Voltage-Gated Calcium Channels

Introduction

In the heart, the influx of Ca ²⁺ , acting as a multidimensional signaling molecule, is essential for the activation of excitation-contraction coupling (E-C coupling) and also contributes to the plateau phase of the cardiac action potential, pacemaker activity in nodal cells, and the modulation of cellular processes (gene expression). The major pathway for entry of Ca ²⁺ into excitable cells is through voltage-gated Ca ²⁺ channels. Modulating Ca ²⁺ influx through these channels is an important regulator of cardiac contractility during β-adrenergic activation. Thus voltage-gated Ca ²⁺ channels are empowered to transduce membrane depolarization into cellular functions and physiologic stress into increased cardiac contractility. Dysfunctions of Ca ²⁺ channels have been associated with cardiac diseases.

Electrophysiologic and pharmacologic studies have defined six classes of voltage-gated Ca ²⁺ channels: T-, L-, N-, P-, Q-, and R-type. These channel classes can be grouped by low membrane voltage activation (LVA) versus high membrane voltage activation (HVA), susceptibility to pharmacologic antagonists, and rate of inactivation ( Table 2.1 ). Of these six classes of Ca ²⁺ channels, only long-lasting (L)-type and transient (T)-type Ca ²⁺ channels are expressed in cardiomyocytes. Additionally, several non–voltage-gated Ca ²⁺ permeable transient receptor potential (TRP) channels are expressed in the heart. ^, This chapter reviews the roles of these ion channels in the function of the heart in health and disease.

Molecular Composition of Voltage-Gated Ca ²⁺ Channels

Voltage-gated Ca ²⁺ channels are multimers comprised of a core α ₁ pore-forming subunit and several auxiliary subunits, including β, α _2δ , and γ ( Fig. 2.1A ). The α ₁ -subunit contains binding sites for most regulators and drugs, whereas the β, α _2δ , and γ contribute to trafficking, anchorage, and regulatory functions. To date, 10 α ₁ -subunit isoforms exhibiting diverse electrophysiologic properties and response to drugs have been identified. In Ca ²⁺ channel nomenclature, the chemical symbol of calcium, Ca, is followed by a subscript “V,” which denotes voltage as the primary regulator, and two numerical identifiers, which correspond to the α ₁ -subunit gene subfamily and the order of discovery within that subfamily, respectively. ^, Sequence alignment suggests that gene duplication and divergence of an ancestral Ca ²⁺ channel gene gave rise to the LVA and HVA subfamilies (see Fig. 2.1B ). Ca _V 1 and Ca _V 2 subfamilies arose more than 500 million years ago by duplication of the HVA gene. Four Ca _V 1 genes, three Ca _V 2 genes, and three Ca _V 3 genes arose within these subfamilies. The amino acid sequences of these α ₁ -subunits are more than 70% identical within a family but less than 40% identical among families.

Ca _V 1.1, Ca _V 1.2, Ca _V 1.3, and Ca _V 1.4 exhibit relatively long-lasting currents and are referred to as L-type Ca ²⁺ channels. Relative to L-type Ca ²⁺ channels, Ca _V 3.1, Ca _V 3.2, and Ca _V 3.3 exhibit transient Ca ²⁺ currents and are activated at more negative potentials (see Table 2.1 ). These channels, known as T-type Ca ²⁺ channels, contribute to automaticity and are predominantly expressed in the sinoatrial node, atrioventricular node, and Purkinje fiber network. Ca _V 2.1, Ca _V 2.2, and Ca _V 2.3, also known as P/Q-type, N-type, and R-type Ca ²⁺ channels, respectively, are predominantly expressed in nerve terminals, dendrites, and neuronal cell bodies and are characterized by their response to specific pharmacologic antagonists, including snake and spider toxins. ^,

The α ₁ -subunit, which contains approximately 2000 amino acid residues, is organized into four repeated domains (I to IV), each of which contains six transmembrane segments (S1 to S6) with a pore between S5 and S6 (see Fig. 2.1C ), similar to the Na ⁺ channel α-subunit. The alternating positively charged arginine or lysine residues at every third or fourth position in S4 of each domain regulate voltage sensitivity. Four negatively charged glutamate residues on the pore loop located between S5 and S6 are responsible for the Ca ²⁺ selectivity of the channels. Remarkably, the selectivity for Ca ²⁺ can be changed to Na ⁺ by the mutation of only three amino acids in the pore loops of domains I, III, and IV. The pore region also contains overlapping binding sites for all major L-type Ca ²⁺ channel blocking agents, including dihydropyridines, phenylalkylamines, and benzothiazepines.

The 3.6 angstrom resolution cryogenic electron microscopy (cryo-EM) structure of Ca _V 1.1 (see Fig. 2.1A ) has provided important insights into channel function. In the resting-closed state, the pore is occluded toward the intracellular side by S6 and locked in this position by downward positioning of the voltage sensor segments. Membrane depolarization causes upward movement of the voltage sensors, allowing the S6 segments to disengage from the pore. The cryo-EM structure has also been used to elucidate the mechanism of action for dihydropyridine (DHP) and non-DHP calcium channel blockers (CCB). ^,

The α _1C C-terminus contains several protein-protein interaction motifs (see Fig. 2.1C ), and their roles in regulating Ca _V 1.2 trafficking and function in cardiomyocytes will be reviewed. The human α _1C gene contains 50 exons and at least 20 exons that undergo alternative splicing. This contributes to both gene regulation and protein diversity. For instance, Ca _V 1.2b, the smooth muscle splice variant, is more sensitive to inhibition by dihydropyridines than Ca _V 1.2a, the heart variant, because it contains the dihydropyridine-sensitive exon 8B. Ca _V 1.2 containing exon 33L is a nonfunctional splice variant present during embryogenesis, which exhibits a dominant-negative suppression of wild-type Ca _V 1.2 channel via acceleration of ubiquitin-proteasome degradation.

In the heart, α _1C exists as a 240- or 210-kDa protein depending on the extent of C-terminus truncation by posttranslational proteolytic processing. In cardiomyocytes, proteolytic cleavage of α _1C C-terminus occurs in more than 80% of cardiac channels. The cleaved fragment is believed to noncovalently reassemble with the truncated α _1C and regulate Ca _V 1.2 channel function by acting as an inhibitor of channel function. Proteolytic processing has been proposed to occur via an unidentified protease at α _1C Ala ¹⁸⁰⁰ (see Fig. 2.1C ), but this is not recapitulated in heterologous expression systems. Channels with a deletion of the proteolytic cleavage site expressed in mice traffic to the T-tubules, function appropriately, and exhibit normal regulation by β-adrenergic agonists. In heterologous expression studies, truncation of α _1C C-terminus at residue 1821 exhibited a 2.9-fold increase in current at +10 mV compared with full length, ^{, ,} and truncation at residue 1905 moderately reduced Ca _V 1.2 surface expression by around 30% but significantly enhanced single-channel open probability ( P _o ). ^, By contrast, knock-in mice expressing α _1C truncated either at Gly ¹⁷⁹⁶ or Asp ¹⁹⁰⁴ displayed a dramatic reduction in Ca _V 1.2 surface expression and current in cardiomyocytes and displayed cardiac failure and perinatal death. ^, Surprisingly, Ca _V 1.2 expression and function in vascular smooth muscle was relatively unaffected in the knock-in mice, suggesting a cardiomyocyte-specific role for the α _1C distal C-terminus in trafficking and regulation of Ca _V 1.2.

The amino acid sequences of the Ca _V β-subunits, which are encoded by four β-subunit genes (Ca _V β 1 to 4), differ considerably. Among the β-subunits, the guanylate kinase (GK) domain, and Src-homology 3 (SH3) domain are very similar, whereas the N-termini (variable region 1), the linker between SH3 and GK (variable region 2), and the C-termini (variable region 3) are different. All β-subunits, however, increase α _1C trafficking to the plasma membrane, regulate the voltage dependence of activation, and impart unique kinetics of inactivation. β ₂ is the predominantly expressed isoform in the adult murine heart, with β ₁ , β ₃ , and β ₄ not detectable by immunoblot. ^, By contrast, all four βs are detected in canine myocytes, suggesting species dependence in the complement of β isoforms expressed in cardiomyocytes. Ca _V βs primarily interact with an 18-residue sequence in the I-II intracellular linker, termed the α-interacting domain (AID), conserved among all high-voltage activated Ca ²⁺ channel α ₁ -subunits (see Fig. 2.1C ). Crystal structures show that the AID forms an α-helix that binds to the hydrophobic groove present in the Ca _V β-subunit GK domain through 10 AID side chain interactions. Alanine-substitution of the three residues within Ca _V 1.2 AID (Y437, W440, I441) ablates the β binding. In heterologous expression experiments, β binding to α _1C is obligatory for Ca _V 1.2 to traffic to the cell surface; deletion of the AID domain prevents α _1C surface expression, and α _1C expressed without β fails to traffic to the cell surface. In the heart, however, Ca _V 1.2 does not require Ca _V β to traffic to the cell membrane. Cardiomyocyte-specific, conditional expression of dihydropyridine-resistant α _1C -subunits lacking critical residues in the AID site traffic to the membrane and can support excitation-contraction coupling. This is consistent with the observation that cardiomyocyte-specific, conditional deletion of the Cacnb2 gene in adult mice causes an approximately 96% reduction in Ca _V β ₂ protein expression in cardiomyocytes but only a 29% reduction in Ca ²⁺ current and no obvious cardiac impairment. Ca _V β plays an important role in cardiac development as global or cardiac-specific deletion of the Cacnb2 gene leads to abnormal heart development and embryonic death and likely increases open probability of the channel. β1-, β3-, and β4-deficient mice do not demonstrate significant cardiac phenotypes.

The α _2δ subunit is a 175-kDa single transmembrane protein encoded by four genes ( Cacna2d1, Cacna2d2, Cacna2d3, Cacna2d4 ) with multiple splice variants. Although mRNAs of α _2δ 1 to 3 have been identified in human myocardium, only α _2δ 1 is known to bind Ca _V 1.2. The δ-subunit is encoded by the 3′ end of the coding sequence of the same gene as the α2-subunit and is produced by posttranslational proteolytic processing. The α2-subunit has numerous glycosylation sites, is extracellular, and is attached to the membrane through a disulfide linkage to the δ-subunit (see Fig. 2.1C ). It also contains a von Willebrand factor type A (VWA) domain that participates in interactions with Ca _V 1.2. α _2δ associates with multiple regions of the carboxyl-terminal half of α ₁ , including the S5-S6 linker in the third repeat domain. The δ component is required for this interaction. The α ₁ -subunit is sufficient to produce functional channels, albeit with low expression level and abnormal kinetics/voltage dependence. Coexpression of the α _2δ -subunit and especially the β-subunit enhances expression and produces normal gating properties. BK channels may also interact with α _2δ 1, sequestering them from Ca _V 1.2 and reducing Ca _V function in neurons. Mutations in α _2δ 1 are associated with short QT syndrome and Brugada syndrome.

Eight γ-subunit isoforms, encoded by the CACNG1-8 genes , have been identified, although only the γ4, γ6, γ7, and γ8 cDNA are expressed in the human heart. γ-Subunits are composed of four transmembrane domains with intracellular N- and C-terminal ends (see Fig. 2.1C ). Studies on the γ1 isoform in skeletal muscle have localized the interaction site with α ₁ to the first half of the γ-subunit, whereas studies on γ6, which has an inhibitory effect on Ca _V 3.1 current in rat atrial myocytes, have localized it to the first transmembrane domain (TM1). The γ-subunits have variable effects on Ca _V 1.2 function. γ4, γ6, and γ7 cause a leftward shift in the V ₅₀ for channel activation, whereas γ8 inhibits the effects of the α _2δ 1-subunit on channel function. Furthermore, these effects are different depending on what β-subunit isoform is coexpressed (β _1b vs β _2b ).

L-Type Ca ²⁺ Channels

Role of L-Type Ca ²⁺ Channels in the Heart

In early development, Ca _V 1.3 is the predominant L-type Ca ²⁺ channel in the heart, where it contributes to automaticity. Later, during embryogenesis, the expression pattern changes and Ca _V 1.2 becomes the dominant L-type Ca ²⁺ channel in ventricular myocytes. Postnatally, the expression of Ca _V 1.3 is limited to the sinoatrial node and atrioventricular node. Ca _V 1.2 is situated on the T-tubules in close proximity to ryanodine receptors (RyR2), which are intracellular Ca ²⁺ release channels located on the sarcoplasmic reticulum (SR; Fig. 2.2 ). In a process known as Ca ²⁺ -induced Ca ²⁺ release, Ca ²⁺ entry through the L-type Ca ²⁺ channels triggers RyR2 to release Ca ²⁺ from the SR into the cytoplasm where Ca ²⁺ then binds troponin C, enabling actin-myosin cross-linking and cellular contraction. L-type Ca ²⁺ channels are also functional within caveolae, where Ca ²⁺ influx can control signal transduction pathways (see Fig. 2.2 ). In healthy hearts, bridging integrator 1 (BIN1) is a membrane scaffolding protein that causes Ca _V 1.2 to traffic to T-tubules. In failing hearts the expression of BIN1 is decreased, and this reduction impairs Ca _V 1.2 trafficking, Ca ²⁺ transients, and contractility.

The critical contribution of Ca _V 1.2 to myocardial function is highlighted by the observation that Ca _V 1.2 knock-out mice are embryonically lethal by 14 days. Survival in early development is likely because of the presence of Ca _V 1.3. Mice lacking Ca _V 1.3 exhibit reduced rates of sinoatrial node diastolic depolarization and bradycardia as well as atrioventricular node dysfunction. Colocalization of Ca _V 1.3 and RyR2 in the sinoatrial node likely contributes to the large increase in intracellular Ca ²⁺ levels that drives diastolic depolarization.

Increased Ca ²⁺ influx via L-type Ca ²⁺ channels can lead to arrhythmias. When the action potential duration is prolonged, recovery of L-type Ca ²⁺ channel inactivation occurs during the plateau phase, which can cause early afterdepolarizations (EADs). An increased L-type Ca ²⁺ current also contributes to delayed afterdepolarizations (DADs) and Ca ²⁺ -evoked arrhythmias. Mutations in L-type Ca ²⁺ channels have been associated with inherited arrhythmia syndromes, including Timothy syndrome, which is a multisystem disorder characterized by invariant prolonged QT interval and syndactyly, and variably penetrant phenotypes of autism spectrum disorders, craniofacial abnormalities, and hypoglycemia. The biophysical mechanism underlying the cardiac manifestations of Timothy syndrome is the prominent loss of VDI, leading to the failure of L-type Ca ²⁺ channels to close during the plateau phase of the ventricular action potential. Loss-of-function mutants of the pore-forming α _1C , β _2b , and α _2δ1 -subunits have also been linked to Brugada, early repolarization, and short QT syndromes ( Table 2.2 ).

TABLE 2.2

Summary of Variants in Ca _V 1.2 Associated With Arrhythmogenic Disorders

Dx	Localization	Variants
CACNA1C
LQT 8	N-terminus	A28T
	DI-S6	P381S
	Ml loop	M456I
	DII-S2/S3	A582D
	II-III loop	L762F, P857R, R858H, R860G, K834E, P857L
	DIII-S6	11166V, I1166T
	DIV-S6	I1475M
	C-terminus	E1496K, G1783C, R1906Q
BrS3	N-terminus	A39V
	I-II loop	G490R
	DII-S5/S6	R632R
	DIII-S5/S6	E1115K
	C-terminus	E1829_Q1833dup, V2014I, R1880Q, C1873Y, D2130N
TS, SQT	DI-S6, HI loop, C- terminus, DIV-S6, C- terminus	G402S, G406R, R518C/H, R1937P, A1473G, R1977Q
ERS 2	II-III loop	E850del
CACNB2b
BrS4	N-terminus	T11l
	C-terminus	S481L, D601E, T450I, D538E, R571C
	HOOK region	S143F, S160T, K170N
	GK-like domain	L399F
IVF	SH3 domain	A73V
CACNA2D1
SQT6	Extracellular	S755T
ERS4	Extracellular	S956T
BrS9	Cache domain	D550Y
	Extracellular	S709N, Q917H

BrS , Brugada syndrome; ERS , early repolarization syndrome; GK, guanylate kinase; IVF , idiopathic ventricular fibrillation; LQT, long QT syndrome; SH3, Src-homology 3; SQT, short QT syndrome; TS , Timothy syndrome.

Dysfunctional Ca ²⁺ regulation has also been implicated in the mechanisms of cardiac hypertrophy. Transgenic cardiac overexpression of either the β _2a -subunit or the α _1C -subunit is sufficient to activate pathologic hypertrophy signaling. After a myocardial infarction, transgenic overexpression of β _2a caused greater ventricular dilation, myocyte hypertrophy and death, and depressed cardiac function. Initial in vitro studies suggested that the hypertrophic signaling was blocked by a caveolae-targeted L-type Ca ²⁺ channel antagonist, implicating Ca ²⁺ influx via L-type Ca ²⁺ channels within caveolae as a required source for pathologic cardiac hypertrophy. Follow-up in vivo studies, however, demonstrated that L-type Ca ²⁺ channels in the caveolae microdomain do not affect cardiac function and are not necessary for the regulation of hypertrophic signaling in the adult mouse heart. In Ca _V 1.2 heterozygous null mice, modest reduction in L-type Ca ²⁺ current caused greater ventricular dilation, cardiac hypertrophy, and reductions in ventricular performance after pressure overload stimulation, isoproterenol infusion, and swimming. The reduction in L-type Ca ²⁺ current led to activation of the neurohormonal stress pathways and activation of calcineurin/nuclear factor of activated T-cells (NFAT) signaling. Thus both increased and decreased L-type Ca ²⁺ currents are implicated in the development of pathologic remodeling with stress stimuli in some animal models.