Molecular Composition

LTCCs are multimeric proteins consisting of an α₁-subunit that constitutes the pore of the channel and several accessory subunits denoted β, α₂-δ, and γ2,5,6 (Figure 2-1). Currently, four α₁-subunits for L-type Ca²⁺ channels are known, and two of these, Ca_V1.2 (α1C encoded by the CACNA1C gene) and Ca_V1.3 (α1D encoded by the CACNA1D gene), are expressed in the heart^3,5,7 (see Table 2-1). These α₁-subunits form the channel pore and contain the voltage sensor that controls channel gating, as well as drug binding sites and regulatory sites targeted by second messengers. Ca²⁺ channel α₁-subunits have a similar structure to voltage-gated Na⁺ and K⁺ channels, whereby they are organized into four domains (I through IV), each containing six transmembrane segments (S1 through S6). The voltage sensor is located in the S4 transmembrane segment of each domain, and the pore loops are located between S5 and S6. Alternative splice variants of Ca_V1.2 have been documented and can impact regulation by second messengers and drug binding. Recently, alternative splice variants of Ca_V1.3 have also been described.^8,9 These variants result in the expression of long and short forms of Ca_V1.3, which are characterized by differences in biophysical properties such as the activation midpoint; however, not all of the short forms of Ca_V1.3 are expressed in the heart.⁸

Ca²⁺ channel β-subunits (Ca_Vβ₁ through Ca_Vβ₄) are encoded by four genes (CACNB1-4) and, like the α₁-subunits, can be alternatively spliced to generate a number of isoforms.10,11 Crystal structures of LTCC β-subunits demonstrate that these subunits contain conserved Src homology 3 (SH3) and guanylate kinase domains, as seen in scaffolding proteins in the membrane-associated guanylate kinase (MAGUK) family.^12,13 Ca_Vβ-subunits are intracellular proteins (see Figure 2-1) that bind to a single site, called the α interaction domain (AID), on the loop connecting domains I and II on the α₁-subunit of the LTCC. Ca_Vβ acts as a chaperone protein that facilitates the trafficking of LTCCs to the plasma membrane and also modulates the biophysical properties of these channels. Specifically, coexpression of Ca_Vβ2 with Ca_V1.2 increases I_Ca,L amplitude, accelerates activation and inactivation kinetics, and shifts the steady inactivation curve to more negative membrane potentials. Ca_Vβ also profoundly enhances the affinity of dihydropyridines (blockers of LTCCs) for the α₁-subunit by decreasing their dissociation from the channel.¹⁴

Four genes (CACNA2D1-4), which can be alternatively spliced, encode the α₂-δ-subunit of the LTCC.15,16 The α₂-δ₁ and α₂-δ₃ isoforms are expressed in the heart. The protein is cleaved post-translationally and then is relinked via disulfide interactions. This subunit is primarily extracellular, with the δ-subunit portion anchored in the plasma membrane (see Figure 2-1). CaVα₂-δ-subunits facilitate the targeting of LTCCs to the plasma membrane and increase I_Ca,L by shifting the voltage dependence of activation and inactivation to hyperpolarized membrane potentials.

Ca²⁺ channel γ-subunits are membrane-bound proteins encoded by eight genes (CACNG1-8) with several isoforms, including γ₄, γ₆, γ₇, and γ₈, which are present in cardiac muscle. These isoforms associate with Ca_V1.2 and alter activation and inactivation properties of the channel.¹⁷

Biophysical Properties of I_Ca,L

As their name suggests, LTCCs are highly selective for Ca²⁺ over monovalent cations. The channel pore is approximately 6 Å in diameter at its narrowest point,¹⁸ indicating that size is not the main factor in determining selectivity. Rather, a series of four glutamate residues (EEEE motif) is responsible for conferring ion selectivity through the ability of this motif to bind divalent cations (Ca²⁺ and Mg²⁺), which block the passage of monovalent cations.^19,20 As additional divalent ions enter the channel, bound Ca²⁺ ions are displaced from the EEEE motif and are pushed through the pore, which generates I_Ca,L.

At the single-channel level, I_Ca,L conductance in ventricular myocytes (i.e., Ca_V1.2 dependent) has been reported to be approximately 5 pS in 2 mM Ca²⁺ and approximately 15 pS with Ba²⁺ as the charge carrier.²¹ Similarly, single-channel conductance for Ca_V1.3 has been determined in heterologous expression systems and has been found to be approximately 15 pS with Ba²⁺ as the charge carrier.⁸

I_Ca,L is mediated by Ca_V1.2 and Ca_V1.3 in the heart, and these two channel isoforms are distinguished by their unique biophysical properties7,22 (Figure 2-2). Ca_V1.2-mediated I_Ca,L, which is expressed throughout the myocardium (atrium, ventricles, and conduction system), has a bell-shaped current-voltage (I-V) relationship in which the current activates at membrane potentials positive to −40 mV and peaks between 0 and +10 mV. The V_1/2 of channel activation (V_1/2(act) ) is typically between −10 and −15 mV. In contrast, recombinant Ca_V1.3–dependent I_Ca,L activates at more negative membrane potentials (i.e., the V_1/2(act) is hyperpolarized) and displays slower inactivation kinetics.^8,23,24 Ca_V1.3 is also less sensitive to dihydropyridines than is Ca_V1.2.

Ca_V1.2 is the primary determinant of I_Ca,L in the ventricular myocardium, where it plays a prominent role in excitation-contraction coupling and the Ca²⁺ induced–Ca²⁺ release process.¹ Approximately 75% of these LTCCs are located in dyads, in close proximity to the ryanodine receptors located in the junctional sarcoplasmic reticulum, within the t-tubular system (see Figure 2-4). Consistent with the critical role of Ca_V1.2 in the ventricles, Ca_V1.2 knockout mice die before birth in association with cardiac failure.²⁵ Ca_V1.2 is also expressed in the SAN, AVN, and atrial myocardium, along with Ca_V1.3.^22,26 As a result, total I_Ca,L in these supraventricular tissues is dependent on both α1C and α1D pore-forming subunits and demonstrates biophysical characteristics that are different from those of the ventricles. The generation of Ca_V1.3 knockout mice has been important in determining the biophysical properties of these channels and in distinguishing them from Ca_V1.2-mediated I_Ca,L (and I_Ca,T).^23,27 Specifically, these Ca_V1.3 knockouts have been used to show that I_Ca,L in SAN and atrial myocytes activates between −60 and −50 mV in association with a left shift in the V_1/2(act) and peaks at membrane potentials around −10 mV. The steady state I_Ca,L inactivation curve is also shifted to the left when Ca_V1.3 contributes to total I_Ca,L. Together, available data show that Ca_V1.3-dependent I_Ca,L has intermediate biophysical properties compared with Ca_V1.2-dependent I_Ca,L and I_Ca,T.

The unique biophysical properties of Ca_V1.3 enable I_Ca,L to play a prominent role in pacemaker activity in the SAN and in the electrical conduction in the atrial myocardium. In the SAN, Ca_V1.3-mediated I_Ca,L contributes to the diastolic depolarization phase of the action potential and thus is a major determinant of heart rate in vivo. Ca_V1.2, on the other hand, contributes more prominently to the action potential upstroke in SAN myocytes. Consistent with this, Ca_V1.3 knockout mice display sinus bradycardia in association with a reduced diastolic depolarization slope.26–28 Ca_V1.3 knockout mice also show increased susceptibility to atrial fibrillation,^24,29 a very common cardiac arrhythmia, confirming that these Ca²⁺ channels play an important role in atrial conduction.

I_Ca,L Inactivation

I_Ca,L undergoes decay during sustained depolarization, which is termed inactivation (Figure 2-3, A). This inactivation process is time, voltage, and calcium dependent.^30–32 Voltage-dependent activation is relatively slow, as is indicated by the degree of inactivation with Ba²⁺ as the charge carrier, or when monovalent cations permeate LTCCs in the absence of divalent cations.³³ In the presence of extracellular Ca²⁺, inactivation is much faster, and this Ca²⁺-dependent inactivation is further accelerated in the presence of functional SR Ca²⁺ release (i.e., during normal excitation-contraction coupling).³⁴ This indicates that both the Ca²⁺ entering the myocytes through the LTCC and the Ca²⁺ released from the SR during the Ca²⁺ transient contribute to LTCC inactivation.

Ca²⁺-dependent inactivation of I_Ca,L is a calmodulin (CaM) dependent process.35,36 CaM is bound to the C-terminus of the channel via the IQ domain, and when local Ca²⁺ is elevated, an increase in Ca²⁺ binding to CaM occurs. This enhances the interaction of CaM with the IQ domain, thereby causing inactivation of the channel. Ca²⁺-dependent inactivation may serve as a protective negative feedback mechanism to prevent Ca²⁺ overload in cardiomyocytes.

Steady state inactivation (i.e., channel availability), like voltage-dependent activation, follows a sigmoidal relationship, with a V_1/2(inact) of ≈−35 mV for Ca_V1.2-dependent I_Ca,L and −45 mV for Ca_V1.3-dependent I_Ca,L.²² The activation and inactivation curves for I_Ca,L can overlap, resulting in a “window current,” which has been postulated to play a role in the generation of arrhythmogenic early afterdepolarizations (EADs; see Figure 2-3, B).^37,38

LTCCs are also known to undergo a process called facilitation, whereby a progressive increase in I_Ca,L amplitude and the time constant of inactivation can occur during increases in pacing frequency.34,39 The facilitation process occurs as the result of a reduction in Ca²⁺-dependent inactivation and is mediated by calmodulin-dependent kinase II (CaMKII) phosphorylation.⁴⁰

Organization and Localization of LTCCs

It is now appreciated that LTCC can be organized into distinct subcellular compartments through the actions of scaffolding proteins, including A-kinase anchoring proteins (AKAPs),41,42 within cardiomyocytes⁴³ (Figure 2-4). Examples of subpopulations of LTCCs include those found in dyads within the T-tubules and those in separate plasma membrane domains such as caveolae and lipid rafts.² This pattern of organization is critical for the precise spatio-temporal modulation and regulation of LTCCs in different physiological functions such as excitation-contraction coupling, transcriptional regulation, and responses to β-adrenergic (β-AR) receptor activation.

Within dyads, LTCCs from a complex with β-ARs, adenylyl cyclase (AC), protein kinase A (PKA), and AKAPs.2,44,45 These proteins area also in close apposition to a complex of proteins associated with the SR, including ryanodine receptors, PKA, protein phosphatase 2A (PP2A), phosphodiesterases, and AKAPs (AKAP5 and AKAP15).^41,42 It is the AKAPs that are thought to be responsible for the organization of these signaling complexes. Within caveolae, LTCCs associate with β₂-ARs, AC, PKA, PP2A, and caveolin 3, all of which enable LTCCs in caveolae to be locally stimulated by β₂-AR activation.⁴⁶

LTCC also demonstrate a property called coupled gating, which occurs when LTCCs are grouped together into clusters in the plasma membrane, resulting in localized regions with significantly elevated intracellular Ca²⁺ concentrations. Coupled gating is enhanced by the scaffolding protein AKAP5 and by protein kinase C (PKC), which form the signaling complex with the clusters of LTCCs.43,47

Molecular Composition and Biophysical Properties

Although L-type Ca²⁺ channels get the lion’s share of the attention in both cardiac and vascular electrophysiology, low voltage-activated T-type Ca²⁺ channels also play important roles in normal and diseased hearts. Three T-type Ca²⁺ channels exist in mammals: Cav3.1 (α1G), Cav3.2 (α1H), and Cav3.3 (α1I), with Cav3.1 and Cav3.2 channels being the major channels in heart48,49 (see Table 2-1). Although L-type Ca²⁺ channels are complex oligomeric proteins that are heavily regulated by many distinct signal transduction mechanisms, much less is known about the auxiliary subunit structure for T-type Ca²⁺ channels.⁵⁰ The electrophysiological properties of T-type α1-subunits expressed alone are very similar to those observed in native channels, suggesting that T-type Ca²⁺ channels do not require auxiliary subunits, as do L-type Ca²⁺ channels, for proper function. Consistent with this, antisense depletion of Ca_Vβ-subunits has no effect on I_Ca,T (in neurons),^51,52 and coexpression of cloned Ca_Vβ-subunits has no major effect on I_Ca,T in heterologous expression systems.⁵³ Ca_Vγ-subunits have been found to have little or no effect on Ca_V3.3 currents⁵⁴; however, they have been found to accelerate inactivation and negatively shift steady state inactivation of the Ca_V3.1 current.⁵⁵ Coexpression of α₂δ-subunits with α₁G doubled the size of I_Ca,T, possibly through increased expression of α₁-α₂δ complexes at the plasma membrane.^53,56 T-type Ca²⁺ channels have been shown to interact with, and be modulated by, other proteins such as Kelch-like 1 protein⁵⁷ and caveolin-3.⁵⁸ In summary, available data indicate that Ca_V3 α₁-subunits can interact with some auxiliary subunits, but additional studies are needed to determine the physiological role for these interactions, particularly in the heart.

The biophysical properties of T-type Ca²⁺ channels in heart are distinct from those of L-type Ca²⁺ channels.⁵⁹ For example, T-type Ca²⁺ channels activate and inactivate at lower (more negative) membrane potentials with thresholds for activation observed at about −70 mV while inactivation begins to occur at approximately −90 mV⁵⁰ (see Figure 2-2). T-type Ca²⁺ channels also show much faster entry and exit from inactivation^50,60 that is independent of Ca²⁺. An analysis of the steady state activation and inactivation curves reveals that T-type Ca²⁺ channels have relatively large steady state window currents at membrane voltages between −80 and −40 mV,⁶¹ which are expected to facilitate depolarization in myocytes of the SAN and the conducting system, thereby promoting spontaneous action potential firing. Although the permeation properties of T-type Ca²⁺ channels have been challenging to quantify, the single-channel conductance is reported to be 2.5-fold smaller than that of L-type Ca²⁺ channels (when Ba²⁺ is the charge carrier).⁶²

T-type Ca²⁺ channels have distinct pharmacologic properties compared with L-type Ca²⁺ channels, and these differences have been exploited for dissecting the physiologic role of I_Ca,T in the myocardium. Relative to L-type channels, T-type channels have a very high sensitivity to block by Ni²⁺

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