New Determinant for the CaVbeta2 subunit modulation of the CaV1.2 calcium channel

Ca(v)beta subunits support voltage gating of Ca(v)1.2 calcium channels and play important role in excitation-contraction coupling. The common central membrane-associated guanylate kinase (MAGUK) region of Ca(v)beta binds to the alpha-interaction domain (AID) and the IQ motif of the pore-forming alpha(1C) subunit, but these two interactions do not explain why the cardiac Ca(v)beta(2) subunit splice variants differentially modulate inactivation of Ca(2+) currents (I(Ca)). Previously we described beta(2Deltag), a functionally active splice variant of human Ca(v)beta(2) lacking MAGUK. By deletion analysis of beta(2Deltag), we have now identified a 41-amino acid C-terminal essential determinant (beta(2)CED) that stimulates I(Ca) in the absence of Ca(v)beta subunits and conveys a +20-mV shift in the peak of the I(Ca)-voltage relationship. The beta(2)CED is targeted by alpha(1C) to the plasma membrane, forms a complex with alpha(1C) but does not bind to AID. Electrophysiology and binding studies point to the calmodulin-interacting LA/IQ region in the alpha(1C) subunit C terminus as a functionally relevant beta(2)CED binding site. The beta(2)CED interacts with LA/IQ in a Ca(2+)- and calmodulin-independent manner and need LA, but not IQ, to activate the channel. Deletion/mutation analyses indicated that each of the three Ca(v)beta(2)/alpha(1C) interactions is sufficient to support I(Ca). However, beta(2)CED does not support Ca(2+)-dependent inactivation, suggesting that interactions of MAGUK with AID and IQ are crucial for Ca(2+)-induced inactivation. The beta(2)CED is conserved only in Ca(v)beta(2) subunits. Thus, beta(2)CED constitutes a previously unknown integrative part of the multifactorial mechanism of Ca(v)beta(2)-subunit differential modulation of the Ca(v)1.2 calcium channel that in beta(2Deltag) occurs without MAGUK.     

Analysis of the complex between Ca2+ channel beta-subunit and the Rem GTPase

Voltage-gated calcium channels are multiprotein complexes that regulate calcium influx and are important contributors to cardiac excitability and contractility. The auxiliary beta-subunit (CaV beta) binds a conserved domain (the alpha-interaction domain (AID)) of the pore-forming CaV alpha1 subunit to modulate channel gating properties and promote cell surface trafficking. Recently, members of the RGK family of small GTPases (Rem, Rem2, Rad, Gem/Kir) have been identified as novel contributors to the regulation of L-type calcium channel activity. Here, we describe the Rem-association domain within CaV beta2a. The Rem interaction module is located in a approximately 130-residue region within the highly conserved guanylate kinase domain that also directs AID binding. Importantly, CaV beta mutants were identified that lost the ability to bind AID but retained their association with Rem, indicating that the AID and Rem association sites of CaV beta2a are structurally distinct. In vitro binding studies indicate that the affinity of Rem for CaV beta2a interaction is lower than that of AID for CaV beta2a. Furthermore, in vitro binding studies indicate that Rem association does not inhibit the interaction of CaV beta2a with AID. Instead, CaV beta can simultaneously associate with both Rem and CaV alpha1-AID. Previous studies had suggested that RGK proteins may regulate Ca2+ channel activity by blocking the association of CaV beta subunits with CaV alpha1 to inhibit plasma membrane trafficking. However, surface biotinylation studies in HIT-T15 cells indicate that Rem can acutely modulate channel function without decreasing the density of L-type channels at the plasma membrane. Together these data suggest that Rem-dependent Ca2+ channel modulation involves formation of a Rem x CaV beta x AID regulatory complex without the need to disrupt CaV alpha1 x CaV beta association or alter CaV alpha1 expression at the plasma membrane.     

A specific tryptophan in the I-II linker is a key determinant of beta-subunit binding and modulation in Ca(V)2.3 calcium channels

The ancillary beta subunits modulate the activation and inactivation properties of high-voltage activated (HVA) Ca(2+) channels in an isoform-specific manner. The beta subunits bind to a high-affinity interaction site, alpha-interaction domain (AID), located in the I-II linker of HVA alpha1 subunits. Nine residues in the AID motif are absolutely conserved in all HVA channels (QQxExxLxGYxxWIxxxE), but their contribution to beta-subunit binding and modulation remains to be established in Ca(V)2.3. Mutations of W386 to either A, G, Q, R, E, F, or Y in Ca(V)2.3 disrupted [(35)S]beta3-subunit overlay binding to glutathione S-transferase fusion proteins containing the mutated I-II linker, whereas mutations (single or multiple) of nonconserved residues did not affect the protein-protein interaction with beta3. The tryptophan residue at position 386 appears to be an essential determinant as substitutions with hydrophobic (A and G), hydrophilic (Q, R, and E), or aromatic (F and Y) residues yielded the same results. beta-Subunit modulation of W386 (A, G, Q, R, E, F, and Y) and Y383 (A and S) mutants was investigated after heterologous expression in Xenopus oocytes. All mutant channels expressed large inward Ba(2+) currents with typical current-voltage properties. Nonetheless, the typical hallmarks of beta-subunit modulation, namely the increase in peak currents, the hyperpolarization of peak voltages, and the modulation of the kinetics and voltage dependence of inactivation, were eliminated in all W386 mutants, although they were preserved in part in Y383 (A and S) mutants. Altogether these results suggest that W386 is critical for beta-subunit binding and modulation of HVA Ca(2+) channels.     

The alpha1-beta-subunit interaction that modulates calcium channel activity is reversible and requires a competent alpha-interaction domain

High voltage-gated calcium channels consist of a pore-forming subunit (alpha(1)) and three nonhomologous subunits (alpha(2)/delta, beta, and gamma). Although it is well established that the beta-subunit promotes traffic of channels to the plasma membrane and modifies their activity, the reversible nature of the interaction with the alpha(1)-subunit remains controversial. Here, we address this issue by examining the effect of purified beta(2a) protein on Ca(V)1.2 and Ca(V)2.3 channels expressed in Xenopus oocytes. The beta(2a)-subunit binds to the alpha(1)-interaction domain (AID) in vitro, and when injected into oocytes, it shifts the voltage dependence of activation and increases charge movement to ionic current coupling of Ca(V)1.2 channels. This increase depended on the integrity of AID but was not abolished by bafilomycin, demonstrating that the alpha(1)-beta interaction through the AID site can take place at the plasma membrane. Furthermore, injection of beta(2a) protein inhibited inactivation of Ca(V)2.3 channels and converted fast inactivating Ca(V)2.3/beta(1b) channels to slow inactivating channels. Inhibition of inactivation required larger concentration of beta(2a) in oocytes expressing Ca(V)2.3/beta(1b) channels than expressing Ca(V)2.3 alone but reached the same maximal level as expected for a competitive interaction through a single binding site. Together, our data show that the alpha(1)-beta interaction is reversible in intact cells and defines calcium channels beta-subunits as regulatory proteins rather than stoichiometric subunits.     

Dose-dependent and isoform-specific modulation of Ca2+ channels by RGK GTPases

Although inhibition of voltage-gated calcium channels by RGK GTPases (RGKs) represents an important mode of regulation to control Ca(2+) influx in excitable cells, their exact mechanism of inhibition remains controversial. This has prevented an understanding of how RGK regulation can be significant in a physiological context. Here we show that RGKs-Gem, Rem, and Rem2-decreased Ca(V)1.2 Ca(2+) current amplitude in a dose-dependent manner. Moreover, Rem2, but not Rem or Gem, produced dose-dependent alterations on gating kinetics, uncovering a new mode by which certain RGKs can precisely modulate Ca(2+) currents and affect Ca(2+) influx during action potentials. To explore how RGKs influence gating kinetics, we separated the roles mediated by the Ca(2+) channel accessory beta subunit's interaction with its high affinity binding site in the pore-forming alpha(1C) subunit (AID) from its other putative contact sites by utilizing an alpha(1C)*beta3 concatemer in which the AID was mutated to prevent beta subunit interaction. This mutant concatemer generated currents with all the hallmarks of beta subunit modulation, demonstrating that AID-beta-independent interactions are sufficient for beta subunit modulation. Using this construct we found that although inhibition of current amplitude was still partially sensitive to RGKs, Rem2 no longer altered gating kinetics, implicating different determinants for this specific mode of Rem2-mediated regulation. Together, these results offer new insights into the molecular mechanism of RGK-mediated Ca(2+) channel current modulation.     

Essential Ca(V)beta modulatory properties are AID-independent

Voltage-gated Ca(2+) channel beta (Ca(v)beta) subunits have a highly conserved core consisting of interacting Src homology 3 and guanylate kinase domains, and are postulated to exert their effects through AID, the major interaction site in the pore-forming alpha(1) subunit. This stereotypical interaction does not explain how individual Ca(v)beta subunits modulate alpha(1) subunits differentially. Here we show that AID is neither necessary nor sufficient for critical Ca(v)beta regulatory properties. Complete modulation depends on additional contacts that are exclusive of AID and not revealed in recent crystal structures. These data offer a new context for understanding Ca(v)beta modulation, suggesting that the AID interaction orients the Ca(v)beta core so as to permit additional isoform-specific Ca(v)alpha(1)-Ca(v)beta interactions that underlie the particular regulation seen with each Ca(v)alpha(1)-Ca(v)beta pair, rather than as the main site of regulation.     

Several structural domains contribute to the regulation of N-type calcium channel inactivation by the beta 3 subunit

Calcium channel beta subunits are essential regulatory elements of the gating properties of high voltage-activated calcium channels. Co-expression with beta(3) subunits typically accelerates inactivation, whereas co-expression with beta(4) subunits results in a slowly inactivating phenotype. Here, we have examined the molecular basis of the differential effect of these two subunits on the inactivation characteristics of Ca(v)2.2 + alpha(2)-delta(1) N-type calcium channels by creating a series of 22 chimeric beta subunits that are based on various combinations of variable and conserved regions of the parent beta subunit isoforms. Our data show that replacement of the N terminus region of beta(4) with a corresponding 14-amino acid stretch of beta(3) sequence accelerates the inactivation kinetics to levels seen with wild type beta(3). A similar kinetic speeding is observed by a concomitant substitution of the second conserved and variable regions, but not when these regions are substituted individually, suggesting that 1) the second variable and conserved regions cooperatively regulate N-type calcium channel inactivation and 2) that there are two redundant mechanisms that allow the beta(3) subunit to accelerate N-type channel inactivation. In contrast with previous reports in Ca(v)2.1 calcium channels, deletion of the C-terminal region of Ca(v)2.2 did not alter the regulation of the channel by wild type and chimeric beta subunits. Hence, the molecular underpinnings of beta subunit regulation of voltage-gated calcium channels appear to vary with calcium channel subtype.     

Molecular determinants of inactivation within the I-II linker of alpha1E (CaV2.3) calcium channels

Voltage-dependent inactivation of CaV2.3 channels was investigated using point mutations in the beta-subunit-binding site (AID) of the I-II linker. The quintuple mutant alpha1E N381K + R384L + A385D + D388T + K389Q (NRADK-KLDTQ) inactivated like the wild-type alpha1E. In contrast, mutations of alpha1E at position R378 (position 5 of AID) into negatively charged residues Glu (E) or Asp (D) significantly slowed inactivation kinetics and shifted the voltage dependence of inactivation to more positive voltages. When co-injected with beta3, R378E inactivated with tau(inact) = 538 +/- 54 ms (n = 14) as compared with 74 +/- 4 ms (n = 21) for alpha1E (p < 0.001) with a mid-potential of inactivation E(0.5) = -44 +/- 2 mV (n = 10) for R378E as compared with E(0.5) = -64 +/- 3 mV (n = 9) for alpha1E. A series of mutations at position R378 suggest that positively charged residues could promote voltage-dependent inactivation. R378K behaved like the wild-type alpha1E whereas R378Q displayed intermediate inactivation kinetics. The reverse mutation E462R in the L-type alpha1C (CaV1.2) produced channels with inactivation properties comparable to alpha1E R378E. Hence, position 5 of the AID motif in the I-II linker could play a significant role in the inactivation of Ca(V)1.2 and CaV2.3 channels.     

Differential role of the alpha1C subunit tails in regulation of the Cav1.2 channel by membrane potential, beta subunits, and Ca2+ ions

Voltage-gated Ca(v)1.2 channels are composed of the pore-forming alpha1C and auxiliary beta and alpha2delta subunits. Voltage-dependent conformational rearrangements of the alpha1C subunit C-tail have been implicated in Ca2+ signal transduction. In contrast, the alpha1C N-tail demonstrates limited voltage-gated mobility. We have asked whether these properties are critical for the channel function. Here we report that transient anchoring of the alpha1C subunit C-tail in the plasma membrane inhibits Ca2+-dependent and slow voltage-dependent inactivation. Both alpha2delta and beta subunits remain essential for the functional channel. In contrast, if alpha1C subunits with are expressed alpha2delta but in the absence of a beta subunit, plasma membrane anchoring of the alpha1C N terminus or its deletion inhibit both voltage- and Ca2+-dependent inactivation of the current. The following findings all corroborate the importance of the alpha1C N-tail/beta interaction: (i) co-expression of beta restores inactivation properties, (ii) release of the alpha1C N terminus inhibits the beta-deficient channel, and (iii) voltage-gated mobility of the alpha1C N-tail vis a vis the plasma membrane is increased in the beta-deficient (silent) channel. Together, these data argue that both the alpha1C N- and C-tails have important but different roles in the voltage- and Ca2+-dependent inactivation, as well as beta subunit modulation of the channel. The alpha1C N-tail may have a role in the channel trafficking and is a target of the beta subunit modulation. The beta subunit facilitates voltage gating by competing with the N-tail and constraining its voltage-dependent rearrangements. Thus, cross-talk between the alpha1C C and N termini, beta subunit, and the cytoplasmic pore region confers the multifactorial regulation of Ca(v)1.2 channels.     

Negatively charged residues in the N-terminal of the AID helix confer slow voltage dependent inactivation gating to CaV1.2

The E462R mutation in the fifth position of the AID (alpha1 subunit interaction domain) region in the I-II linker is known to significantly accelerate voltage-dependent inactivation (VDI) kinetics of the L-type CaV1.2 channel, suggesting that the AID region could participate in a hinged-lid type inactivation mechanism in these channels. The recently solved crystal structures of the AID-CaVbeta regions in L-type CaV1.1 and CaV1.2 channels have shown that in addition to E462, positions occupied by Q458, Q459, E461, K465, L468, D469, and T472 in the rabbit CaV1.2 channel could also potentially contribute to a hinged-lid type mechanism. A mutational analysis of these residues shows that Q458A, Q459A, K465N, L468R, D469A, and T472D did not significantly alter VDI gating. In contrast, mutations of the negatively charged E461, E462, and D463 to neutral or positively charged residues increased VDI gating, suggesting that the cluster of negatively charged residues in the N-terminal end of the AID helix could account for the slower VDI kinetics of CaV1.2. A mutational analysis at position 462 (R, K, A, G, D, N, Q) further confirmed that E462R yielded faster VDI kinetics at +10 mV than any other residue with E462R > E462K approximately E462A > E462N > wild-type approximately E462Q approximately E462G > E462D (from the fastest to the slowest). E462R was also found to increase the VDI gating of the slow CEEE chimera that includes the I-II linker from CaV1.2 into a CaV2.3 background. The fast VDI kinetics of the CaV1.2 E462R and the CEEE + E462R mutants were abolished by the CaVbeta2a subunit and reinstated when using the nonpalmitoylated form of CaVbeta2a C3S + C4S (CaVbeta2a CS), confirming that CaVbeta2a and E462R modulate VDI through a common pathway, albeit in opposite directions. Altogether, these results highlight the unique role of E461, E462, and D463 in the I-II linker in the VDI gating of high-voltage activated CaV1.2 channels.     

Mechanism of beta4 subunit modulation of BK channels

Large-conductance (BK-type) Ca(2+)-activated potassium channels are activated by membrane depolarization and cytoplasmic Ca(2+). BK channels are expressed in a broad variety of cells and have a corresponding diversity in properties. Underlying much of the functional diversity is a family of four tissue-specific accessory subunits (beta1-beta4). Biophysical characterization has shown that the beta4 subunit confers properties of the so-called "type II" BK channel isotypes seen in brain. These properties include slow gating kinetics and resistance to iberiotoxin and charybdotoxin blockade. In addition, the beta4 subunit reduces the apparent voltage sensitivity of channel activation and has complex effects on apparent Ca(2+) sensitivity. Specifically, channel activity at low Ca(2+) is inhibited, while at high Ca(2+), activity is enhanced. The goal of this study is to understand the mechanism underlying beta4 subunit action in the context of a dual allosteric model for BK channel gating. We observed that beta4's most profound effect is a decrease in P(o) (at least 11-fold) in the absence of calcium binding and voltage sensor activation. However, beta4 promotes channel opening by increasing voltage dependence of P(o)-V relations at negative membrane potentials. In the context of the dual allosteric model for BK channels, we find these properties are explained by distinct and opposing actions of beta4 on BK channels. beta4 reduces channel opening by decreasing the intrinsic gating equilibrium (L(0)), and decreasing the allosteric coupling between calcium binding and voltage sensor activation (E). However, beta4 has a compensatory effect on channel opening following depolarization by shifting open channel voltage sensor activation (Vh(o)) to more negative membrane potentials. The consequence is that beta4 causes a net positive shift of the G-V relationship (relative to alpha subunit alone) at low calcium. At higher calcium, the contribution by Vh(o) and an increase in allosteric coupling to Ca(2+) binding (C) promotes a negative G-V shift of alpha+beta4 channels as compared to alpha subunits alone. This manner of modulation predicts that type II BK channels are downregulated by beta4 at resting voltages through effects on L(0). However, beta4 confers a compensatory effect on voltage sensor activation that increases channel opening during depolarization.     

Role of the C terminus of the alpha 1C (CaV1.2) subunit in membrane targeting of cardiac L-type calcium channels

We have previously demonstrated that formation of a complex between L-type calcium (Ca(2+)) channel alpha(1C) (Ca(V)1.2) and beta subunits was necessary to target the channels to the plasma membrane when expressed in tsA201 cells. In the present study, we identified a region in the C terminus of the alpha(1C) subunit that was required for membrane targeting. Using a series of C-terminal deletion mutants of the alpha(1C) subunit, a domain consisting of amino acid residues 1623-1666 ("targeting domain") in the C terminus of the alpha(1C) subunit has been identified to be important for correct targeting of L-type Ca(2+) channel complexes to the plasma membrane. Although cells expressing the wild-type alpha(1C) and beta(2a) subunits exhibited punctate clusters of channel complexes along the plasma membrane with little intracellular staining, co-expression of deletion mutants of the alpha(1C) subunit that lack the targeting domain with the beta(2a) subunit resulted in an intracellular localization of the channels. In addition, three other regions in the C terminus of the alpha(1C) subunit that were downstream of residues 1623-1666 were found to contribute to membrane targeting of the L-type channels. Deletion of these domains in the alpha(1C) subunit resulted in a reduction of plasma membrane-localized channels, and a concomitant increase in channels localized intracellularly. Taken together, these results have demonstrated that a targeting domain in the C terminus of the alpha(1C) subunit was required for proper plasma membrane localization of the L-type Ca(2+) channels.     

Modulation of voltage- and Ca2+-dependent gating of CaV1.3 L-type calcium channels by alternative splicing of a C-terminal regulatory domain

Low voltage activation of Ca(V)1.3 L-type Ca(2+) channels controls excitability in sensory cells and central neurons as well as sinoatrial node pacemaking. Ca(V)1.3-mediated pacemaking determines neuronal vulnerability of dopaminergic striatal neurons affected in Parkinson disease. We have previously found that in Ca(V)1.4 L-type Ca(2+) channels, activation, voltage, and calcium-dependent inactivation are controlled by an intrinsic distal C-terminal modulator. Because alternative splicing in the Ca(V)1.3 alpha1 subunit C terminus gives rise to a long (Ca(V)1.3(42)) and a short form (Ca(V)1.3(42A)), we investigated if a C-terminal modulatory mechanism also controls Ca(V)1.3 gating. The biophysical properties of both splice variants were compared after heterologous expression together with beta3 and alpha2delta1 subunits in HEK-293 cells. Activation of calcium current through Ca(V)1.3(42A) channels was more pronounced at negative voltages, and inactivation was faster because of enhanced calcium-dependent inactivation. By investigating several Ca(V)1.3 channel truncations, we restricted the modulator activity to the last 116 amino acids of the C terminus. The resulting Ca(V)1.3(DeltaC116) channels showed gating properties similar to Ca(V)1.3(42A) that were reverted by co-expression of the corresponding C-terminal peptide C(116). Fluorescence resonance energy transfer experiments confirmed an intramolecular protein interaction in the C terminus of Ca(V)1.3 channels that also modulates calmodulin binding. These experiments revealed a novel mechanism of channel modulation enabling cells to tightly control Ca(V)1.3 channel activity by alternative splicing. The absence of the C-terminal modulator in short splice forms facilitates Ca(V)1.3 channel activation at lower voltages expected to favor Ca(V)1.3 activity at threshold voltages as required for modulation of neuronal firing behavior and sinoatrial node pacemaking.     

Structural determinants of L-type channel activation in segment IIS6 revealed by a retinal disorder

The mechanism of channel opening for voltage-gated calcium channels is poorly understood. The importance of a conserved isoleucine residue in the pore-lining segment IIS6 has recently been highlighted by functional analyses of a mutation (I745T) in the Ca(V)1.4 channel causing severe visual impairment (Hemara-Wahanui, A., Berjukow, S., Hope, C. I., Dearden, P. K., Wu, S. B., Wilson-Wheeler, J., Sharp, D. M., Lundon-Treweek, P., Clover, G. M., Hoda, J. C., Striessnig, J., Marksteiner, R., Hering, S., and Maw, M. A. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 7553-7558). In the present study we analyzed the influence of amino acids in segment IIS6 on gating of the Ca(V)1.2 channel. Substitution of Ile-781, the Ca(V)1.2 residue corresponding to Ile-745 in Ca(V)1.4, by residues of different hydrophobicity, size and polarity shifted channel activation in the hyperpolarizing direction (I781P > I781T > I781N > I781A > I781L). As I781P caused the most dramatic shift (-37 mV), substitution with this amino acid was used to probe the role of other residues in IIS6 in the process of channel activation. Mutations revealed a high correlation between the midpoint voltages of activation and inactivation. A unique kinetic phenotype was observed for residues 779-782 (LAIA) located in the lower third of segment IIS6; a shift in the voltage dependence of activation was accompanied by a deceleration of activation at hyperpolarized potentials, a deceleration of deactivation at all potentials (I781P and I781T), and decreased inactivation. These findings indicate that Ile-781 substitutions both destabilize the closed conformation and stabilize the open conformation of Ca(V)1.2. Moreover there may be a flexible center of helix bending at positions 779-782 of Ca(V)1.2. These four residues are completely conserved in high voltage-activated calcium channels suggesting that these channels may share a common mechanism of gating.     

Alanine-scanning mutagenesis defines a conserved energetic hotspot in the CaValpha1 AID-CaVbeta interaction site that is critical for channel modulation

Voltage-gated calcium channels (CaVs) are large, multisubunit complexes that control cellular calcium entry. CaV pore-forming (CaValpha1) and cytoplasmic (CaVbeta) subunits associate through a high-affinity interaction between the CaValpha1 alpha interaction domain (AID) and CaVbeta alpha binding pocket (ABP). Here we analyze AID-ABP interaction thermodynamics using isothermal titration calorimetry. We find that commensurate with their strong sequence similarity, all CaV1 and CaV2 AID peptides bind CaVbeta with similar nanomolar affinities. Although the AID-ABP interface encompasses 24 side chains, alanine-scanning mutagenesis reveals that the binding energy is focused in two complementary hotspots comprising four deeply conserved residues. Electrophysiological experiments show that hotspot interaction disruption prevents trafficking and functional modulation of CaV1.2 by CaVbeta. Together, the data support the primacy of the AID-ABP interface for CaValpha1-CaVbeta association, underscore the idea that hotspots dominate protein-protein interaction affinities, and uncover a target for strategies to control cellular excitability by blocking CaValpha1-CaVbeta complex formation.     

The beta subunit increases Ca2+ currents and gating charge movements of human cardiac L-type Ca2+ channels.

The properties of the gating currents (nonlinear charge movements) of human cardiac L-type Ca2- channels and their relationship to the activation of the Ca2+ channel (ionic) currents were studied using a mammalian expression system. Cloned human cardiac alpha1 + rabbit alpha 2 subunits or human cardiac alpha 1 + rabbit alpha 2 + human beta 3 subunits were transiently expressed in HEK293 cells. The maximum Ca2+ current density increased from -3.9 +/- 0.9 pA/pF for the alpha 1 + alpha 2 subunits to -11.6 +/- 2.2 pA/pF for alpha 1 + alpha 2 + beta 3 subunits. Calcium channel gating currents were recorded after the addition of 5 mM Co2+, using a -P/5 protocol. The maximum nonlinear charge movement (Qmax) increased from 2.5 +/- 0.3 nC/muF for alpha 1 + alpha 2 subunit to 12.1 +/- 0.3 nC/muF for alpha 1 + alpha 2 + beta 3 subunit expression. The QON was equal to the QOFF for both subunit combinations. The QON-Vm data were fit by a sum of two Boltzmann expressions and ranged over more negative potentials, as compared with the voltage dependence for activation of the Ca2+ conductance. We conclude that 1) the beta subunit increases the number of functional alpha 1 subunits expressed in the plasma membrane of these cells and 2) the voltage-dependent activation of the human cardiac L-type calcium channel involves the movements of at least two nonidentical and functionally distinct gating structures.     

The role of the GX9GX3G motif in the gating of high voltage-activated Ca2+ channels

The putative hinge point revealed by the crystal structure of the MthK potassium channel is a glycine residue that is conserved in many ion channels. In high voltage-activated (HVA) Ca(V) channels, the mid-S6 glycine residue is only present in IS6 and IIS6, corresponding to G422 and G770 in Ca(V)1.2. Two additional glycine residues are found in the distal portion of IS6 (Gly(432) and Gly(436) in Ca(V)1.2) to form a triglycine motif unique to HVA Ca(V) channels. Lethal arrhythmias are associated with mutations of glycine residues in the human L-type Ca(2+) channel. Hence, we undertook a mutational analysis to investigate the role of S6 glycine residues in channel gating. In Ca(V)1.2, alpha-helix-breaking proline mutants (G422P and G432P) as well as the double G422A/G432A channel did not produce functional channels. The macroscopic inactivation kinetics were significantly decreased with Ca(V)1.2 wild type > G770A > G422A congruent with G436A > G432A (from the fastest to the slowest). Mutations at position Gly(432) produced mostly nonfunctional mutants. Macroscopic inactivation kinetics were markedly reduced by mutations of Gly(436) to Ala, Pro, Tyr, Glu, Arg, His, Lys, or Asp residues with stronger effects obtained with charged and polar residues. Mutations within the distal GX(3)G residues blunted Ca(2+)-dependent inactivation kinetics and prevented the increased voltage-dependent inactivation kinetics brought by positively charged residues in the I-II linker. In Ca(V)2.3, mutation of the distal glycine Gly(352) impacted significantly on the inactivation gating. Altogether, these data highlight the role of the GX(3)G motif in the voltage-dependent activation and inactivation gating of HVA Ca(V) channels with the distal glycine residue being mostly involved in the inactivation gating.     

A novel Ca(V)1.2 N terminus expressed in smooth muscle cells of resistance size arteries modifies channel regulation by auxiliary subunits

Voltage-dependent L-type Ca(2+) (Ca(V)1.2) channels are the principal Ca(2+) entry pathway in arterial myocytes. Ca(V)1.2 channels regulate multiple vascular functions and are implicated in the pathogenesis of human disease, including hypertension. However, the molecular identity of Ca(V)1.2 channels expressed in myocytes of myogenic arteries that regulate vascular pressure and blood flow is unknown. Here, we cloned Ca(V)1.2 subunits from resistance size cerebral arteries and demonstrate that myocytes contain a novel, cysteine rich N terminus that is derived from exon 1 (termed "exon 1c"), which is located within CACNA1C, the Ca(V)1.2 gene. Quantitative PCR revealed that exon 1c was predominant in arterial myocytes, but rare in cardiac myocytes, where exon 1a prevailed. When co-expressed with alpha(2)delta subunits, Ca(V)1.2 channels containing the novel exon 1c-derived N terminus exhibited: 1) smaller whole cell current density, 2) more negative voltages of half activation (V(1/2,act)) and half-inactivation (V(1/2,inact)), and 3) reduced plasma membrane insertion, when compared with channels containing exon 1b. beta(1b) and beta(2a) subunits caused negative shifts in the V(1/2,act) and V(1/2,inact) of exon 1b-containing Ca(V)1.2alpha(1)/alpha(2)delta currents that were larger than those in exon 1c-containing Ca(V)1.2alpha(1)/alpha(2)delta currents. In contrast, beta(3) similarly shifted V(1/2,act) and V(1/2,inact) of currents generated by exon 1b- and exon 1c-containing channels. beta subunits isoform-dependent differences in current inactivation rates were also detected between N-terminal variants. Data indicate that through novel alternative splicing at exon 1, the Ca(V)1.2 N terminus modifies regulation by auxiliary subunits. The novel exon 1c should generate distinct voltage-dependent Ca(2+) entry in arterial myocytes, resulting in tissue-specific Ca(2+) signaling.     

Isoform-specific effects of the beta2 subunit on voltage-gated sodium channel gating

Voltage-gated sodium channels (Nav) are complex glycoproteins comprised of an alpha subunit and often one to several beta subunits. We have shown that sialic acid residues linked to Nav alpha and beta1 subunits alter channel gating. To determine whether beta2-linked sialic acids similarly impact Nav gating, we co-expressed beta2 with Nav1.5 or Nav1.2 in Pro5 (complete sialylation) and in Lec2 (essentially no sialylation) cells. Beta2 sialic acids caused a significant hyperpolarizing shift in Nav1.5 voltage-dependent gating, thus describing for the first time an effect of beta2 on Nav1.5 gating. In contrast, beta2 caused a sialic acid-independent depolarizing shift in Nav1.2 gating. A deglycosylated mutant, beta(2-DeltaN), had no effect on Nav1.5 gating, indicating further the impact of beta2 N-linked sialic acids on Nav1.5 gating. Conversely, beta(2-DeltaN) modulated Nav1.2 gating virtually identically to beta2, confirming that beta2 N-linked sugars have no impact on Nav1.2 gating. Thus, beta2 modulates Nav gating through multiple mechanisms possibly determined by the associated alpha subunit. Beta1 and beta2 were expressed together with Nav1.5 or Nav1.2 in Pro5 and Lec2 cells. Together beta1 and beta2 produced a significantly larger sialic acid-dependent hyperpolarizing shift in Nav1.5 gating. Under fully sialylating conditions, the Nav1.2.beta1.beta2 complex behaved like Nav1.2 alone. When sialylation was reduced, only the sialic acid-independent depolarizing effects of beta2 on Nav1.2 gating were apparent. Thus, the varied effects of beta1 and beta2 on Nav1.5 and Nav1.2 gating are apparently synergistic and highlight the complex manner, through subunit- and sugar-dependent mechanisms, by which Nav activity is modulated.