Reversibility of the Ca(2+) channel alpha(1)-beta subunit interaction

The auxiliary beta subunit importantly regulates voltage-dependent Ca(2+) channel activity through an interaction with the AID domain, a binding site located in the cytoplasmic I-II linker of the ion-conducting alpha(1) subunit. In the present study, we used two synthetic peptides corresponding to partial sequences of the I-II linker of alpha(1A) (AID(A)-peptides) as tools to disrupt the alpha(1)-beta interaction. In vitro binding experiments confirmed that these peptides exhibit a reasonable affinity to the neuronal beta(3) subunit to serve this purpose, although they failed to prevent immunoprecipitation of native N- and P/Q-type channels by anti-beta(3) antibodies. Together, our results (i) provide evidence for the reversibility of channel subunit association suggesting that the disruption of the alpha(1)-beta interaction may be a possible mechanism for Ca(2+) channel regulation in vivo, and (ii) support a model whereby the alpha(1)-beta association is based on multiple interaction sites.     

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.     

Structural analysis of the voltage-dependent calcium channel beta subunit functional core and its complex with the alpha 1 interaction domain

Voltage-dependent calcium channels (VDCC) are multiprotein assemblies that regulate the entry of extracellular calcium into electrically excitable cells and serve as signal transduction centers. The alpha1 subunit forms the membrane pore while the intracellular beta subunit is responsible for trafficking of the channel to the plasma membrane and modulation of its electrophysiological properties. Crystallographic analyses of a beta subunit functional core alone and in complex with a alpha1 interaction domain (AID) peptide, the primary binding site of beta to the alpha1 subunit, reveal that beta represents a novel member of the MAGUK protein family. The findings illustrate how the guanylate kinase fold has been fashioned into a protein-protein interaction module by alteration of one of its substrate sites. Combined results indicate that the AID peptide undergoes a helical transition in binding to beta. We outline the mechanistic implications for understanding the beta subunit's broad regulatory role of the VDCC, particularly via the AID.     

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.     

Interacting effects of N-terminal variation and strex exon splicing on slo potassium channel regulation by calcium,phosphorylation, and oxidation

We have investigated the structural basis for the phenotype of a native rat Slo (rSlo) potassium channel (BK(Ca); KCNMA1) in a rat pituitary cell line, GH(4)C(1). Opposing regulation of these calcium- and voltage-activated potassium channels by cAMP- and cGMP-dependent protein kinases requires an alternatively spliced exon (strex) of 59 amino acids in the cytoplasmic C terminus of the pore-forming alpha subunit encoded by rslo. However, inclusion of this cysteine-rich exon produces a 10-fold increase in the sensitivity of the channels to inhibition by oxidation. Inclusion of the strex exon also increases channel sensitivity to stimulation by calcium, but responses in the physiological ranges of calcium and voltage require coassembly with beta(1) subunits. With strex present, however, beta(1) subunits only stimulated channels assembled from rSlo alpha subunits with a truncated N terminus beginning MDALI-. Thus N-terminal variation and strex exon splicing in rSlo interact to produce BK(Ca) channels with a physiologically relevant phenotype.     

Mutations of nonconserved residues within the calcium channel alpha1-interaction domain inhibit beta-subunit potentiation

Voltage-dependent calcium channels consist of a pore-forming subunit (Ca(V)alpha(1)) that includes all the molecular determinants of a voltage-gated channel, and several accessory subunits. The ancillary beta-subunit (Ca(V)beta) is a potent activator of voltage-dependent calcium channels, but the mechanisms and structural bases of this regulation remain elusive. Ca(V)beta binds reversibly to a conserved consensus sequence in Ca(V)alpha(1), the alpha(1)-interaction domain (AID), which forms an alpha-helix when complexed with Ca(V)beta. Conserved aromatic residues face to one side of the helix and strongly interact with a hydrophobic pocket on Ca(V)beta. Here, we studied the effect of mutating residues located opposite to the AID-Ca(V)beta contact surface in Ca(V)1.2. Substitution of AID-exposed residues by the corresponding amino acids present in other Ca(V)alpha(1) subunits (E462R, K465N, D469S, and Q473K) hinders Ca(V)beta's ability to increase ionic-current to charge-movement ratio (I/Q) without changing the apparent affinity for Ca(V)beta. At the single channel level, these Ca(V)1.2 mutants coexpressed with Ca(V)beta(2a) visit high open probability mode less frequently than wild-type channels. On the other hand, Ca(V)1.2 carrying either a mutation in the conserved tryptophan residue (W470S, which impairs Ca(V)beta binding), or a deletion of the whole AID sequence, does not exhibit Ca(V)beta-induced increase in I/Q. In addition, we observed a shift in the voltage dependence of activation by +12 mV in the AID-deleted channel in the absence of Ca(V)beta, suggesting a direct participation of these residues in the modulation of channel activation. Our results show that Ca(V)beta-dependent potentiation arises primarily from changes in the modal gating behavior. We envision that Ca(V)beta spatially reorients AID residues that influence the channel gate. These findings provide a new framework for understanding modulation of VDCC gating by Ca(V)beta.     

Binding of G alpha(o) N terminus is responsible for the voltage-resistant inhibition of alpha(1A) (P/Q-type, Ca(v)2.1) Ca(2+) channels

G-protein-mediated inhibition of presynaptic voltage-dependent Ca(2+) channels is comprised of voltage-dependent and -resistant components. The former is caused by a direct interaction of Ca(2+) channel alpha(1) subunits with G beta gamma, whereas the latter has not been characterized well. Here, we show that the N terminus of G alpha(o) is critical for the interaction with the C terminus of the alpha(1A) channel subunit, and that the binding induces the voltage-resistant inhibition. An alpha(1A) C-terminal peptide, an antiserum raised against G alpha(o) N terminus, and a G alpha(o) N-terminal peptide all attenuated the voltage-resistant inhibition of alpha(1A) currents. Furthermore, the N terminus of G alpha(o) bound to the C terminus of alpha(1A) in vitro, which was prevented either by the alpha(1A) channel C-terminal or G alpha(o) N-terminal peptide. Although the C-terminal domain of the alpha(1B) channel showed similar ability in the binding with G alpha(o) N terminus, the above mentioned treatments were ineffective in the alpha(1B) channel current. These findings demonstrate that the voltage-resistant inhibition of the P/Q-type, alpha(1A) channel is caused by the interaction between the C-terminal domain of Ca(2+) channel alpha(1A) subunit and the N-terminal region of G alpha(o).     

The neuronal beta 4 subunit increases the unitary conductance of L-type voltage-gated calcium channels in PC12 cells

beta subunits of voltage-gated calcium channels influence channel behavior in numerous ways, including enhancing the targeting of alpha1 subunits to the plasma membrane and shifting the voltage dependence of activation and inactivation. Of the four beta subunits that have been identified, beta 4 is of particular interest because mutation of its alpha1 subunit interaction domain produces severe neurological defects. Its differential distribution in the hippocampus prompted us to examine whether this subunit was responsible for the heterogeneity of hippocampal L-type calcium channels. To study the functional effects of the beta 4 subunit on native L-type calcium channels, we transfected beta 4 cDNA subcloned out of embryonic hippocampal neurons into PC12 cells, a cell line that contains the beta 1, beta 2, and beta 3 subunits but not the beta 4 subunit. Cell-attached single-channel recordings of L-type channel activity from untransfected and transfected PC12 cells compared with recordings obtained from hippocampal neurons revealed an effect of the beta 4 subunit on single-channel conductance. L-type channels in untransfected PC12 cells had a significantly smaller conductance (19.8 picosiemens (pS)) than L-type channels in hippocampal neurons (22 pS). After transfection of beta 4, however, L-type single-channel conductance was indistinguishable between the two cell types. Our data suggest that calcium channel beta 4 subunits affect the conductance of L-type calcium channels and that native hippocampal L-type channels contain the beta 4 subunit.     

The voltage-dependent calcium channel beta subunit contains two stable interacting domains

Voltage-dependent calcium channels selectively enable Ca2+ ion movement through cellular membranes. These multiprotein complexes are involved in a wide spectrum of biological processes such as signal transduction and cellular homeostasis. alpha1 is the membrane pore-forming subunit, whereas beta is an intracellular subunit that binds to alpha1, facilitating and modulating channel function. We have expressed, purified, and characterized recombinant beta3 and beta2a using both biochemical and biophysical methods, including electrophysiology, to better understand the beta family's protein structural and functional correlates. Our results indicate that the beta protein is composed of two distinct domains that associate with one another in a stable manner. The data also suggest that the polypeptide regions outside these domains are not structured when beta is not in complex with the channel. In addition, the beta structural core, comprised of just these two domains without other sequences, binds tightly to the alpha interaction domain (AID) motif, a sequence derived from the alpha1 subunit and the principal anchor site of beta. Domain II is responsible for this binding, but domain I enhances it.     

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 Rem2 - voltage dependant calcium channel beta subunit interaction and Rem2 interaction with phosphorylated phosphatidylinositide lipids

Voltage dependant calcium channels (VDCC) play a critical role in coupling electrical excitability to important physiological events such as secretion by neuronal and endocrine cells. Rem2, a GTPase restricted to neuroendocrine cell types, regulates VDCC activity by a mechanism that involves interaction with the VDCC beta subunit (Ca(V)beta). Mapping studies reveal that Rem2 binds to the guanylate kinase domain (GK) of the Ca(V)beta subunit that also contains the high affinity binding site for the pore forming and voltage sensing VDCC alpha subunit (Ca(V)alpha) interaction domain (AID). Moreover, fine mapping indicates that Rem2 binds to the GK domain in a region distinct from the AID interaction site, and competitive inhibition studies reveal that Rem2 does not disrupt Ca(V)alpha - Ca(V)beta binding. Instead, the Ca(V)beta subunit appears to serve a scaffolding function, simultaneously binding both Rem2 and AID. Previous studies have found that in addition to Ca(V)beta binding, Rem2 must be localized to the plasma membrane to inhibit VDCC function. Plasma membrane localization requires the C-terminus of Rem2 and binding studies indicate that this domain directs phosphorylated phosphatidylinositide (PIP) lipids association. Plasma membrane localization may provide a unique point of regulation since the ability of Rem2 to bind PIP lipids is inhibited by the phosphoserine dependant binding of 14-3-3 proteins. Thus, in addition to Ca(V)beta binding, VDCC blockade by Rem2 is likely to be controlled by both the localized concentration of membrane PIP lipids and direct 14-3-3 binding to the Rem2 C-terminus.     

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.     

Solution structure of the N-terminal A domain of the human voltage-gated Ca2+channel beta4a subunit

Ca2+ channel beta subunits regulate trafficking and gating (opening and closing) of voltage-dependent Ca2+ channel alpha1 subunits. Based on primary sequence comparisons, they are thought to be modular structures composed of five domains (A-E) that are related to the large family of membrane associated guanylate-kinase (MAGUK) proteins. The crystal structures of the beta subunit core, B-D, domains have recently been reported; however, very little is known about the structures of the A and E domains. The N-terminal A domain is a hypervariable region that differs among the four subtypes of Ca2+ channel beta subunits (beta1-beta4). Furthermore, this domain undergoes alternative splicing to create multiple N-terminal structures within a given gene class that have distinct effects on gating. We have solved the solution structure of the A domain of the human beta4a subunit, a splice variant that we have shown previously to have alpha1 subunit subtype-specific effects on Ca2+ channel trafficking and gating.     

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.     

The NH(2) terminus of the epithelial sodium channel contains an endocytic motif.

An epithelial sodium channel (ENaC) is composed of three homologous subunits: alpha, beta, and gamma. To elucidate the function of the cytoplasmic, NH(2) terminus of rat ENaC (rENaC) subunits, a series of mutant cDNAs was constructed and the cRNAs for all three subunits were expressed in Xenopus oocytes. Amiloride-sensitive Na(+) currents (I(Na)) were measured by the two-electrode voltage clamp technique. Deletion of the cytoplasmic, NH(2) terminus of alpha (Delta2-109), beta (Delta2-49), or gamma-rENaC (Delta2-53) dramatically reduced I(Na). A series of progressive, NH(2)-terminal deletions of alpha-rENaC were constructed to identify motifs that regulate I(Na). Deletion of amino acids 2-46 had no effect on I(Na): however, deletion of amino acids 2-51, 2-55, 2-58, and 2-67 increased I(Na) by approximately 4-fold. By contrast, deletion of amino acids 2-79, 2-89, 2-100, and 2-109 eliminated I(Na). To evaluate the mechanism whereby Delta2-67-alpha-rENaC increased I(Na), single channels were evaluated by patch clamp. The single-channel conductance and open probability of alpha,beta,gamma-rENaC and Delta2-67-alpha,beta,gamma-rENaC were similar. However, the number of active channels in the membrane increased from 6 +/- 1 channels per patch with alpha,beta,gamma-rENaC to 11 +/- 1 channels per patch with Delta2-67-alpha,beta,gamma-rENaC. Laser scanning confocal microscopy confirmed that there were more Delta2-67-alpha,beta, gamma-rENaC channels in the plasma membrane than alpha,beta, gamma-rENaC channels. Deletion of amino acids 2-67 in alpha-rENaC reduced the endocytic retrieval of channels from the plasma membrane and increased the half-life of the channel in the membrane from 1.1 +/- 0.2 to 3.5 +/- 1.1 h. We conclude that the cytoplasmic, NH(2) terminus of alpha-, beta-, and gamma-rENaC is required for channel activity. The cytoplasmic, NH(2) terminus of alpha-rENaC contains two key motifs. One motif regulates the endocytic retrieval of the channel from the plasma membrane. The second motif is required for channel activity.     

Voltage-dependent facilitation of a neuronal alpha 1C L-type calcium channel.

Calcium entry into excitable cells through voltage-gated calcium channels can be influenced by both the rate and pattern of action potentials. We report here that a cloned neuronal alpha 1C L-type calcium channel can be facilitated by positive pre-depolarization. Both calcium and barium were effective as charge carriers in eliciting voltage-dependent facilitation. The induction of facilitation was shown to be independent of intracellular calcium levels, G-protein interaction and the level of phosphatase activity. Facilitation was reduced by the injection of inhibitors of protein kinase A and required the coexpression of a calcium channel beta subunit. In contrast, three neuronal non-L-type calcium channels, alpha 1A, alpha 1B and alpha 1E, were not subject to voltage-dependent facilitation when coexpressed with a beta subunit. The results indicate that the mechanism of neuronal L-type calcium channel facilitation involves the interaction of alpha 1 and beta subunits and is dependent on protein kinase A activity. The selective voltage-dependent modulation of L-type calcium channels is likely to play an important role in neuronal physiology and plasticity.     

Calcium channel beta subunit promotes voltage-dependent modulation of alpha 1 B by G beta gamma

Voltage-dependent calcium channels (VDCCs) are heteromultimers composed of a pore-forming alpha1 subunit and auxiliary subunits, including the intracellular beta subunit, which has a strong influence on the channel properties. Voltage-dependent inhibitory modulation of neuronal VDCCs occurs primarily by activation of G-proteins and elevation of the free G beta gamma dimer concentration. Here we have examined the interaction between the regulation of N-type (alpha 1 B) channels by their beta subunits and by G beta gamma dimers, heterologously expressed in COS-7 cells. In contrast to previous studies suggesting antagonism of G protein inhibition by the VDCC beta subunit, we found a significantly larger G beta gamma-dependent inhibition of alpha 1 B channel activation when the VDCC alpha 1 B and beta subunits were coexpressed. In the absence of coexpressed VDCC beta subunit, the G beta gamma dimers, either expressed tonically or elevated via receptor activation, did not produce the expected features of voltage-dependent G protein modulation of N-type channels, including slowed activation and prepulse facilitation, while VDCC beta subunit coexpression restored all of the hallmarks of G beta gamma modulation. These results suggest that the VDCC beta subunit must be present for G beta gamma to induce voltage-dependent modulation of N-type calcium channels.     

A mutation in the beta interaction domain of the Ca(2+) channel alpha(1C) subunit reduces the affinity of the (+)-[(3)H]isradipine binding site

The molecular mechanisms of how alpha(1) and beta subunits of voltage-gated Ca(2+) channels interact with one another are still controversial. Here we show that despite a mutation in the beta interaction domain that has previously been shown to disrupt binding, alpha(1C)Y467S and beta(1a-myc) still formed immunoprecipitable complexes when coexpressed in tsA201 cells. However, the alpha(1C)Y467S-beta(1a-myc) complexes had a decreased affinity to (+)-[(3)H]isradipine. This indicates that the beta interaction domain in the I-II loop of the alpha(1) subunit is not merely an anchor required for the functional interaction of the two Ca(2+) channel subunits but is itself part of the effector pathway for beta-induced channel modulation.     

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.     

Gamma 1 subunit interactions within the skeletal muscle L-type voltage-gated calcium channels

Voltage-gated calcium channels mediate excitationcontraction coupling in the skeletal muscle. Their molecular composition, similar to neuronal channels, includes the pore-forming alpha(1) and auxiliary alpha(2)delta, beta, and gamma subunits. The gamma subunits are the least characterized, and their subunit interactions are unclear. The physiological importance of the neuronal gamma is emphasized by epileptic stargazer mice that lack gamma(2). In this study, we examined the molecular basis of interaction between skeletal gamma(1) and the calcium channel. Our data show that the alpha(1)1.1, beta(1a), and alpha(2)delta subunits are still associated in gamma(1) null mice. Reexpression of gamma(1) and gamma(2) showed that gamma(1), but not gamma(2), incorporates into gamma(1) null channels. By using chimeric constructs, we demonstrate that the first half of the gamma(1) subunit, including the first two transmembrane domains, is important for subunit interaction. Interestingly, this chimera also restores calcium conductance in gamma(1) null myotubes, indicating that the domain mediates both subunit interaction and current modulation. To determine the subunit of the channel that interacts with gamma(1), we examined the channel in muscular dysgenesis mice. Cosedimentation experiments showed that gamma(1) and alpha(2)delta are not associated. Moreover, alpha(1)1.1 and gamma(1) subunits form a complex in transiently transfected cells, indicating direct interaction between the gamma(1) and alpha(1)1.1 subunits. Our data demonstrate that the first half of gamma(1) subunit is required for association with the channel through alpha(1)1.1. Because subunit interactions are conserved, these studies have broad implications for gamma heterogeneity, function and subunit association with voltage-gated calcium channels.