Characterization of CaV1.2 exon 33 heterozygous knockout mice and negative correlation between Rbfox1 and CaV1.2 exon 33 expressions in human heart failure

Recently, we reported that homozygous deletion of alternative exon 33 of Ca_V1.2 calcium channel in the mouse resulted in ventricular arrhythmias arising from increased Ca_V1.2_Δ33 I_CaL current density in the cardiomyocytes. We wondered whether heterozygous deletion of exon 33 might produce cardiac phenotype in a dose-dependent manner, and whether the expression levels of RNA splicing factors known to regulate alternative splicing of exon 33 might change in human heart failure. Unexpectedly, we found that exon 33^+/? cardiomyocytes showed similar Ca_V1.2 channel properties as wild-type cardiomyocyte, even though Ca_V1.2_Δ33 channels exhibit a gain-in-function. In human hearts, we found that the mRNA level of splicing factor Rbfox1, but not Rbfox2, was downregulated in dilated cardiomyopathy, and CACNA1C mRNA level was dramatically decreased in the both of dilated and ischemic cardiomyopathy. These data imply Rbfox1 may be involved in the development of cardiomyopathies via regulating the alternative splicing of Ca_V1.2 exon 33. (149 words)     

Galectin-1 attenuates cardiomyocyte hypertrophy through splice-variant specific modulation of CaV1.2 calcium channel

Pressure overload-induced cardiac hypertrophy occurs in response to chronic blood pressure increase, and dysfunction of Ca_V1.2 calcium channel involves in cardiac hypertrophic processes by perturbing intracellular calcium concentration ([Ca²⁺]_i) and calcium-dependent signaling. As a carbohydrate-binding protein, galectin-1 (Gal-1) is found to bind with Ca_V1.2 channel, which regulates vascular Ca_V1.2 channel functions and blood pressure. However, the potential roles of Gal-1 in cardiac Ca_V1.2 channel (Ca_V1.2_CM) and cardiomyocyte hypertrophy remain elusive. By whole-cell patch clamp, we find Gal-1 decreases the I_Ca,L with or without isoproterenol (ISO) application by reducing the channel membrane expression in neonatal rat ventricular myocytes (NRVMs). Moreover, Gal-1 could inhibit the current densities of Ca_V1.2_CM by an alternative exon 9*-dependent manner in heterologously expressed HEK293 cells. Of significance, overexpression of Gal-1 diminishes ISO or KCl-induced [Ca²⁺]_i elevation and attenuates ISO-induced hypertrophy in NRVMs. Mechanistically, Gal-1 decreases the ISO or Bay K8644-induced phosphorylation of intracellular calcium-dependent signaling proteins δCaMKII and HDAC4, and inhibits ISO-triggered translocation of HDAC4 in NRVMs. Pathologically, we observe that the expressions of Gal-1 and Ca_V1.2_E9* channels are synchronously increased in rat hypertrophic cardiomyocytes and hearts. Taken together, our study indicates that Gal-1 reduces the channel membrane expression to inhibit the currents of Ca_V1.2_CM in a splice-variant specific manner, which diminishes [Ca²⁺]_i elevation, and attenuates cardiomyocyte hypertrophy by inhibiting the phosphorylation of δCaMKII and HDAC4. Furthermore, our work suggests that dysregulated Gal-1 and Ca_V1.2 alternative exon 9* might be attributed to the pathological processes of cardiac hypertrophy, and provides a potential anti-hypertrophic target in the heart.     

T-type Channels Become Highly Permeable to Sodium Ions Using an Alternative Extracellular Turret Region (S5-P) Outside the Selectivity Filter

T-type (Ca_v3) channels are categorized as calcium channels, but invertebrate ones can be highly sodium-selective channels. We illustrate that the snail LCa_v3 T-type channel becomes highly sodium-permeable through exon splicing of an extracellular turret and descending helix in domain II of the four-domain Ca_v3 channel. Highly sodium-permeable T-type channels are generated without altering the invariant ring of charged residues in the selectivity filter that governs calcium selectivity in calcium channels. The highly sodium-permeant T-type channel expresses in the brain and is the only splice isoform expressed in the snail heart. This unique splicing of turret residues offers T-type channels a capacity to serve as a pacemaking sodium current in the primitive heart and brain in lieu of Na_v1-type sodium channels and to substitute for voltage-gated sodium channels lacking in many invertebrates. T-type channels would also contribute substantially to sodium leak conductances at rest in invertebrates because of their large window currents.     

Alternative Splicing at N Terminus and Domain I Modulates CaV1.2 Inactivation and Surface Expression

The Ca_V1.2 L-type calcium channel is a key conduit for Ca²⁺ influx to initiate excitation-contraction coupling for contraction of the heart and vasoconstriction of the arteries and for altering membrane excitability in neurons. Its α_1C pore-forming subunit is known to undergo extensive alternative splicing to produce many Ca_V1.2 isoforms that differ in their electrophysiological and pharmacological properties. Here, we examined the structure-function relationship of human Ca_V1.2 with respect to the inclusion or exclusion of mutually exclusive exons of the N-terminus exons 1/1a and IS6 segment exons 8/8a. These exons showed tissue selectivity in their expression patterns: heart variant 1a/8a, one smooth-muscle variant 1/8, and a brain isoform 1/8a. Overall, the 1/8a, when coexpressed with Ca_Vβ_2a, displayed a significant and distinct shift in voltage-dependent activation and inactivation and inactivation kinetics as compared to the other three splice variants. Further analysis showed a clear additive effect of the hyperpolarization shift in V_1/2inact of Ca_V1.2 channels containing exon 1 in combination with 8a. However, this additive effect was less distinct for V_1/2act. However, the measured effects were β-subunit-dependent when comparing Ca_Vβ_2a with Ca_Vβ₃ coexpression. Notably, calcium-dependent inactivation mediated by local Ca²⁺-sensing via the N-lobe of calmodulin was significantly enhanced in exon-1-containing Ca_V1.2 as compared to exon-1a-containing Ca_V1.2 channels. At the cellular level, the current densities of the 1/8a or 1/8 variants were significantly larger than the 1a/8a and 1a/8 variants when coexpressed either with Ca_Vβ_2a or Ca_Vβ₃ subunit. This finding correlated well with a higher channel surface expression for the exon 1-Ca_V1.2 isoform that we quantified by protein surface-expression levels or by gating currents. Our data also provided a deeper molecular understanding of the altered biophysical properties of alternatively spliced human Ca_V1.2 channels by directly comparing unitary single-channel events with macroscopic whole-cell currents.     

Splice-variant changes of the CaV3.2 T-type calcium channel mediate voltage-dependent facilitation and associate with cardiac hypertrophy and development

Low voltage-activated T-type calcium (Ca) channels contribute to the normal development of the heart and are also implicated in pathophysiological states such as cardiac hypertrophy. Functionally distinct T-type Ca channel isoforms can be generated by alternative splicing from each of three different T-type genes (Ca_V3.1, Ca_V3.2, Ca_V3.3), although it remains to be described whether specific splice variants are associated with developmental states and pathological conditions. We aimed to identify and functionally characterize Ca_V3.2 T-type Ca channel alternatively spliced variants from newborn animals and to compare with adult normotensive and spontaneously hypertensive rats (SHR). DNA sequence analysis of full-length Ca_V3.2 cDNA generated from newborn heart tissue identified ten major regions of alternative splicing, the more common variants of which were analyzed by quantitative real-time PCR (qRT-PCR) and also subject to functional examination by whole-cell patch clamp. The main findings are that: (1) cardiac Ca_V3.2 T-type Ca channels are subject to considerable alternative splicing, (2) there is preferential expression of Ca_V3.2(−25) splice variant channels in newborn rat heart with a developmental shift in adult heart that results in approximately equal levels of expression of both (+25) and (−25) exon variants, (3) in the adult stage of hypertensive rats there is both an increase in overall Ca_V3.2 expression and a shift towards expression of Ca_V3.2(+25) containing channels as the predominant form and (4) alternative splicing confers a variant-specific voltage-dependent facilitation of Ca_V3.2 channels. We conclude that Ca_V3.2 alternative splicing generates significant T-type Ca channel structural and functional diversity with potential implications relevant to cardiac developmental and pathophysiological states.Key words: voltage-dependent facilitation, alternative splicing, T-type calcium channel, hypertension, cardiac hypertrophy     

Impairment of ubiquitin-proteasome system by E334K cMyBPC modifies channel proteins, leading to electrophysiological dysfunction

Cardiac arrhythmogenesis is regulated by channel proteins whose protein levels are in turn regulated by the ubiquitin-proteasome system (UPS). We have previously reported on UPS impairment induced by E334K cardiac myosin-binding protein C (cMyBPC), which causes hypertrophic cardiomyopathy (HCM) accompanied by arrhythmia. We hypothesized that UPS impairment induced by E334K cMyBPC causes accumulation of cardiac channel proteins, leading to electrophysiological dysfunction. Wild-type or E334K cMyBPC was overexpressed in HL-1 cells and primary cultured neonatal rat cardiac myocytes. The expression of E334K cMyBPC suppressed cellular proteasome activities. The protein levels of K_v1.5, Na_v1.5, Hcn4, Ca_v3.2, Ca_v1.2, Serca, RyR2, and Ncx1 were significantly higher in cells expressing E334K cMyBPC than in wild type. They further increased in cells pretreated with MG132 and had longer protein decays. The channel proteins retained the correct localization. Cells expressing E334K cMyBPC exhibited higher Ca²⁺ transients and longer action potential durations (APDs), accompanied by afterdepolarizations and higher apoptosis. Those augments of APD and Ca²⁺ transients were recapitulated by a simulation model. Although a Ca²⁺ antagonist, azelnidipine, neither protected E334K cMyBPC from degradation nor affected E334K cMyBPC incorporation into the sarcomere, it normalized the APD and Ca²⁺ transients and partially reversed the levels of those proteins regulating apoptosis, thereby attenuating apoptosis. In conclusion, UPS impairment caused by E334K cMyBPC may modify the levels of channel proteins, leading to electrophysiological dysfunction. Therefore, UPS impairment due to a mutant cMyBPC may partly contribute to the observed clinical arrhythmias in HCM patients.     

Cooperative regulation of Cav1.2 channels by intracellular Mg2+, the proximal C-terminal EF-hand,and the distal C-terminal domain

L-type Ca²⁺ currents conducted by Ca_v1.2 channels initiate excitation–contraction coupling in cardiac myocytes. Intracellular Mg²⁺ (Mg_i) inhibits the ionic current of Ca_v1.2 channels. Because Mg_i is altered in ischemia and heart failure, its regulation of Ca_v1.2 channels is important in understanding cardiac pathophysiology. Here, we studied the effects of Mg_i on voltage-dependent inactivation (VDI) of Ca_v1.2 channels using Na⁺ as permeant ion to eliminate the effects of permeant divalent cations that engage the Ca²⁺-dependent inactivation process. We confirmed that increased Mg_i reduces peak ionic currents and increases VDI of Ca_v1.2 channels in ventricular myocytes and in transfected cells when measured with Na⁺ as permeant ion. The increased rate and extent of VDI caused by increased Mg_i were substantially reduced by mutations of a cation-binding residue in the proximal C-terminal EF-hand, consistent with the conclusion that both reduction of peak currents and enhancement of VDI result from the binding of Mg_i to the EF-hand (K_D ≈ 0.9 mM) near the resting level of Mg_i in ventricular myocytes. VDI was more rapid for L-type Ca²⁺ currents in ventricular myocytes than for Ca_v1.2 channels in transfected cells. Coexpression of Ca_vβ_2b subunits and formation of an autoinhibitory complex of truncated Ca_v1.2 channels with noncovalently bound distal C-terminal domain (DCT) both increased VDI in transfected cells, indicating that the subunit structure of the Ca_v1.2 channel greatly influences its VDI. The effects of noncovalently bound DCT on peak current amplitude and VDI required Mg_i binding to the proximal C-terminal EF-hand and were prevented by mutations of a key divalent cation-binding amino acid residue. Our results demonstrate cooperative regulation of peak current amplitude and VDI of Ca_v1.2 channels by Mg_i, the proximal C-terminal EF-hand, and the DCT, and suggest that conformational changes that regulate VDI are propagated from the DCT through the proximal C-terminal EF-hand to the channel-gating mechanism.     

Splice-variant changes of the CaV3.2 T-type calcium channel mediate voltage-dependent facilitation and associate with cardiac hypertrophy and development

Low voltage-activated T-type calcium (Ca) channels contribute to the normal development of the heart and are also implicated in pathophysiological states such as cardiac hypertrophy. Functionally distinct T-type Ca channel isoforms can be generated by alternative splicing from each of three different T-type genes (Ca_V3.1, Ca_V3.2,Ca_V3 .3), although it remains to be described whether specific splice variants are associated with developmental states and pathological conditions. We aimed to identify and functionally characterize Ca_V3.2 T-type Ca channel alternatively spliced variants from newborn animals and to compare with adult normotensive and spontaneously hypertensive rats (SHR). DNA sequence analysis of full-length Ca_V3.2 cDNA generated from newborn heart tissue identified ten major regions of alternative splicing, the more common variants of which were analyzed by quantitative real-time PCR (qRT-PCR) and also subject to functional examination by whole-cell patch clamp. The main findings are that: (1) cardiac Ca_V3.2 T-type Ca channels are subject to considerable alternative splicing, (2) there is preferential expression ofCa_V3 .2(-25) splice variant channels in newborn rat heart with a developmental shift in adult heart that results in approximately equal levels of expression of both (+25) and (-25) exon variants, (3) in the adult stage of hypertensive rats there is a both an increase in overallCa_V3 .2 expression and a shift towards expression of Ca_V3.2(+25) containing channels as the predominant form, and (4) alternative splicing confers a variant-specific voltage-dependent facilitation ofCa_V3 .2 channels. We conclude that Ca_V3.2 alternative splicing generates significant T-type Ca channel structural and functional diversity with potential implications relevant to cardiac developmental and pathophysiological states.     

C-Terminal Modulatory Domain Controls Coupling of Voltage-Sensing to Pore Opening in Cav1.3 L-type Ca Channels

Activity of voltage-gated Ca_v1.3 L-type Ca²⁺ channels is required for proper hearing as well as sinoatrial node and brain function. This critically depends on their negative activation voltage range, which is further fine-tuned by alternative splicing. Shorter variants miss a C-terminal regulatory domain (CTM), which allows them to activate at even more negative potentials than C-terminally long-splice variants. It is at present unclear whether this is due to an increased voltage sensitivity of the Ca_v1.3 voltage-sensing domain, or an enhanced coupling of voltage-sensor conformational changes to the subsequent opening of the activation gate. We studied the voltage-dependence of voltage-sensor charge movement (Q_ON-V) and of current activation (I_Ca-V) of the long (Ca_v1.3_L) and a short Ca_v1.3 splice variant (Ca_v1.3_42A) expressed in tsA-201 cells using whole cell patch-clamp. Charge movement (Q_ON) of Ca_v1.3_L displayed a much steeper voltage-dependence and a more negative half-maximal activation voltage than Ca_v1.2 and Ca_v3.1. However, a significantly higher fraction of the total charge had to move for activation of Ca_v1.3 half-maximal conductance (Ca_v1.3: 68%; Ca_v1.2: 52%; Ca_v3.1: 22%). This indicated a weaker coupling of Ca_v1.3 voltage-sensor charge movement to pore opening. However, the coupling efficiency was strengthened in the absence of the CTM in Ca_v1.3_42A, thereby shifting I_Ca-V by 7.2 mV to potentials that were more negative without changing Q_ON-V. We independently show that the presence of intracellular organic cations (such as n-methyl-D-glucamine) induces a pronounced negative shift of Q_ON-V and a more negative activation of I_Ca-V of all three channels. These findings illustrate that the voltage sensors of Ca_v1.3 channels respond more sensitively to depolarization than those of Ca_v1.2 or Ca_v3.1. Weak coupling of voltage sensing to pore opening is enhanced in the absence of the CTM, allowing short Ca_v1.3_42A splice variants to activate at lower voltages without affecting Q_ON-V.     

C-Terminal Modulatory Domain Controls Coupling of Voltage-Sensing to?Pore Opening in Cav1.3 L-type Ca2+ Channels

Activity of voltage-gated Ca_v1.3 L-type Ca²⁺ channels is required for proper hearing as well as sinoatrial node and brain function. This critically depends on their negative activation voltage range, which is further fine-tuned by alternative splicing. Shorter variants miss a C-terminal regulatory domain (CTM), which allows them to activate at even more negative potentials than C-terminally long-splice variants. It is at present unclear whether this is due to an increased voltage sensitivity of the Ca_v1.3 voltage-sensing domain, or an enhanced coupling of voltage-sensor conformational changes to the subsequent opening of the activation gate. We studied the voltage-dependence of voltage-sensor charge movement (Q_ON-V) and of current activation (I_Ca-V) of the long (Ca_v1.3_L) and a short Ca_v1.3 splice variant (Ca_v1.3_42A) expressed in tsA-201 cells using whole cell patch-clamp. Charge movement (Q_ON) of Ca_v1.3_L displayed a much steeper voltage-dependence and a more negative half-maximal activation voltage than Ca_v1.2 and Ca_v3.1. However, a significantly higher fraction of the total charge had to move for activation of Ca_v1.3 half-maximal conductance (Ca_v1.3: 68%; Ca_v1.2: 52%; Ca_v3.1: 22%). This indicated a weaker coupling of Ca_v1.3 voltage-sensor charge movement to pore opening. However, the coupling efficiency was strengthened in the absence of the CTM in Ca_v1.3_42A, thereby shifting I_Ca-V by 7.2 mV to potentials that were more negative without changing Q_ON-V. We independently show that the presence of intracellular organic cations (such as n-methyl-D-glucamine) induces a pronounced negative shift of Q_ON-V and a more negative activation of I_Ca-V of all three channels. These findings illustrate that the voltage sensors of Ca_v1.3 channels respond more sensitively to depolarization than those of Ca_v1.2 or Ca_v3.1. Weak coupling of voltage sensing to pore opening is enhanced in the absence of the CTM, allowing short Ca_v1.3_42A splice variants to activate at lower voltages without affecting Q_ON-V.     

Ca(v)1.2 calcium channel is glutathionylated during oxidative stress in guinea pig and ischemic human heart

Glutathionylation as a posttranslational modification of proteins is becoming increasingly recognized, but its role in many diseases has not been demonstrated. Oxidative stress and alterations in calcium homeostasis are associated with the development of cardiac hypertrophy. Because the cardiac L-type Ca²⁺ channel can be persistently activated after exposure to H₂O₂, the aim of this study was to determine whether alterations in channel function were associated with glutathionylation of the α_1C subunit (Ca_v1.2) channel protein. Immunoblot analysis indicated that Ca_v1.2 protein is significantly glutathionylated after exposure to H₂O₂ and glutathione in vitro and after ischemia-reperfusion injury. L-type Ca²⁺ channel macroscopic current and intracellular calcium were significantly increased in myocytes after exposure to oxidized glutathione and reversed by glutaredoxin. The increase in current correlated with an increase in open probability of the channel assessed as changes in single-channel activity after exposing the human long N-terminal Ca_v1.2 to H₂O₂ or oxidized glutathione. We also demonstrate that the Ca_v1.2 channel is significantly glutathionylated in ischemic human heart. We conclude that oxidative stress is associated with an increase in glutathionylation of the Ca_v1.2 channel protein. We suggest that the associated constitutive activity contributes to the development of pathology in ischemic heart disease.     

Characterization of Cav1.4 Complexes (α11.4, β2, and α2δ4) in HEK293T Cells and in the Retina

In photoreceptor synaptic terminals, voltage-gated Ca_v1.4 channels mediate Ca²⁺ signals required for transmission of visual stimuli. Like other high voltage-activated Ca_v channels, Ca_v1.4 channels are composed of a main pore-forming Ca_v1.4 α₁ subunit and auxiliary β and α₂δ subunits. Of the four distinct classes of β and α₂δ, β₂ and α₂δ₄ are thought to co-assemble with Ca_v1.4 α₁ subunits in photoreceptors. However, an understanding of the functional properties of this combination of Ca_v subunits is lacking. Here, we provide evidence that Ca_v1.4 α₁, β₂, and α₂δ₄ contribute to Ca_v1.4 channel complexes in the retina and describe their properties in electrophysiological recordings. In addition, we identified a variant of β₂, named here β_2X13, which, along with β_2a, is present in photoreceptor terminals. Ca_v1.4 α_1, β₂, and α₂δ₄ were coimmunoprecipitated from lysates of transfected HEK293 cells and mouse retina and were found to interact in the outer plexiform layer of the retina containing the photoreceptor synaptic terminals, by proximity ligation assays. In whole-cell patch clamp recordings of transfected HEK293T cells, channels (Ca_v1.4 α₁ + β_2X13) containing α₂δ₄ exhibited weaker voltage-dependent activation than those with α₂δ₁. Moreover, compared with channels (Ca_v1.4 α₁ + α₂δ₄) with β_2a, β_2X13-containing channels exhibited greater voltage-dependent inactivation. The latter effect was specific to Ca_v1.4 because it was not seen for Ca_v1.2 channels. Our results provide the first detailed functional analysis of the Ca_v1.4 subunits that form native photoreceptor Ca_v1.4 channels and indicate potential heterogeneity in these channels conferred by β_2a and β_2X13 variants.     

Calmodulin overexpression does not alter Cav1.2 function or oligomerization state

Interactions between calmodulin (CaM) and voltage-gated calcium channels (Ca_vs) are crucial for Ca_v activity-dependent feedback modulation. We recently reported an X-ray structure that shows two Ca²⁺/CaM molecules bound to the Ca_v1.2 C terminal tail, one at the PreIQ region and one at the IQ domain. Surprisingly, the asymmetric unit of the crystal showed a dimer in which Ca²⁺/CaM bridged two PreIQ helixes to form a 4:2 Ca²⁺/CaM:Ca_v C-terminal tail assembly. Contrary to previous proposals based on a similar crystallographic dimer, extensive biochemical analysis together with subunit counting experiments of full-length channels in live cell membranes failed to find evidence for multimers that would be compatible with the 4:2 crossbridged complex. Here, we examine this possibility further. We find that CaM over-expression has no functional effect on Ca_v1.2 inactivation or on the stoichiometry of full-length Ca_v1.2. These data provide further support for the monomeric Ca_v1.2 stoichiometry. Analysis of the electrostatic surfaces of the 2:1 Ca²⁺/CaM:CaV C-terminal tail assembly reveals notable patches of electronegativity. These could influence various forms of channel modulation by interacting with positively charged elements from other intracellular channel domains.     

Ca<Subscript>v</Subscript>1.3 and BK Channels for Timing and Regulating Cell Firing

L-type Ca²⁺ channels (LTCCs, Ca_v1) open readily during membrane depolarization and allow Ca²⁺ to enter the cell. In this way, LTCCs regulate cell excitability and trigger a variety of Ca²⁺-dependent physiological processes such as: excitation–contraction coupling in muscle cells, gene expression, synaptic plasticity, neuronal differentiation, hormone secretion, and pacemaker activity in heart, neurons, and endocrine cells. Among the two major isoforms of LTCCs expressed in excitable tissues (Ca_v1.2 and Ca_v1.3), Ca_v1.3 appears suitable for supporting a pacemaker current in spontaneously firing cells. It has steep voltage dependence and low threshold of activation and inactivates slowly. Using Ca_v1.3^−/− KO mice and membrane current recording techniques such as the dynamic and the action potential clamp, it has been possible to resolve the time course of Ca_v1.3 pacemaker currents that regulate the spontaneous firing of dopaminergic neurons and adrenal chromaffin cells. In several cell types, Ca_v1.3 is selectively coupled to BK channels within membrane nanodomains and controls both the firing frequency and the action potential repolarization phase. Here we review the most critical aspects of Ca_v1.3 channel gating and its coupling to large conductance BK channels recently discovered in spontaneously firing neurons and neuroendocrine cells with the aim of furnishing a converging view of the role that these two channel types play in the regulation of cell excitability.     

Functional diversity in neuronal voltage-gated calcium channels by alternative splicing of Ca(v)alpha1

Alternative splicing is a critical mechanism used extensively in the mammalian nervous system to increase the level of diversity that can be achieved by a set of genes. This review focuses on recent studies of voltage-gated calcium (Ca) channel Ca_vα₁ subunit splice isoforms in neurons. Voltage-gated Ca channels couple changes in neuronal activity to rapid changes in intracellular Ca levels that in turn regulate an astounding range of cellular processes. Only ten genes have been identified that encode Ca_vα₁ subunits, an insufficient number to account for the level of functional diversity among voltage-gated Ca channels. The consequences of regulated alternative splicing among the genes that comprise voltage-gated Ca channels permits specialization of channel function, optimizing Ca signaling in different regions of the brain and in different cellular compartments. Although the full extent of alternative splicing is not yet known for any of the major subtypes of voltage-gated Ca channels, it is already clear that it adds a rich layer of structural and functional diversity”.