of L-type Ca²⁺ channel currents, which results in a faster and stronger heartbeat. The activity of HVA Ca²⁺ channels is regulated by a variety of protein kinases and phosphatases. While Ca_vα₁ is often the target of phosphorylation, Ca_vβ can nevertheless modulate the effect of such phosphorylation, and in some cases, Ca_vβ itself is the target. HVA Ca²⁺ channels can also be regulated by membrane lipids and their metabolic products. This section discusses the role of Ca_vβ in phospho- and lipid regulation of HVA Ca²⁺ channels.

A. Ca²⁺/Calmodulin-Dependent Kinase II

Ca²⁺/calmodulin-dependent kinase II (CaMKII) is among the most abundant enzymes in many cell types. Recent studies show that CaMKII can interact directly with Ca_vα₁ of HVA Ca²⁺ channels and regulate their activities (242, 273, 358). In cardiac and smooth muscle cells, CaMKII plays a role in the facilitation of L-type Ca²⁺ channels (133, 192, 308). In cardiomyocytes, the molecular mechanism partly involves CaMKII-mediated phosphorylation of the β_2a subunit (192). CaMKII binds to the COOH terminus of β_2a and phosphorylates it at T498, which leads to an upregulation of L-type currents (192). This upregulation is not observed in the absence of β_2a or when a nonphosphorylatable β_2a mutant, β_2a (T498A), is coexpressed in tsA201 cells. This mutant can also act as a dominant negative, preventing CaMKII-mediated facilitation of endogenous Ca²⁺ currents. Moreover, T498 phosphorylation promotes the dissociation of CaMKII from β_2a, which may serve as a negative-feedback mechanism (193). Since most β₂ splice variants have a common COOH terminus identical to that of β_2a (Fig. 6), it would be interesting to examine whether CaMKII also interacts with and phosphorylates these β₂ variants.

CaMKII has also been shown to associate with β_1b in vitro, but not with β₃ and β₄ (193). A recent study further shows that CaMKII coimmunoprecipitates with forebrain L-type Ca²⁺ channel complexes containing β₁ or β₂ but not β₄ (1). β_1b, β₃, and β₄ have also been shown to be phosphorylated by CaMKII (193), but the physiological consequences remain to be determined.

B. Mitogen-Activated Protein Kinase

Mitogen-activated protein kinase (MAPK) is a member of a signaling network that responds to extracellular stimuli and induces diverse physiological and pathological processes. The small monomeric G protein, Ras, can upregulate Ca²⁺ currents in dorsal root ganglion neurons through the activation of the MAPK signaling pathway (160). In COS-7 cells, this upregulation is shown to require Ca_vβ, because in the absence of Ca_vβ, MAPK-dependent upregulation is abolished (159). Furthermore, different Ca_vβs support different degrees of upregulation. For example, in the presence of β_2a, but not other Ca_vβs, Ca²⁺ channels are partially resistant to inhibition by an antagonist of MAPKK, the exclusive activator of MAPK (159). It is speculated that MAPK directly phosphorylates the channel complex (159). While both Ca_vα₁ and Ca_vβ have consensus MAPK phosphorylation sites, it is unclear which, if any, are phosphorylated in cells.

C. Phosphoinositide 3-Kinase and Protein Kinase B (or Akt)

Some external stimuli (e.g., insulin-like growth factor) activate receptors that are associated with tyrosine kinases and upregulate L- and N-type Ca²⁺ channel currents (42). The mechanism likely involves phosphoinositide 3-kinase (PI3K) activation and subsequent production of phosphatidylinositol 4,5-bisphosphate (PIP₂) and phosphatidylinositol 3,4,5-trisphosphate (PIP₃), known regulators of HVA Ca²⁺ channels (for reviews, see Refs. 122, 318). Increased PIP₃ levels recruit protein kinase B (PKB) to the membrane, which can phosphorylate β_2a at S574 (454). This results in an upregulation of currents conducted by channels containing β_2a and Ca_v1.2 or Ca_v2.2, mainly by increasing surface expression (454). Ca²⁺ channels containing an unphosphorylatable β_2a (bearing the S574A mutation) are resistant to upregulation by PI3K/PKB, whereas those containing a phosphorylation-mimic β_2a (bearing the S574E mutation) exhibit tonically increased current and are insensitive to upregulation by PI3K. The permissive role of β_2a is not shared by other Ca_vβs and does not depend on palmitoylation (454).

A similar PI3K/PKB signaling pathway may play a role in maintaining normal cardiac function. Thus, in the absence of active PKB (created in a cardiac-specific conditional knockout), L-type Ca²⁺ channel surface expression is greatly reduced, leading to severe cardiomyopathy (70). This deficit results from lysosomal degradation of L-type channels, initiated by conserved PEST sequences (signals for rapid protein degradation) in Ca_v1.2. When PKB is active, it binds and phosphorylates β₂, which in turn masks the degradation signals and leads to an increased channel surface expression (70).

D. cAMP-Dependent Protein Kinase

cAMP-dependent protein kinase (PKA)-mediated upregulation of cardiac L-type Ca²⁺ channel currents was one of the first examples of ion channel modulation. During the “fight-or-flight” response, β-adrenergic stimulation, through G proteins and adenylate cyclase, results in increased levels of cAMP and subsequent activation of PKA, eventually leading to dramatic increases of cardiac L-type Ca²⁺ currents and the consequent faster and stronger heartbeat (for reviews, see Refs. 72, 309). In spite of intense research, the target of PKA phosphorylation that underlies L-type current upregulation still remains obscure, partly owing to the difficulty of reconstituting this regulation in heterologous expression systems. In vivo, this upregulation is accompanied by increased phosphorylation of both Ca_v1.2 and β₂ (54, 72, 114, 115, 180, 197). A PKA phosphorylation site on Ca_v1.2 (S1928) has been proposed to mediate the β-adrenergic response (115). However, a recent study using the Ca_v1.2 (S1928A) knock-in mice shows that basal L-type currents and the upregulation of L-type currents by PKA and β-adrenergic receptor stimulation are unchanged (275), indicating that PKA phosphorylation of S1928 is not the underlying cause for L-type current upregulation.

On the other hand, it has been shown that, in vitro, PKA phosphorylates three sites on β_2a: S459, S478, and S479 (180). It was initially proposed that β_2a phosphorylation at S478 and S479 is critical for L-type channel upregulation (54, 173). This was based on the lack of or reduced upregulation of currents conducted by L-type channels containing β_2a (S478A/S479A) (54, 173). A caveat is that these studies were done either with a truncated Ca_v1.2 (54) or without comparative experiments with WT β_2a (173). The role of β_2a phosphorylation in L-type current upregulation has been strongly challenged by a recent study, which shows that in cardiac muscle cells L-type channels containing β_2a(S459A/S478A/S479A) exhibit the same degree of PKA-mediated upregulation as channels containing WT β_2a (321). This study further demonstrates that the extent of PKA modulation is influenced by the associated Ca_vβ. Thus channels containing β_1b show the strongest upregulation, followed by those containing β₃ and β₄, whereas channels containing β_2a show the least modulation (probably because β_2a dramatically increases channel P_o in the first place, as mentioned in sect. VIA). Since β_1b, β₃, and β₄ do not share the aforementioned PKA phosphorylation sites in β_2a, the latter observation further strengthens the notion that PKA phosphorylation of β_2a does not play an essential role in the upregulation of cardiac L-type currents. Thus identifying the site(s) of PKA phosphorylation that give rise to this event remains a stubborn challenge.

Lastly, Ca_v1.3 channels can also be potentiated by PKA phosphorylation (287). The extent and duration of this potentiation also depend on the identity of the associated Ca_vβ (287).

E. Protein Kinase C

Protein kinase C (PKC) can enhance some neuronal L- and N-type Ca²⁺ channel currents (483). In Ca_v2 channels, the enhancement partly results from the disruption of G_βγ inhibition (19, 21, 96, 202, 426, 496). This is achieved by phosphorylating the I–II loop of Ca_vα₁, which may block G_βγ binding, considering that the I–II loop contributes to form the G_βγ-binding pocket (see sect. VIIID). Since Ca_vβ also binds to the I–II loop, it is conceivable that its binding is sensitive to PKC phosphorylation, or vice versa. In fact, pharmacological activation of PKC can increase Ca_v2.2 and Ca_v2.3 channel currents, but only in the presence of Ca_vβ, and transferring the I–II loop from these channels to the unresponsive Ca_v2.1 and Ca_v1.2 channels can transfer PKC sensitivity (49, 413). Thus it seems that Ca_vβ is permissive for PKC modulation of certain Ca_vα₁. However, the effects of PKC phosphorylation of Ca_vβ itself (e.g., β2a; Ref. 180) are unknown.

F. cGMP-Dependent Protein Kinase

cGMP-dependent protein kinase (PKG) is activated by cGMP and can phosphorylate both Ca_v1.2 and β_2a-Ca_v1.2 at S1928 (the same residue that can also be phsophorylated by PKA) and β_2a at S496 (484, 485). Activation of PKG results in inhibition of channels containing Ca_v1.2 and β_2a in HEK cells (484). This inhibition is abolished by the β_2a S496A mutation, suggesting that PKG phosphorylation of β_2a is critical for this process. The physiological consequences of this inhibition remain to be determined.

G. Arachidonic Acid

Like many other types of ion channels, the activity of HVA Ca²⁺ channels can be regulated by membrane lipids and their metabolic products (for reviews, see Refs. 46, 122, 172, 318, 373). One example is the voltage-independent inhibition of HVA Ca²⁺ channels upon PIP₂ depletion from the plasma membrane (171, 475). Another example is channel inhibition by arachidonic acid (AA), an unsaturated fatty acid released from phospholipids (including PIP₂) by the action of some phospholipases (18, 289, 374, 403). The magnitude of AA inhibition depends on the partnered Ca_vβ in the channel (374). In particular, β_2a seems to dampen AA inhibition of Ca_v1.3 channels. This unique effect of β_2a is abolished when palmitoylation is eliminated in a mutant β_2a (β_2a C3,4S). Attaching a transmembrane segment of an unrelated membrane protein to the NH₂ terminus of this mutant β_2a does not restore the dampening effect, suggesting that palmitoylation rather than membrane anchorage per se is responsible for the antagonizing effect of β_2a on AA-mediated inhibition. It is proposed that the palmitoyl groups of β_2a compete with AA for a common binding site on Ca_v1.3 (374). This unique ability of β_2a has also been proposed to underlie the enhancement of Ca_v2.2 channels containing β_2a by the stimulation of G_q-coupled receptors, which, in contrast, causes inhibition of channels containing β_1b, β₃, or β₄ (210).

XI. INTERACTION OF Ca_vβ WITH OTHER PROTEINS

For many years, Ca_vα₁ was the only known interacting partner for Ca_vβ. In recent years, however, a growing number of proteins have been found to interact with Ca_vβ, in some cases with significant functional impact. This section reviews some of these interactions and their functional consequences. Two more examples are discussed in section XII, in the context of a potential role of Ca_vβ in transcriptional regulation.

A. Ryanodine Receptors

In skeletal muscle, Ca²⁺ channel complexes (DHPRs), which are made up of Ca_v1.1, β_1a, α₂δ₁, and γ₁ subunits, are arranged on the plasma membrane of t tubules in tetrads, which are in contact with ordered RyRs in the membrane of adjoining sarcoplasmic reticulum (SR) (for reviews, see Refs. 167, 363). This physical arrangement is required for efficient excitation-contraction (EC) coupling. It has been shown that β_1a, which is expressed exclusively in skeletal muscle, is indispensible for EC coupling (for reviews, see Refs. 100, 164). This is because β_1a is essential not only for the surface expression of DHPRs (37, 38, 191, 393, 394, 402) but also for the tetrad formation (393, 394). It seems that β_1a allosterically primes Ca_v1.1 to properly interact with RyRs to form the tetrads (393). Expression of exogenous Ca_vβ in skeletal muscle cells isolated from β_1a-null mice or zebrafish can fully rescue L-type Ca²⁺ channel current, but only β_1a (and β_1c) can normalize EC coupling (38, 393, 402). This unique ability is due to the presence of a heptad repeat (L478-V485-L492) in the distal COOH terminus of β_1a (393, 401, 402). When this region is deleted from β_1a, EC coupling is lost, and when it is transferred to β_2a, EC coupling is observed. It is unclear, however, where the heptad repeat binds (402). What is certain is that β_1a can bind directly to the RyR (85, 164). On the RyR, β_1a binds to a highly charged region (KKKRRxxR), whose mutation attenuates EC coupling (85). Interestingly, two pathogenic mutations, R3348H and P3527S, occur in this region of the RyR and cause malignant hyperthermia susceptibility (384) and multi-minicore congenital myopathy (148), respectively. It remains to be determined whether the loss of interaction with β_1a is the underlying cause.

B. Ahnak

Ahnak, a ubiquitous large (700 kDa) signaling and scaffolding protein involved in diverse aspects of cell physiology and pathophysiology (reviewed in Refs. 7, 195), has been shown to interact with Ca_vβ in several distinct cell types, including cardiac cells, osteoblasts, and T lymphocytes (6, 199, 223, 305, 398). This interaction provides a potential link between Ca²⁺ channels, the cytoskeleton, and cellular organelles. Multiple regions in the COOH terminus of Ahnak can bind β_2a in vitro, with apparent affinities ranging from 50 to ~300 nM (196, 199, 223). The Ahnak-interacting region on β_2a is unknown, but since Ahnak coimmunoprecipitates with β_1b, β₃, and β_2a (6, 199, 398), it is likely to be in the conserved GK or SH3 domains.

It has been proposed that the Ahnak-β_2a interaction plays a role in PKA-mediated upregulation of cardiac L-type currents (195, 196). Both β_2a and Ahnak can be phosphorylated by PKA, and this phosphorylation weakens the binding between the two proteins by ~50% (196). This effect is accompanied by L-type current upregulation, a hyperpolarizing shift in the voltage dependence of activation, and an occlusion of subsequent PKA effects (196, 199). Thus it was suggested that under basal conditions, L-type channel activity is suppressed by the Ahnak-β_2a interaction and that PKA phosphorylation of both proteins disengages β_2a from Ahnak and hence relieves this tonic inhibition (195). However, a challenge to this proposal is that PKA phosphorylation of β_2a does not appear to play a role in the β-adrenergic receptor stimulation-induced upregulation of cardiac L-type currents, as discussed in section XD.

In the immune system, T cells are central in cell-mediated immunity. T cells are nonexcitable cells, yet they express all four Ca_v1 channels and all four Ca_vβs (241, 419). Activation of T-cell antigen receptors (TCR) causes Ca²⁺ influx, which is key for T-cell activation. This Ca²⁺ influx is thought to be mediated in part by Ca_v1 channels, although the mechanism of channel activation remains unclear (11, 241,305, 419). CD4⁺ T lymphocytes isolated from β₃- and β₄-null mice and CD8⁺ T cells isolated from β₃-null mice display impaired TCR-triggered Ca²⁺ response (11, 241), presumably because of deficient surface expression of Ca_v1 channels. T cells from Ahnak1 knockout mice also respond poorly to TRC stimulation and have impaired Ca²⁺ influx (305). It is proposed that this deficit results from decreased plasma membrane expression of Ca_v1 channels, owing to the lack of the Ahnak1-Ca_vβ interaction (305); however, this hypothesis needs further testing.

C. BK_Ca Potassium Channels

Large-conductance Ca²⁺-activated K⁺ channels (BK_Ca) are synergistically activated by membrane depolarization and intracellular Ca²⁺ and play important roles in various physiological processes (383). Even though BK_Ca channels have their own auxiliary subunits, it has been shown recently that BK_Ca channels bind directly to the Ca²⁺ channel β₁ subunit (508). In HEK 293T cells, this interaction dampens the Ca²⁺ sensitivity of BK_Ca channels and slows their activation and deactivation. The GK domain of β₁ is necessary and sufficient for these effects, suggesting that other Ca_vβs may share this β₁ function, but this remains to be determined. It is also unknown whether the Ca_vβ-BK_Ca channel interaction occurs in native cells, and whether and how this interaction affects HVA Ca²⁺ channels.

D. Bestrophin

Bestrophin (Best1) is a 585– 604 amino acid chloride channel expressed in the retinal pigment epithelium (RPE) whose mutations cause Best’s and other retinopathies (see review in Ref. 205). It modulates L-type Ca²⁺ channel gating and blocks L-type channel-mediated rises in [Ca²⁺]_i in RPE (301, 378). Recently, it was shown that Best1 binds the Ca²⁺ channel β₄ subunit and that its effects disappear in the absence of β₄, suggesting that Best1 may be acting through β₄ on Ca_v1.3 channels (493). Befittingly, β₄ knockout mice also have rethinopathies (301). The COOH terminus of Best1, which on its own does not generate Cl⁻ currents, can also inhibit L-type channels. It contains a predicted proline-rich domain (PRD) whose mutation abolishes the effects of Best1. It is proposed that this PRD binds to the SH3 domain of β₄, disrupts the GK-SH3 interaction, and causes L-type channel inhibition (493). However, direct evidence for such a mechanism is still lacking. It would be interesting to examine whether the PxxP-binding region of β₄ binds Best1. As discussed in section IIIB, although this region is occluded in the Ca_vβ crystal structures (84, 341, 447), it could conceivably become accessible when Ca_vβ is bound to Ca_vα₁ and other proteins.

E. Dynamin

A recent study reported that full-length β_2a interacts in vitro with dynamin, a multi-partner GTPase involved in endocytosis (186). This interaction was presumed to involve a PRD of dynamin and the SH3 domain of β_2a, since a purified β_2a fragment (amino acids 24–136) containing the four contiguous β sheets of the SH3 domain was found to interact with dynamin and this interaction was partially blocked by the dynamin PRD. This β_2a fragment was able to markedly suppress the surface expression of Ca_v1.2 channels, and this suppression depended on dynamin. It was proposed that the dynamin-SH3 domain interaction links HVA Ca²⁺ channels to the endocytotic machinery (186). It should be noted, however, that the β_2a fragment used in this study lacks the fifth (i.e., the last) β sheet of the SH3 domain and the HOOK region, which hinders access to the PxxP-binding region of Ca_vβ (84, 341, 447). To determine whether the PxxP-binding region of β_2a is involved in the interaction between dynamin and full-length β_2a, it would be useful to examine whether this interaction is abolished by selective mutations of β_2a residues that are presumably directly involved in binding PRDs.

F. Synaptic Proteins: Synaptotagmin I and RIM1

The α₁ subunit of presynaptic HVA Ca²⁺ channels physically interacts with presynaptic proteins, including syntaxin, SNAP-25, and synaptotagmin I; these interactions are important for synaptic vesicle docking and fusion (reviewed in Ref. 400). Recent studies show that Ca_vβ can also interact with synaptic proteins (257, 452). For example, the NH₂ terminus of β₃ and β_4a (but not β_4b) binds to synaptotagmin I, and this interaction is abolished by a high concentration (10 mM) of Ca²⁺ (452). However, the physiological importance of this interaction is yet unknown. Another study shows that RIM1, a presynaptic protein critical for synaptic transmission and plasticity (69, 392), binds directly and with a high affinity (35 nM) to β_4b and β_2a. This interaction is mediated by the COOH terminus of RIM1, and the SH3-HOOK-GK module of Ca_vβ is sufficient for binding to occur. The most prominent effect of the RIM1-Ca_vβ interaction on HVA Ca²⁺ channels is the slowing of VDI and a hyperpolarizing shift of the voltage dependence of inactivation. This effect is observed on recombinant L-, N-, P/Q-, and R-type channels containing β_4b and on recombinant P/Q-type channels containing β_1a, β_2a, β₃, or β_4b. RIM1 may also play a role in anchoring synaptic vesicles to presynaptic VGCCs through binding to the synaptic vesicle protein Rab3. Consequently, overexpression of a mutant Ca_vβ that is unable to bind Ca_vα₁ can attenuate vesicle docking at the presynaptic membrane in PC12 cells, presumably by competing with the WT Ca_vα₁/β complex for RIM1 binding. Furthermore, overexpression of RIM1 in PC12 cells and cultured cerebellar neurons enhances neurotransmitter release. This study establishes a direct role of Ca_vβ in the physical organization of the synaptic vesicle release machinery (257).

G. Zinc Transporter 1

Zinc transporter 1 (ZnT-1), a ubiquitous transmembrane protein involved in zinc transport and metabolism, binds directly to β_2a (280) and has been shown to inhibit L-type Ca²⁺ channels in heterologous expression systems and native cells (29, 280,337, 396). When coexpressed with Ca_v1.2 and β_2a in Xenopus oocytes, ZnT-1 reduced Ca_v1.2 channel currents (280). This inhibitory effect disappeared in the absence of β_2a or in the presence of an excess amount of β_2a. ZnT-1 reduced the surface expression of Ca_v1.2 without changing its total expression level when they were coexpressed in HEK 293T cells (280). The authors proposed that ZnT-1, through direct binding to Ca_vβ, inhibits L-type channels by reducing their trafficking to the plasma membrane (280). It remains to be examined whether ZnT-1 physically interacts with other types of Ca_vβs and whether it inhibits Ca_v2 channels.

XII. Ca²⁺ CHANNEL-INDEPENDENT FUNCTIONS OF Ca_vβ

Until recently, the functions of Ca_vβ have been exclusively linked to VGCCs. However, a stream of recent studies suggests that Ca_vβ may possess functions independent of their association with VGCCs. This line of inquiry began with the cloning of various short isoforms of Ca_vβ, some of which lacked the GK domain. This inability to engage in the high-affinity AID-GK domain interaction with Ca_vα₁ raised questions about their functions (92, 166,204, 217, 229). The first study examining possible alternative functions of truncated splice variants of Ca_vβ centered on a β₄ splice variant expressed in chicken cochlea and brain, termed β_4c (217); to avoid confusion, we refer to it as cβ_4c. cβ_4c is truncated after exon 8 and thus lacks 90% of the GK domain and the entire COOH terminus (Fig. 6). As expected, cβ_4c barely affects Ca_v2.1 channels coexpressed with α₂δ in Xenopus oocytes. However, cβ_4c interacts directly with the scaffolding domain of heterochromatin protein 1 (HP1), a nuclear protein involved in gene silencing and transcriptional regulation. Both proteins are colocalized in the nuclei of cochlear hair cells, and their coexpression in tsA201 cells causes translocation of cβ_4c from the cytoplasm to the nucleus. Moreover, cβ_4c attenuates the repressor function of HP1 in a dose-response manner. The effects on HP1 are specific since a longer isoform, β_4a, has no effect. These findings suggest that cβ_4c may function as a transcription regulator.

In a recent study, Zhang et al. (504) reported that full-length β₃ could directly interact with a new splicing isoform of Pax6, a transcription factor critical for the development of the eye and nervous system. The new isoform, named Pax6(S), has a truncated COOH terminus with a unique serine-rich tail. The interaction between Pax6(S) and β₃ is conferred mainly by the S tail and the SH3-HOOK-GK module of β₃. Since the other three subtypes of Ca_vβ can also interact with Pax6(S), the binding site for the S tail likely resides in the conserved SH3 or GK domain. Coexpression of Pax6(S) with Ca_v2.1 channels containing β₃ in Xenopus oocytes does not alter channel properties; however, the in vitro transcriptional activity of Pax6(S) is markedly suppressed by β₃. Furthermore, co-expression of β₃ and Pax6(S) in HEK 293T cells results in the translocation of β₃ from the cytoplasm to the nucleus. These results suggest that full-length Ca_vβs may function as transcription regulators (504).

This notion is further supported by other recent studies (16, 427), which show that, upon neuronal differentiation, full-length β_4a physically interacts with B56δ, a nuclear regulatory subunit of phosphatase 2A (PP2A). The β_4a/B56δ complex relocates to the nucleus, where it associates with nucleosomes and regulates the dephosphorylation of histones, a key mechanism in transcriptional regulation. A mutant β₄ lacking the last 38 COOH-terminal residues and associated with a case of juvenile myoclonic epilepsy (142), can neither associate with B56δ nor translocate to the nucleus, and its in vitro transcriptional regulation activity is different from that of WT β₄ (16, 427). Formation of the β_4a/B56δ complex requires an intact intramolecular SH3-GK interaction.

Consistent with the notion that full-length Ca_vβs may function as transcription regulators, it has been shown that full-length Ca_vβs can be targeted to the nucleus in native cells. For example, β₄, and to a lesser extent, β_1b and β₃, are translocated into the nucleus when they are exogenously expressed in cardiac cells (93). A recent study reports that endogenous β₄ is present in the nuclei of cerebellar granule cells and Purkinje cells (425). When heterologously expressed in skeletal myotubes or cultured hippocampal neurons, β_4b is robustly targeted to the nucleus, whereas other Ca_vβs are not. Nuclear localization of β_4b is dependent on an Arg-Arg-Ser motif in the NH₂ terminus, which is necessary since deleting this motif decreases nuclear targeting of β_4b. This motif is also sufficient since fusing it to β_4a increases nuclear targeting of the resulting chimera. Importantly, nuclear targeting of β_4b is diminished upon increased electrical activity and Ca²⁺ influx through L-type Ca²⁺ channels, suggesting a potential physiological function (425).

Another possible VGCC-independent function for β₄ is demonstrated by a recent study in zebrafish (135), which express all four subtypes of Ca_vβs (506). Morpholino knockdown of zebrafish β₄ abolishes or retards epiboly, an early development process, due to disturbances in mitotic and postmitotic cytoskeletal rearrangements. Epiboly can be rescued by coinjecting full-length human β_4a or β_4b cRNA. Interestingly, epiboly can also be rescued upon coinjection of a mutant β_4a, which contains a triple mutation (M204A/L208A/L350A) in its AID-binding pocket and cannot bind Ca_vα₁ or enhance Ca²⁺ channel currents in Xenopus oocytes. These results suggest an involvement of β₄ in zebrafish early development, probably through VGCC-independent actions. It remains to be determined how β₄ is involved and whether this function is shared by other Ca_vβs.

In a study using β₃ knockout mice, high glucose conditions caused pancreatic β cells to produce twofold more insulin than their WT counterparts (31). No change in VGCC currents was detected, but the β₃-deficient cells exhibited a higher frequency of glucose-induced intracellular [Ca²⁺] oscillations, accompanied by increased IP₃ production and increased Ca²⁺ release from intracellular stores. On the basis of the colocalization of these proteins in pancreatic β cells, it was hypothesized that β₃ may directly interact with IP₃ receptors to cause some of these effects (31).

In snails (Lymnaea), the expression of the sole Ca_vβ (LCa_vβ) is temporally and spatially uncoupled from the expression of LCa_v2, a Lymnaea homolog of the mammalian Ca_v2 family of VGCCs (409). Functionally, LCa_vβ does not modulate the surface expression or gating of LCa_v2 when they are coexpressed in tsA201 cells, even though they show current upregulation and gating modulation when they are partnered with rat Ca_vα₁ and Ca_vβ, respectively (409). Furthermore, knockdown of LCa_v2, but not LCa_vβ, alters neurite morphology. These results suggest that LCa_vβ may have VGCC-independent functions, which remain to be elucidated. This work illustrates that studies in simple model organisms might be beneficial to our understanding of the full spectrum of Ca_vβ functions.

XIII. Ca_vβ KNOCKOUTS AND PATHOPHYSIOLOGY

As expected, because of the essential role of Ca_vβ in the surface expression and functional modulation of HVA Ca²⁺ channels, Ca_vβ knockouts or mutations can produce severe functional deficits and, in some cases, are lethal. The phenotype of Ca_vβ knockout mice depends on the ability of the remaining three Ca_vβ genes to compensate. This section discusses the phenotypes and pathophysiology of Ca_vβ knockouts and mutations.

A. Gene Knockouts and Mutants

1. β₁

As mentioned in section XIA, β_1a is irreplaceable in partnering with Ca_v1.1 channels to enable skeletal muscle EC coupling. Thus β₁ knockout mice, similar to Ca_v1.1 knockouts, are born motionless and die immediately from asphyxiation (191). Skeletal muscles isolated from β₁-null mice are twitchless upon electrical stimulation, and action potentials do not elicit Ca²⁺ transients. L-type Ca²⁺ channel currents and the surface expression of Ca_v1.1 subunits are much reduced in these muscles, but caffeine can still cause contractions, indicating that internal Ca²⁺ stores are intact (191). Transgenic expression of β_1a exclusively in the skeletal muscle of β₁-null mice rescues the mice, which exhibit no obvious phenotype, suggesting that the remaining Ca_vβ genes can compensate for the functions of β₁ in other tissues (14).

Zebrafish β₁ knockouts also exist (393, 394). They have the Relaxed phenotype and die paralyzed days after hatching, with completely deficient EC coupling. Skeletal muscles from β₁-null zebrafish have no tetrads and show reduced depolarization-induced Ca²⁺ transients, but exhibit normal caffeine-induced Ca²⁺ transients (394). Unlike in β₁-null mice, targeting of Ca_v1.1 subunits to t tubules and the formation of triads are preserved (394), suggesting a nonessential role of β₁ in these processes in zebrafish. It remains to be determined whether other Ca_vβs are expressed in β₁-null zebrafish skeletal muscles or whether zebrafish Ca_v1.1 is able to traffic to the plasma membrane on its own.

2. β₂

Several β₂ splice variants are the predominant Ca_vβs expressed in the heart (Table 1). Thus it is no surprise that β₂ knockouts have no cardiac contractions and are nonviable beyond embryonic day 10.5 (14, 468). This is due to diminished L-type Ca²⁺ channel currents in cardiomyocytes and cardiac failure-associated defective remodeling of blood vessels. The β₂-null phenotype can be rescued by the expression of β₂ under a cardiac muscle-specific promoter (14). These partial knockouts revealed an essential role of β₂ in tissues besides the heart: such mice (lacking β₂ in all but cardiac tissues) are deaf due to a dramatic reduction in the membrane expression of Ca_v1.3 channels in inner hair cells, coupled with decreased exocytosis, improper hair cell development, and defective cochlear amplification (331). These “rescued” mice also have defects in vision with a phenotype similar to human patients with congenital stationary night blindness (13).

Given the knockout results, genetic mutations in β₁ and β₂ are expected to affect mainly skeletal and cardiac muscles, respectively. While no β₁ mutations have been associated with genetic diseases thus far, β₂ mutations have. Thus a mutation in the COOH terminus of β_2b (CACNB2b), S481L, contributes to a type of sudden death syndrome characterized by a short QT interval and an elevated ST segment (8), which are categorized into a group of genetic heart diseases called the Brugada syndrome. This mutation decreases Ca_v1.2 currents by ~75% in an expression system (CHO-K1 cells). Another mutation, in the β_2b NH₂ terminus (T11I), causes accelerated inactivation of cardiac L-type channels and is also linked to the Brugada syndrome (97). This mutation occurs in exon 2C of the CACNB2 gene and only affects β_2b, the most abundant Ca_vβ isoform in the heart (93). A recent study suggests that variations in CACNB2 may be also associated with a heightened risk for Alzheimer’s disease (286).

3. β₃

β₃ Knockouts are viable and were initially found to be normal (328, 330). Later studies, however, uncovered a wide spectrum of abnormalities, especially under stress conditions. For example, at high glucose concentrations, the frequency of [Ca²⁺]_i oscillations and the resulting insulin secretion from pancreatic β cells are potentiated (31). This is likely due to the attenuation of β₃-mediated inhibition of IP₃ production (31, 339). Also, a high-salt diet causes elevation of blood pressure, reduction in plasma catecholamine levels, and a hypertrophy of heart and aortic smooth muscle (328, ). The latter effects, as well as observations from mice overexpressing β₃ (327), are consistent with a function of β₃ in sympathetic control. In this regard, β₃ knockouts resemble N-type channel (Ca_v2.2) knockouts (428, ), which is not surprising since N-type channels preferably partner with β₃ (294, 325–327, 395). Indeed, sympathetic neurons from β₃-null mice have reduced N- and L-type channels activity (330). N-type current is also decreased in dorsal root neurons, which dampens inflammatory pain, but not mechanical or thermal pain (325). In the brain, N-type channel expression is reduced by ~40% (326); in the hippocampus, expression of NR2B (an NMDA receptor subunit), NMDA receptor currents, and long-term potentiation are all increased (239). Some forms of hippocampus-dependent learning and memory appear to be enhanced, but working memory is impaired (239, ). Furthermore, pruning of visual retinocollicular pathways is developmentally reduced and delayed (98). Behaviorally, β₃-null mice have lower anxiety, increased aggression, and increased night-time activity (326). Finally, β₃-null mice show abnormal signaling in CD4 T-cells, where receptor-mediated Ca²⁺ responses, nuclear translocation of NFAT, and cytokine production are all attenuated (11).

4. β₄

Mutated β₄ was first reported in lethargic mice (55, 130, 131). The naturally occurring null mutation is a four nucleotide insertion in Cacnb4, causing a translational frame shift and a premature stop codon. Lethargic mice have ataxia, seizures, absence epilepsy, and paroxysmal dyskinesia (17, 55, 227). The abnormal phenotype appears after postnatal day 15, a time when WT animals have an increase in β₄ expression in the brain (311). The upregulation of β₄ in WT mice is particularly robust in cerebellar granule and Purkinje neurons, which likely explains the ataxia in null mice (55). T-type Ca²⁺ channel upregulation (by ~50%) in thalamic neurons of lethargic mice likely contributes to the seizures (503). It is not clear why the remaining Ca_vβs fail to compensate for the lack of β₄, but this could be partly because of the unique interactions between the NH₂ and COOH termini of β₄ with other proteins (for examples, see Refs. 51, 121, 420, 460, 461, 474). Nevertheless, in lethargic mice, there is increased pairing of Ca_v2.2 and Ca_v2.1 with other Ca_vβs; in particular, both β_1b and Ca_v2.2/β_1b complexes are upregulated, similar to what is found in the developing brain (310, 311). Some other characteristics of lethargic mice include lower N-type channel expression in the forebrain and cerebellum (310), reduced excitatory neurotransmission in the thalamus (57), a modified electro-oculogram (301), splenic and thymic involution (130, 131), and renal cysts (130). Similar to β₃ knockouts, CD4⁺ T-cells have attenuated receptor-mediated Ca²⁺ responses, nuclear translocation of NFAT, and cytokine production (11).

Since β₄ is the predominant partner for P/Q-type (Ca_v2.1) channels in brain (311), it is no surprise that tottering mice (161), with mutations in Ca_v2.1, have a phenotype very similar to lethargic mice (356). Both tottering and lethargic mice are also models for epilepsy (17, 226). Indeed, there are examples where mutations in β₄ precipitate epilepsy and ataxia in humans. In one case, an R468Q mutation in CACNB4, which enhances Ca_v2.1 current, was associated with a history of febrile seizures (338). In another, truncated β₄ (R482x) that only has a very minor effect on HVA Ca²⁺ channel properties was found in a juvenile myoclonic epilepsy patient (142). In yet another case, a mutation in the SH3 domain of β₄ (C104F) causes different symptoms in two different families: episodic ataxia in one and generalized epilepsy and praxis-induced seizures in the other, presumably as a result of different genetic backgrounds (142).

B. Ca_vβ in Pathophysiology

Changes in the expression level of various Ca_vβs have been reported in certain pathological conditions. For example, in hypertrophic obstructive cardiomyopathy, β₂ is upregulated, which likely drives the observed increase in Ca²⁺ channels (198, 465). Downregulation of Ca_vβ is observed in allografts from diastolically failing hearts (228), in pancreatic islets from type 2 diabetic rats (235), and during atrial fibrillation (188). However, these observations are only correlative, and it remains unclear whether these changes are causative or incidental to the disease.

In the Lambert-Eaton myasthenic syndrome (LEMS), an autoimmune disease, autoantibodies against the extracellular loops of presynaptic Ca²⁺ channels disrupt channel arrays at the neuromuscular junction and impair synaptic transmission (269, 299). However, antibodies against β₃ and β₄ are also common in sera from LEMS patients (55% of the time), including in five of five LEMS patients who also had small-cell lung carcinoma (368). In some instances, the Ca_vβ autoantibodies can prevent Ca_vα₁-Ca_vβ binding (368, 377). It is unclear, however, how Ca_vβ autoantibodies contribute to the disease, since Ca_vβ is an intracellular protein and is unlikely to be a target of the Ca_vβ autoantibodies in the intact muscle. Indeed, immunization of rats with a purified Ca_vβ protein causes no neuropathy in spite of the induction of high antibody titers (453). These observations and considerations suggest that Ca_vβ autoantibodies do not directly contribute to the pathology of the disease, but their presence can serve as an additional diagnostic tool (368).

Finally, schistosomiasis, or bilharzia, is a parasitic disease caused by Schistosoma flatworms, which infect ~200 million people in the developing world, damaging the nervous system and internal organs. It is relatively successfully cured with Praziquantel (PZQ). The exact mechanism of action is still unclear, but PZQ seems to target a variant of Schistostoma Ca_vβ (reviewed in Refs. 124, 190, 240). Schistosomas have one “conventional” Ca_vβ and one, named β_var, with a long COOH terminus and nonconserved changes in both the SH3 and GK domains (240, 263,264, 334). When expressed with a mammalian Ca_vα₁, β_var modulates gating as expected for a Ca_vβ, but it causes a decrease in current amplitude (263). PZQ recovers current amplitude (263), consistent with results showing that PZQ causes a Ca²⁺ influx into worms, followed by a sustained muscular contraction and paralysis (240). It is not clear, however, whether β_var associates with Schistosoma Ca_vα₁ since expressing them has been difficult (190). A new study shows that Ca_vβ knockdown in Schistosomas, using siRNA, confers resistance to PZQ, further implicating Ca_vβ (334). Thus PZQ likely targets Schistosoma Ca_vβ, but the downstream events remain to be elucidated (124, 190). They may involve an increase in Ca²⁺ channel currents but may also include other pathways. Recently, it was suggested that Ca²⁺ influx on its own is not sufficient to kill Schistosomas, because cytochalasin D, an inhibitor of actin polymerization, can rescue Schistosomas from PZQ, in spite of cellular Ca²⁺ overload (354).

XIV. PERSPECTIVES

Great strides have been made in the last two decades in our understanding of the molecular biology, structure, function, and regulation of Ca_vβ. An emerging theme is that Ca_vβ is a multifunction protein, acting primarily as a Ca²⁺ channel regulatory subunit but also performing Ca²⁺ channel-independent functions. Although much is known, many important questions and issues remain to be elucidated, some of which we highlight here.

1. Although it is well established that Ca_vβ is essential for the surface expression of HVA Ca²⁺ channels, it is yet unclear why Ca_vβ is required. The traditional view is that Ca_vβ facilitates the export of Ca_vα₁ from the ER. However, no definitive ER retention signals have been found on Ca_vα₁ that are blocked by the binding of Ca_vβ. An alternative possibility is that Ca_vα₁ can traffic to the plasma membrane on its own, but its continued presence there requires Ca_vβ. Further studies on the role of Ca_vβ in Ca_vα₁ internalization, ubiquitination, and proteasomal degradation may shed light on this issue.

2. Given that many isoforms of Ca_vβ exist, that the association between Ca_vβ and Ca_vα₁ is promiscuous, and that Ca_vβ regulates channel gating in a Ca_vα₁-Ca_vβ pair-specific manner, there is enormous combinatorial complexity. Furthermore, the reversible nature of Ca_vα₁-Ca_vβ association provides a means for dynamic regulation of HVA Ca²⁺ channel activity. Thus, to better understand the function and regulation of HVA Ca²⁺ channels in native cells, it is necessary to examine the spatial and temporal expression of different Ca_vβ isoforms, not only at cellular levels but also at subcellular levels, as exemplified by the work in neurons (317, 336). Currently, we know very little about the molecular mechanisms governing the splicing and expression of Ca_vβ in different tissues, cell types, and subcellular locations (e.g., soma vs. dendrites vs. axon terminals).

3. Although the high-affinity interaction between the Ca_vβ GK domain and the AID is essential for Ca_vβ regulation of HVA Ca²⁺ channel gating, it is the interactions among other regions of Ca_vβ and Ca_vα₁ that confer distinct Ca_vα₁-Ca_vβ pair-specific characteristics to Ca_vβ regulation. We do not yet have a full grasp of the molecular determinants involved in these interactions, owing to their intrinsically low affinity. Characterizing these low-affinity interactions will remain a difficult challenge, as conventional biochemical approaches may not be sufficient to uncover the underlying molecular components and mechanisms.

4. Useful knowledge has been gained from Ca_vβ knockouts; however, lethal phenotypes and/or compensation by other Ca_vβs have limited the amount of information gleaned from systemic knockouts. It would be desirable to achieve inducible and tissue-specific knockout, knockdown, or overexpression of a particular Ca_vβ. A recent study (90) suggests that such approaches may even become useful venues for gene therapy of certain forms of cardiovascular or neurological disorders.

5. The list of Ca_vβ-interacting proteins continues to grow, but in most cases, the physiological importance of their interactions with Ca_vβ in native cells remains unclear. Some VGCC-independent functions of Ca_vβ are presented in this review, but almost certainly more are to be discovered. In this regard, it would be particularly interesting to investigate whether, and under what conditions, full-length Ca_vβs participate in regulating gene expression in native cells.

6. To better understand the function of Ca_vβ, it would be valuable to obtain high-resolution structures of full-length Ca_vβs, by themselves and in complex with their various interacting partners. These are clearly long-term goals, and there will undoubtedly be technical challenges in such endeavors, but the successful determination of the crystal structure of three different Ca_vβ cores’ and two different AID-Ca_vβ core complexes warrants optimism.