Biochemical Studies of Voltage-Gated Ca2+ Channels

William A. Catterall

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Biochemical Studies of Voltage-Gated Ca²⁺ Channels

William A. Catterall.

Author Information and Affiliations

Voltage-Gated Calcium Channels edited by Gerald W. Zamponi.

Voltage-gated Ca²⁺ channels mediate Ca²⁺ entry into cells in response to membrane depolarization. Electrophysiological studies reveal different Ca²⁺ currents designated L-, N-, P-, Q-, R-, and T-type. The high-voltage-activated Ca²⁺ channels that have been characterized biochemically are complexes of a pore-forming α1 subunit of about 190 to 250 kDa, a transmembrane, disulfide-linked complex of α2 and δ subunits, an intracellular β subunit, and in some cases a transmembrane γsubunit.The α1 subunits form the transmembrane pore. The α2 and δ subunits are glycoproteins encoded by the same gene and produced posttranslational proteolytic processing. The γ subunits are transmembrane glycoproteins, whereas the β subunits are hydrophilic subunits located on the cytosolic face of the channel. The Ca_V1 family of α1 subunits conduct L-type Ca²⁺ currents, which initiate muscle contraction, endocrine secretion, and gene transcription. The Ca_V2 family of α1 subunits conduct N-type, P/Q-type, and Rtype Ca²⁺ currents, which initiate rapid synaptic transmission. Both of these families of Ca²⁺ channels are regulated by protein phosphorylation and interact with intracellular signal transduction proteins that mediate Ca²⁺-dependent regulatory events and in turn regulate the channels.

Introduction

Ca²⁺ channels have been studied intensively by electrophysiological methods since the initial recordings of Ca²⁺ currents by Reuter in 1967.¹ In the early 1980's, stimulated in part by success at purification and reconstitution of sodium channels,² a new focus of Ca²⁺ channel research developed aimed at identification of the Ca²⁺ channel proteins and analysis of their biochemical properties. In this chapter, I briefly review the biochemical studies that led to the first identification of the Ca²⁺ channel proteins and provided the foundation for the subsequent analysis of the structure and function of the channel subunits by molecular biological methods. I have restricted consideration here primarily to experiments in which biochemical methods were applied to purified protein preparations. For this reason, only the Ca²⁺ channels of the Ca_v1 and Ca_v2 families are considered. Readers are directed to subsequent chapters in this book for consideration of more recent studies that have employed a combination of molecular biological and protein expression methods to further analyze these questions.

Purification and Biochemical Characterization of Skeletal Muscle Ca²⁺ Channels

The Ca²⁺ channels in the transverse tubule membranes of skeletal muscle have served as a primary biochemical preparation for studies of Ca²⁺ channels because of their abundance. These channels serve two critical physiological roles. Like other Ca²⁺ channels, they mediate Ca2+ entry in response to depolarization. The primary voltage-gated Ca²⁺ currents in skeletal muscle are L-type,³^-⁵ characterized by slow voltage-dependent inactivation, large single channel conductance (about 25 pS), high voltage of activation, and specific inhibition by dihydropyridine Ca²⁺ channel antagonists. These channels activate very slowly, and the Ca²⁺ entering vertebrate skeletal muscle through voltage-gated Ca²⁺ channels is not required for muscle contraction. It appears to replenish cellular Ca²⁺ during periods of rapid activity and to increase intracellular Ca²⁺ in response to tetanic stimulation, leading to increased contractile force. The primary physiological role for the skeletal muscle Ca²⁺ channel is to serve as a voltage sensor in excitation-contraction coupling. Voltage-gated Ca²⁺ channels in the transverse tubule membranes are thought to interact physically with the Ca²⁺ release channels located in the sarcoplasmic reticulum membrane. Voltage-driven conformational changes in the voltage-gated Ca²⁺ channels then activate the Ca²⁺ release from the sarcoplasmic reticulum by protein-protein interactions.⁶^-⁸

Purification of Ca²⁺ channels from skeletal muscle began with isolation of the transverse tubule membranes, which are highly enriched in Ca²⁺ channel protein, and specific labeling by high affinity binding to dihydropyridine Ca²⁺ channel antagonists to identify the channel protein. ⁹^,¹⁰ Ca²⁺ channels were solubilized in the mild detergent digitonin to retain native subunit associations and purified by a combination of ion exchange chromatography, affinity chromatography on wheat germ agglutinin-Sepharose, and sedimentation through sucrose gradients.¹⁰ A heterogeous a subunit band⁹^,¹⁰ and associated β subunits of 50 kD and γ subunits of 33 kD¹⁰ were identified as components of the Ca²⁺ channel in the initial purification studies, as assessed by comigration during column chromatography and sucrose gradient sedimentation. Subsequent experiments demonstrated that the heterogenous a subunit band contained not only the principal α1 subunits with an apparent molecular mass of 175 kD but also a disulfidelinked dimer of α2-δ subunits with apparent molecular masses of 143 kD and 27 kD, respectively, as illustrated in the SDS-PAGE results in Figure 1A.¹¹^-¹⁵ The specific association of these proteins as a multisubunit complex was supported by the copurification of each subunit with the dihyropyridine binding activity and Ca²⁺ conductance activity of the Ca²⁺ channel,¹⁰^,¹⁴^,¹⁶^,¹⁷ by coimmunoprecipitation of all these proteins by antibodies directed against the α1 subunits, ¹²^,¹⁴^,¹⁸ and by co-immunoprecipitation of the Ca²⁺ channel complex by antibodies against each auxiliary subunit.¹⁹^,²⁰^,²¹ Estimates of stoichiometry indicated that each mol of Ca²⁺ channel complex contains approximately 1 mol of each of the 5 subunits. The biochemical and molecular properties of each of the subunits of skeletal muscle Ca²⁺ channels are considered below.

α1 Subunit

The α1 subunit of skeletal muscle Ca²⁺ channels was cloned by library screening based on amino acid sequence²² (fig. 2). The cDNA predicts a protein of 1873 amino acids with a molecular weight of 212 kD, considerably larger than the estimate of 175 kD for the α1 subunits of purified Ca²⁺ channels. Analysis of the α1 subunits of purified Ca²⁺ channels and Ca²⁺ channels in transverse tubule membranes using sequence-directed antibodies showed that most (>90%) contained α1 subunits that were truncated in their carboxyl terminal domain between residues 1685 and 1699 resulting in a 190 kD form that runs anomalously in SDS gels at 175 kD.²³^,²⁴ Only a small fraction (<10%) of skeletal muscle Ca²⁺ channels contained the full length α1 subunit encoded by the cDNA. Both forms were detected in rat skeletal muscle cells in culture suggesting that both may be present in vivo.²⁵ Since no mRNA encoding the more abundant, truncated form has been identified, the truncated form is probably produced by specific proteolytic processing in vivo. A similar cleavage product can be produced by calpain treatment, suggesting calpain as a candidate for in vivo processing of the a1 subunits.²⁶

Figure 2

Structures of the Subunits of Ca_V Channels. Transmembrane folding models and site of regulation of the Ca²⁺ channel subunits. Predicted alpha helices are depicted as cylinders. The lengths of lines correspond approximately to the lengths of the polypeptide (more...)

α2 Subunit

The α2 subunit of skeletal muscle Ca²⁺ channels is a hydrophobic glycoprotein with an apparent molecular mass of 143 kD before deglycosylation and 105 kD after deglycosylation (fig.1A, lanes 1 and 2).¹⁴^,²⁷^,²⁸ It contains both high mannose and complex carbohydrate chains (fig. 1A, lanes 4 and 5). Cloning and sequencing cDNAs encoding the α2 subunit defined a protein of 1106 amino acids with a molecular mass of 125 kD, multiple potential transmembrane segments, and multiple consensus sites for N-linked glycosylation²⁹ (fig. 2). The predicted α2 protein was 20 kD larger than the apparent molecular mass of the deglycosylated alpha;2 subunit suggesting that a portion of the protein encoded by the α2 cDNAs may not be present in the mature α2 subunit that had been characterized biochemically. Subsequent studies showed that both α2-δ subunits are encoded by the same mRNA (see below).

Figure 1

Biochemical properties of skeletal muscle Ca²⁺ channels. A. Summary of the biochemical properties of purified skeletal muscle Ca²⁺ channels. Lanes 1 and 2, silver stain of polypeptides; lane 3, staining with an antibody against the α1 subunit; (more...)

β Subunit

The β subunits are hydrophilic proteins that are not glycosylated and therefore are likely to be located on the intracellular side of the membrane (fig. 1).¹⁴^,²⁰ cDNA cloning and sequencing revealed a protein of 524 amino acids with a predicted molecular mass of 58 kD.³⁰ In agreement with biochemical data, the primary structure does not include any potential transmembrane segments but contains multiple consensus sites for phosphorylation by different protein kinases (fig. 2B).

γ Subunit

The γ subunit of skeletal muscle Ca²⁺ channels is a hydrophobic glycoprotein with an apparent molecular mass of 30 kD without deglycosylation and 20 kD following deglycosylation.¹⁴^,²¹ Cloning and sequencing cDNAs encoding γ subunits revealed a protein of 222 amino acid residues with a molecular mass of 25 kD.³¹^,³² The deduced primary structure contained four predicted hydrophobic transmembrane segments and multiple sites for N-linked glycosylation.

δ Subunit

The δ subunit appears on SDS gels as a doublet of 24 and 27 kD proteins, which are both hydrophobic and glycosylated.¹⁴^,²⁸ Determination of the amino acid sequences of peptides derived from the δ subunit showed that it was encoded by the same mRNA as the alpha;2 subunit. ³³^,³⁴ The mature α2 subunit is truncated at alanine 934 of the α2δ precursor protein; residues 935-1106 constitute the disulfide-linked δ subunit. This sequence comprises a protein of 16 kD and contains a single transmembrane segment and three consensus sequences for Nlinked glycosylation. The doublet on SDS gels represents two differently glycosylated forms of the δ subunit.

Membrane Association of Subunits

The transmembrane organization of the Ca²⁺ channel complex was intially investigated by labeling of potential transmembrane segments with a hydrophobic photoaffinity probe.¹⁴ By this criterion, the α1, α2δ complex, and γ subunits were identified as probable integral membrane proteins, with the labeling of the α2 subunits the weakest despite its large size (fig.1A, lane 7, and B).¹⁴ Subsequent hydropathy analysis of the primary structures revealed 24 transmembrane segments in the α1 subunit, four in the γ subunit, and one in the δ subunit (fig.2), but the prediction of transmembrane alpha helices in the α2 subunits has been uncertain.²⁹ Analysis of association of the α2 subunit with the membrane by expression and biochemical extraction procedures led to the conclusion that the α2 subunit does not have a true transmembrane segment.³⁵ Instead, it is likely to be associated with the membrane primarily by virtue of its disulfide linkage to the δ subunit and perhaps also by superficial ionic and hydrophobic interactions with the membrane bilayer (fig.1B).

Sites of Protein Phosphorylation

Activation of skeletal muscle L-type Ca²⁺ channels is enhanced by phosphorylation by cAMPdependent protein kinase.³⁶^,³⁷ In cultured skeletal muscle cells, repetitive depolarization causes a dramatic enhancement of Ca²⁺ currents,³⁸^,³⁹ up to 10-fold in the critical membrane potential range near -20 mV. This potentiation of Ca²⁺ currents is strongly voltage-dependent and also is dependent on the activity of cAMP-dependent protein kinase.³⁹ This may result from interaction between voltage-dependent gating and phosphorylation of the Ca²⁺ channel itself. This novel regulatory mechanism greatly increases Ca²⁺ channel activity during tetanic stimulation of skeletal muscle cells and may play a critical role in the regulation of contractile force of skeletal muscle in response to hormones and to the frequency of stimulation of the motor nerve.

The α1 subunit and the β subunit of skeletal muscle Ca²⁺ channels are substrates for phosphorylation by cAMP-dependent protein kinase and a number of other protein kinases (fig.1A, lane 8).¹⁴^,⁴⁰^-⁴⁴ Ca²⁺ flux through the purified skeletal muscle Ca²⁺ channel is regulated by cAMP-dependent protein phosphorylation.¹⁷^,⁴⁵^-⁴⁷ Ion flux studies in reconstituted phospholipid vesicles show that phosphorylation of the a1 and β subunits can greatly increase the number of functional Ca²⁺ channels in purified preparations.⁴⁶^,⁴⁷ Single channel recording experiments in planar bilayer membranes detect both increases in the number of functional Ca²⁺ channels and increases in the activity of single Ca²⁺ channels after phosphorylation by cAMP-dependent protein kinase.¹⁷^,⁴⁵ Thus, the α1 and β subunits of the purified Ca²⁺ channel contain the sites at which cAMP-dependent protein phosphorylation modulates channel function in vitro.

Two size forms of the α1 subunit of approximately 190 kDa and 212 kDa are present in purified preparations of skeletal muscle Ca²⁺ channels, T-tubule membranes, and intact skeletal muscle cells in culture, and both are phosphorylated by PKA in intact cells.²³^-²⁵ Antibody mapping of the C-terminal region of α₁₁₉₀ placed the C-terminus between residues 1685 and 1699.²⁴ The most rapidly phosphorylated site in the truncated form of the α1 subunit in purified Ca²⁺ channel preparations is Ser 687, located in the intracellular loop between domains II and III.⁴⁸^,⁴⁹ In contrast, time course experiments indicated that Ser 1854 near the Cterminal portion of full-length α1212 is the most intensely and rapidly phosphorylated.⁴⁹^,⁵⁰ To date, the site(s) of phosphorylation that regulate skeletal muscle Ca²⁺ channel activity have not been directly identified.

Phosphorylation of the β Subunit of Skeletal Muscle Ca²⁺ Channels

Like the α1 subunit of the skeletal muscle Ca²⁺ channel, the β subunit is stoichiometrically phosphorylated by cAMP-dependent protein kinase in purified preparations⁴⁰ and in reconstituted Ca²⁺ channels that are regulated by cAMP-dependent protein kinase.¹⁷^,⁴⁷ Both serine 182 and threonine 205 have been shown to be phosphorylated in vitro.²³^,³⁰ Since phosphorylation of both α1-β subunits have been shown to be correlated with regulation of the ion conductance activity of skeletal muscle Ca²⁺ channels, both are candidates for sites of channel modulation by phosphorylation.

Sites of Drug Binding

A defining feature of Ca_v1 channels and the L-type Ca²⁺ currents they conduct is block by dihydropyridines, phenylalkylamines, and other Ca²⁺ channel antagonist drugs. Photoaffinity labeling studies established that the binding sites of dihydropyridines,¹³^,¹⁴^,¹⁵^,²¹ phenylalkylamines,⁵¹ and benzothiazepines⁵² are located in the α1 subunits. Detailed mapping of the dihydropyridine receptor site by proteolytic digestion and mapping of the digested fragments with sequence-directed antibodies identified the IIIS6 and IVS6 segments as important components of the binding site.⁵³ These results led to a domain interface model of drug binding of dihydropyridines, in which these drugs bind at the interface between domain III and IV at the point of interaction of the S6 segments.⁵⁴ Subsequent extensive mapping of the dihydropyridine receptor site by mutagenesis and functional analysis has given a detailed view of this important receptor site (see Chapter 17) and allowed construction of high affinity dihydropyridine receptor in non-L-type Ca²⁺ channels.⁵⁵^-⁵⁷ Similarly, the receptor sites of phenylalkylamines and benzothiazepines have been extensively mapped by photoaffininty labeling and peptide mapping⁵⁸^,⁵⁹ and subsequently by mutagenesis and functional analysis (see Chapter 17). All three receptor sites are close in the expected three-dimensional structure of the channel and have both distinct and overlapping molecular determinants of their binding.

Biochemical Properties of Other Ca_v1 Channels

Subunits of Purified Cardiac Ca²⁺ Channels

Like the skeletal muscle Ca²⁺ channel, the principal cardiac Ca²⁺ channel conducts L-type Ca²⁺ currents.⁶⁰^,⁶¹ Antibodies against α2d subunits of skeletal muscle Ca²⁺ channels detect corresponding subunits in cardiac preparations.⁶²^,⁶³ Partially purified cardiac Ca²⁺ channels contain a1, α2δ, and β subunits.⁶⁴^-⁶⁸ However, the relatively low abundance of Ca²⁺ channels in cardiac tissue and the difficulty of controlling proteolysis during lengthy purification procedures have frustrated attempts at complete purification of an intact cardiac Ca²⁺ channel complex. The α1 subunit with an apparent mass of 165 kD to 190 kD has been directly identified by photoaffinity labeling with photoreactive dihydropyridines.⁶⁴^,⁶⁶^,⁶⁹ As in skeletal muscle, most of the α1 subunits of cardiac Ca²⁺ channels are truncated at the C-terminus by proteolytic processing.⁷⁰ The primary site for phosphorylation by PKA in vitro and in cultured cells is Ser1928 (in rabbit Cav1.2 sequence numbering), which is located in the distal C-terminus beyond the site of proteolytic cleavage,⁷⁰ as observed for the skeletal muscle Ca²⁺ channel.⁴⁹^,⁵⁰ Therefore, it is possible that there is a functional relationship between proteolytic cleavage of the C-terminal domain and regulation of the cardiac Ca²⁺ channel by protein phosphorylation. Consistent with this idea, truncation of the C-terminal domain by expression of cDNAs with appropriate deletions yields increased Ca²⁺ channel activity,⁷¹ and perfusion of C-terminal peptides into the cells expressing truncated channels inhibits the increase in Ca²⁺ channel activity.⁷²

Molecular Basis for Modulation of Cardiac Ca²⁺ Channels by PKA

Although regulation of the cardiac Ca²⁺ channels by the cAMP pathway was the first example of ion channel regulation through second messenger pathways,⁷³^,⁷⁴ the molecular basis for this regulation is still not clearly resolved. Early biochemical studies of the cardiac Ca²⁺ channel resulted in purification of a short, 160 to 195 kDa form of the α1 subunit that was not a substrate for cAMP-dependent protein kinase.⁶⁴^,⁶⁶ As for skeletal muscle Ca²⁺ channels, more recent results on cardiac Ca²⁺ channels have revealed a full-length a1 subunit form with a molecular mass of approximately 220 kDa, as expected from the cDNA sequence.⁷⁰ This fulllength a1 subunit is phosphorylated on a single serine residue (Ser1928) in the C-terminal domain.⁷⁰ Similarly, expression of the C-terminal domain in bacteria yields a protein that is phosphorylated primarily on Ser1928.⁷⁵ This site is missing in the truncated from of the α1 subunit isolated in early biochemical preparations. Primarily the full-length form of Ca_V1.2 is present in cardiac tissue.⁷⁶^,⁷⁷ Both β1 and β2 isoforms are expressed in cardiac tissue⁷⁶^-⁷⁹ and likely are both associated in Ca_V1.2 channels.

Ca²⁺ channels in a CHO cell line that expresses only the full-length form of the Ca_V1.2 α1 subunit can be regulated by activation of endogenous PKA, by intracellular perfusion of PKA, and by voltage-dependent potentiation, which requires the activity of PKA.⁸⁰^,⁸¹ These results indicate that at least part of the regulation of Ca²⁺ channel activity is due to phosphorylation of the α1 subunit. Since the α1 subunit is only phosphorylated on Ser1928 in vitro or in transfected cells,⁷⁰ this regulation likely involves phosphorylation of this site. In support of this conclusion, mutation of this serine residue to alanine reduces the regulation of Ca²⁺ channel activity caused by activation of PKA with 8-Br-cAMP in transiently transfected human embryonic kidney cells from a 35% increase to a 7% increase.⁷⁶^,⁷⁷ Although these results are promising, the extent of regulation of Ca²⁺ channel activity in these experiments in transfected cells falls well short of the magnitude recorded in native cardiac cells (2- to 4-fold increase), and regulation of cloned Ca²⁺ channels analyzed in this way is not observed in some of the major laboratories in the field.⁸² Therefore, it seems likely that additional regulatory influences that have not yet been reproduced and characterized in transfected cells are important for control of the activity of cardiac Ca²⁺ channels in vivo.

Phosphorylation of the β Subunit of Cardiac Ca²⁺ Channels by PKA

The β subunits of cardiac Ca²⁺ channels are phosphorylated by PKA in intact hearts treated with β-adrenergic agonists.⁸³ Bunemann et al⁸⁴ have reported substantial (greater than 2-fold) regulation of a C-terminal truncated form of Ca_V1.2 lacking Ser1928 by intracellular perfusion of activated PKA in human embryonic kidney cells co-transfected with Ca_V1.2 and β2a subunits. This regulation required phosphorylation of Ser 478 and/or Ser 479, two serine residues in non-classical PKA phosphorylation sites that are specific to the β2a subunit. These results provide the best evidence to date for an important role of the β subunits in regulation of cardiac Ca²⁺ channels. It will be important to further analyze Ca²⁺ channel regulation when full-length α1 subunits are co-expressed with β2a and other β subunits expressed in cardiac myocytes and to restore regulation by physiological stimulus procedures rather than intracellular perfusion of activated PKA.

Subunits of Purified Neuronal Ca_v1 Channels

Because the concentration of Ca²⁺ channels in skeletal muscle transverse tubules is much higher than in neuronal membranes, the biochemical properties of these channels in neurons are not as well established. Immunoprecipitation of Ca²⁺ channel complexes labeled with dihydropyridines using specific antibodies against α2δ subunits revealed a complex of polypeptides with sizes corresponding to α1, α2δ, and β subunits of dihydropyridine-sensitive L-type Ca²⁺ channels in the brain.¹⁹^,⁸⁵^,⁸⁶ A potentially novel 100 kD protein was also identified as a specifically associated component of the L-type Ca²⁺ channel complex from brain.¹⁹ Further biochemical characterization of brain Cav1 channels will require development of a method for purification of larger quantities of channel protein.

Biochemical Properties of the Ca_v2 Family of Ca²⁺ Channels

Multiple types of Ca²⁺ channels, which differ in physiological and pharmacological properties, are expressed in neurons. At least three types of high-voltage-activated Ca²⁺ channels have been distinguished in addition to L-type (see Chapter 3).⁸⁷^-⁹⁰ N-type, P-type, and Q-type channels all have intermediate single channel conductances (about 15 pS) and can mediate Ca²⁺ currents with varying rates of voltage-dependent inactivation depending on their subunit composition and on other factors (see below). They are best distinguished by their pharmacological properties: N-type channels are specifically inhibited by ω-conotoxin GVIA whereas P/ Q-type are most sensitive to ω-agatoxin IVA and ω-conotoxin MVIIC. These specific peptide toxins have provided experimental tools for analysis of the protein subunits of neuronal Ca²⁺ channels.

Ca_v2.2 Channels

The ω-conotoxin-sensitive N-type Ca²⁺ channels purified from rat brain contain an a1 subunit, a 140 kD α2-like subunit, and β subunits of 60 kDa to 70 kD as identified by antibodies against the skeletal muscle forms of these subunits.¹⁹^,⁹¹^-⁹⁴ Both L-type and N-type Ca²⁺ channels from brain appear to lack a γ subunit, but a protein of approximately 100 kD is specifically associated with N-type Ca²⁺ channels as well as L-type Ca²⁺ channels from brain and may be an additional, brain-specific associated protein.¹⁹^,⁹¹^,⁹²^,⁹⁴ The α1 subunit of N-type Ca²⁺ channels was identified as Ca_v2.2 by homology cDNA cloning, co-immunoprecipitation, and functional expression.⁹⁵^,⁹⁶ As for the Cav1 family channels, analysis of the Cav2.2α1 subunit using sequence-specific antibodies revealed two size forms (240 kD and 210 kD) that differ in their C-termini and in their phosphorylation by specific protein kinases.⁹⁷^,⁹⁸ It is unknown at present whether these Cav2.2α1 subunits are derived from proteolytic truncation, alternatively spliced mRNAs or both (see Chapter 5).

Ca_v2.1 Channels

Agatoxin-sensitive P/Q-type Ca²⁺ channels purified from brain are also composed of α1, α2δ, and β subunits.⁹⁹^,¹⁰⁰^,¹⁰¹ In addition, recent experiments have unexpectedly revealed a novel γ subunit, which is the target of the stargazer mutation in mice.¹⁰² This γ-subunit-like protein can modulate the voltage dependence of expressed Ca²⁺ channels containing Cav2.1 subunits, so they may be associated with these Ca²⁺ channels in vivo.¹⁰² If this new γ subunit is indeed associated with neuronal Ca²⁺ channels, their subunit composition would be identical to that of skeletal muscle Ca²⁺ channels defined in biochemical experiments.¹⁴ The cDNA encoding the Ca_v2.1 subunit was isolated by homology cloning and identified by functional expression.¹⁰³^,¹⁰⁴ Analysis of the a1 subunit peptides present in brain has revealed a wide range of size forms, including C-terminal truncations and internal deletions, and these isoforms are differentially phosphorylated by protein kinases.¹⁰⁵,¹⁰⁶ These results continue the theme that all Ca²⁺ channels studied to date have multiple forms due to varying C-terminal domains and that they are differentially phosphorylated by protein kinases.

Interactions of Ca²⁺ Channels with Intracellular Regulatory Proteins

Ca²⁺ entering cells through voltage-gated Ca²⁺ channels initiates many intracellular processes through activation of effector mechanisms. Often these effector mechanisms are highly localized to respond to high local concentrations of Ca²⁺. In addition, local regulation of Ca²⁺ channel function is also modulated by specifically bound regulatory proteins. The interactions of these intracellular effector and regulatory proteins with Ca²⁺ channels have been studied by biochemical approaches as outlined here and by molecular biological methods as described in Chapters 8, 9 and 11. These studies show that a functional Ca²⁺ channel complex not only has four auxiliary subunits but also contains multiple associated regulatory proteins that modify its activity.

Ryanodine Receptors

In skeletal muscle fibers, excitation-contraction coupling involves direct conformational coupling of the voltage-gated Ca²⁺ channel in the transverse tubule membrane to the ryanodinesensitive Ca²⁺ release channel (RyR) of the sarcoplasmic reticulum at the transverse tubule/sarcoplasmic reticulum junction.6^,7^,¹⁰⁷ Conformational coupling is mediated by the intracellular loop connecting domains II and III of the α1 subunit.¹⁰⁸^,¹⁰⁹ Depolarization of the transverse tubules by the conducted action potential activates L-type voltage-gated Ca²⁺ channels. A complex of ryanodine receptor and Ca²⁺ channels can be identified by detergent extraction and resolution by sucrose gradient sedimentation and antibody labeling.¹¹⁰ This complex is required for excitation/contraction coupling and for feedback regulation of Ca²⁺ channel function.¹⁰⁹, ¹¹¹ and Chapter 21.

SNARE Proteins

Ca²⁺ entry through voltage-gated Ca²⁺ channels is thought to initiate exocytosis by triggering the fusion of secretory vesicle membranes with the plasma membrane through actions on the SNARE protein complex of syntaxin, SNAP-25, and VAMP/synaptobrevin (reviewed in refs. 112,113). The function of the SNARE protein complex is regulated by interactions with numerous proteins, including the synaptic vesicle Ca²⁺ -binding protein synaptotagmin. The SNARE proteins syntaxin and SNAP-25 are associated with N-type and P/Q-type Ca²⁺ channels and can be co-purified and co-immunoprecipitated from brain.⁹¹^,¹⁰⁰^,¹¹⁴,¹¹⁵ Presynaptic N-type and P/Q-type Ca²⁺ channels interact directly with the SNARE proteins through a specific synaptic protein interaction (synprint) site in the large intracellular loop connecting domains II and III.¹¹⁶^,¹¹⁷ and Chapter 8. This binding interaction is regulated by Ca²⁺ and protein phosphorylation.¹¹⁷^,¹¹⁸ Synaptotagmin also binds to the synprint site of Ca_V2 Ca²⁺ channels.¹¹⁹-¹²¹ Injection of peptide inhibitors of this SNARE protein interaction into pre-synaptic neurons inhibits synaptic transmission, consistent with the conclusion that this interaction is required to position docked synaptic vesicles near Ca²⁺ channels for effective fast exocytosis.¹¹⁶^,¹²²^,¹²³ SNARE protein interaction at this site is also important for G protein regulation of Ca²⁺ channels (see Chapter 9).

Kinase Anchoring Proteins

cAMP-dependent protein kinase is often anchored to specific subcellular compartments or specific kinase substrates by A Kinase Anchoring Proteins (AKAPs).¹²⁴^,¹²⁵ These proteins contain a targeting domain that directs the AKAP to a specific cellular site and a kinase anchoring domain containing an amphipathic alpha helix that binds the regulatory subunit dimer of cAMP-dependent protein kinase. Because regulation of the skeletal muscle Ca²⁺ channel by membrane depolarization and cAMP-dependent protein kinase is very rapid, with observable effects in 50 ms, it was an attractive candidate for regulation through PKA bound to AKAPs. Biochemical studies of skeletal muscle Ca²⁺ channels revealed a novel 15-kDa AKAP (AKAP- 15) associated with purified Ca²⁺ channels and with specifically immunoprecipitated Ca²⁺ channels.¹²⁶ This AKAP is an 81-residue protein with N-terminal palmitoyl and myristoyl moieties that serve as membrane anchors and an amphipathic helix that binds PKA ¹²⁷ (also designated AKAP-18).¹²⁸ AKAP-15, PKA, and Ca²⁺ channels are co-localized in the specialized junctions formed between sarcoplasmic reticulum and transverse tubule membranes, where excitation-contraction coupling takes place.¹²⁷^,¹²⁹ AKAP-15 binds to the skeletal muscle Ca²⁺ channel through a specific modified leucine zipper motif in the distal C-terminal domain.¹³⁰ In this way, the bound AKAP directly targets PKA to its sites of phosphorylation in the C-terminal of the full-length α1 subunit.

Ca²⁺ Binding Proteins

Ca²⁺ entering cells through Ca²⁺ channels is involved in initiation of intracellular signaling processes, and these often involve Ca²⁺ binding proteins like calmodulin, which is expressed ubiquitously, and neuro-specific Ca²⁺ binding proteins, which are expressed in specific neurons and in distinct subcellular locations. Research reviewed in Chapter 11 shows that both calmodulin and neuro-specific Ca²⁺ binding proteins interact with Ca²⁺ channels at specific binding sites in the C-terminal domains and modulate their function.

Conclusion

Voltage-gated Ca²⁺ channels are complex proteins containing five distinct protein subunits, α, α, β, γ, and δ, that specifically associate with each other. In addition to subunit assembly, their biosynthesis involves extensive glycosylation as well as proteolytic processing and disulfide linkage of subunits. Each α1 subunit that has been studied is present in multiple isoforms that differ in their C-terminal domains and in phosphorylation by specific kinases. Ca²⁺ channels provide intracellular Ca²⁺ to initiate local signaling events, and they are directly associated with both effector proteins that initiate Ca²⁺ -dependent processes and with regulatory proteins that control their activity. Ca²⁺ channel signaling complexes are a crucial element of local regulation of cellular events.

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Catterall WA. Biochemical Studies of Voltage-Gated Ca2+ Channels. In: Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013.

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Introduction
Purification and Biochemical Characterization of Skeletal Muscle Ca²⁺ Channels
Biochemical Properties of Other Ca_v1 Channels
Biochemical Properties of the Ca_v2 Family of Ca²⁺ Channels
Interactions of Ca²⁺ Channels with Intracellular Regulatory Proteins
Conclusion
References

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Madame Curie Bioscience Database [Internet].

Biochemical Studies of Voltage-Gated Ca²⁺ Channels

Authors

Affiliations

Introduction

Purification and Biochemical Characterization of Skeletal Muscle Ca²⁺ Channels

α1 Subunit

Figure 2

α2 Subunit

Figure 1

β Subunit

γ Subunit

δ Subunit

Membrane Association of Subunits

Sites of Protein Phosphorylation

Phosphorylation of the β Subunit of Skeletal Muscle Ca²⁺ Channels

Sites of Drug Binding

Biochemical Properties of Other Ca_v1 Channels

Subunits of Purified Cardiac Ca²⁺ Channels

Molecular Basis for Modulation of Cardiac Ca²⁺ Channels by PKA

Phosphorylation of the β Subunit of Cardiac Ca²⁺ Channels by PKA

Subunits of Purified Neuronal Ca_v1 Channels

Biochemical Properties of the Ca_v2 Family of Ca²⁺ Channels

Ca_v2.2 Channels

Ca_v2.1 Channels

Interactions of Ca²⁺ Channels with Intracellular Regulatory Proteins

Ryanodine Receptors

SNARE Proteins

Kinase Anchoring Proteins

Ca²⁺ Binding Proteins

Conclusion

References

Views

In this Page

Related information

Recent Activity

Bookshelf

Madame Curie Bioscience Database [Internet].

Biochemical Studies of Voltage-Gated Ca2+ Channels

Authors

Affiliations

Introduction

Purification and Biochemical Characterization of Skeletal Muscle Ca2+ Channels

α1 Subunit

Figure 2

α2 Subunit

Figure 1

β Subunit

γ Subunit

δ Subunit

Membrane Association of Subunits

Sites of Protein Phosphorylation

Phosphorylation of the β Subunit of Skeletal Muscle Ca2+ Channels

Sites of Drug Binding

Biochemical Properties of Other Cav1 Channels

Subunits of Purified Cardiac Ca2+ Channels

Molecular Basis for Modulation of Cardiac Ca2+ Channels by PKA

Phosphorylation of the β Subunit of Cardiac Ca2+ Channels by PKA

Subunits of Purified Neuronal Cav1 Channels

Biochemical Properties of the Cav2 Family of Ca2+ Channels

Cav2.2 Channels

Cav2.1 Channels

Interactions of Ca2+ Channels with Intracellular Regulatory Proteins

Ryanodine Receptors

SNARE Proteins

Kinase Anchoring Proteins

Ca2+ Binding Proteins

Conclusion

References

Views

In this Page

Related information

Recent Activity

Biochemical Studies of Voltage-Gated Ca²⁺ Channels

Purification and Biochemical Characterization of Skeletal Muscle Ca²⁺ Channels

Phosphorylation of the β Subunit of Skeletal Muscle Ca²⁺ Channels

Biochemical Properties of Other Ca_v1 Channels

Subunits of Purified Cardiac Ca²⁺ Channels

Molecular Basis for Modulation of Cardiac Ca²⁺ Channels by PKA

Phosphorylation of the β Subunit of Cardiac Ca²⁺ Channels by PKA

Subunits of Purified Neuronal Ca_v1 Channels

Biochemical Properties of the Ca_v2 Family of Ca²⁺ Channels

Ca_v2.2 Channels

Ca_v2.1 Channels

Interactions of Ca²⁺ Channels with Intracellular Regulatory Proteins

Ca²⁺ Binding Proteins