Distinct RGK GTPases differentially use α1- and auxiliary β-binding-dependent mechanisms to inhibit CaV1.2/CaV2.2 channels

Ca(V)1/Ca(V)2 channels, comprised of pore-forming α(1) and auxiliary (β,α(2)δ) subunits, control diverse biological responses in excitable cells. Molecules blocking Ca(V)1/Ca(V)2 channel currents (I(Ca)) profoundly regulate physiology and have many therapeutic applications. Rad/Rem/Rem2/Gem GTPases (RGKs) strongly inhibit Ca(V)1/Ca(V)2 channels. Understanding how RGKs block I(Ca) is critical for insights into their physiological function, and may provide design principles for developing novel Ca(V)1/Ca(V)2 channel inhibitors. The RGK binding sites within Ca(V)1/Ca(V)2 channel complexes responsible for I(Ca) inhibition are ambiguous, and it is unclear whether there are mechanistic differences among distinct RGKs. All RGKs bind β subunits, but it is unknown if and how this interaction contributes to I(Ca) inhibition. We investigated the role of RGK/β interaction in Rem inhibition of recombinant Ca(V)1.2 channels, using a mutated β (β(2aTM)) selectively lacking RGK binding. Rem blocked β(2aTM)-reconstituted channels (74% inhibition) less potently than channels containing wild-type β(2a) (96% inhibition), suggesting the prevalence of both β-binding-dependent and independent modes of inhibition. Two mechanistic signatures of Rem inhibition of Ca(V)1.2 channels (decreased channel surface density and open probability), but not a third (reduced maximal gating charge), depended on Rem binding to β. We identified a novel Rem binding site in Ca(V)1.2 α(1C) N-terminus that mediated β-binding-independent inhibition. The Ca(V)2.2 α(1B) subunit lacks the Rem binding site in the N-terminus and displays a solely β-binding-dependent form of channel inhibition. Finally, we discovered an unexpected functional dichotomy amongst distinct RGKs- while Rem and Rad use both β-binding-dependent and independent mechanisms, Gem and Rem2 use only a β-binding-dependent method to inhibit Ca(V)1.2 channels. The results provide new mechanistic perspectives, and reveal unexpected variations in determinants, underlying inhibition of Ca(V)1.2/Ca(V)2.2 channels by distinct RGK GTPases.     

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.     

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.     

Rem GTPase interacts with the proximal CaV1.2 C-terminus and modulates calcium-dependent channel inactivation

The Rem, Rem2, Rad, and Gem/Kir (RGK) GTPases, comprise a subfamily of small Ras-related GTP-binding proteins, and have been shown to potently inhibit high voltage-activated Ca²⁺ channel current following overexpression. Although the molecular mechanisms underlying RGK-mediated Ca²⁺ channel regulation remains controversial, recent studies suggest that RGK proteins inhibit Ca²⁺ channel currents at the plasma membrane in part by interactions with accessory channel β subunits. In this paper, we extend our understanding of the molecular determinants required for RGK-mediated channel regulation by demonstrating a direct interaction between Rem and the proximal C-terminus of Ca_V1.2 (PCT), including the CB/IQ domain known to contribute to Ca²⁺/calmodulin (CaM)-mediated channel regulation. The Rem2 and Rad GTPases display similar patterns of PCT binding, suggesting that the Ca_V1.2 C-terminus represents a common binding partner for all RGK proteins. In vitro Rem:PCT binding is disrupted by Ca²⁺/CaM, and this effect is not due to Ca²⁺/CaM binding to the Rem C-terminus. In addition, co-overexpression of CaM partially relieves Rem-mediated L-type Ca²⁺ channel inhibition and slows the kinetics of Ca²⁺-dependent channel inactivation. Taken together, these results suggest that the association of Rem with the PCT represents a crucial molecular determinant in RGK-mediated Ca²⁺ channel regulation and that the physiological function of the RGK GTPases must be re-evaluated. Rather than serving as endogenous inhibitors of Ca²⁺ channel activity, these studies indicate that RGK proteins may play a more nuanced role, regulating Ca²⁺ currents via modulation of Ca²⁺/CaM-mediated channel inactivation kinetics.     

Rem-GTPase regulates cardiac myocyte L-type calcium current

Rationale: The L-type calcium channels (LTCC) are critical for maintaining Ca ( 2+) -homeostasis. In heterologous expression studies, the RGK-class of Ras-related G-proteins regulates LTCC function; however, the physiological relevance of RGK-LTCC interactions is untested. Objective: In this report we test the hypothesis that the RGK protein, Rem, modulates native Ca ( 2+) current (ICa,L) via LTCC in murine cardiomyocytes. Methods and Results: Rem knockout mice (Rem (-/-) ) were engineered, and ICa,L and Ca ( 2+) -handling properties were assessed. Rem (-/-) ventricular cardiomyocytes displayed increased ICa,L density. ICa,L activation was shifted positive on the voltage axis, and β-adrenergic stimulation normalized this shift compared with wild-type ICa,L. Current kinetics, steady-state inactivation, and facilitation was unaffected by Rem (-/-) . Cell shortening was not significantly different. Increased ICa,L density in the absence of frank phenotypic differences motivated us to explore putative compensatory mechanisms. Despite the larger ICa,L density, Rem (-/-) cardiomyocyte Ca ( 2+) twitch transient amplitude was significantly less than that compared with wild type. Computer simulations and immunoblot analysis suggests that relative dephosphorylation of Rem (-/-) LTCC can account for the paradoxical decrease of Ca ( 2+) transients. Conclusions: This is the first demonstration that loss of an RGK protein influences ICa,L in vivo in cardiac myocytes.     

Absence of the gamma subunit of the skeletal muscle dihydropyridine receptor increases L-type Ca2+ currents and alters channel inactivation properties

In skeletal muscle the oligomeric alpha(1S), alpha(2)/delta-1 or alpha(2)/delta-2, beta1, and gamma1 L-type Ca(2+) channel or dihydropyridine receptor functions as a voltage sensor for excitation contraction coupling and is responsible for the L-type Ca(2+) current. The gamma1 subunit, which is tightly associated with this Ca(2+) channel, is a membrane-spanning protein exclusively expressed in skeletal muscle. Previously, heterologous expression studies revealed that gamma1 might modulate Ca(2+) currents expressed by the pore subunit found in heart, alpha(1C), shifting steady state inactivation, and increasing current amplitude. To determine the role of gamma1 assembled with the skeletal subunit composition in vivo, we used gene targeting to establish a mouse model, in which gamma1 expression is eliminated. Comparing litter-matched mice with control mice, we found that, in contrast to heterologous expression studies, the loss of gamma1 significantly increased the amplitude of peak dihydropyridine-sensitive I(Ca) in isolated myotubes. Whereas the activation kinetics of the current remained unchanged, inactivation of the current was slowed in gamma1-deficient myotubes and, correspondingly, steady state inactivation of I(Ca) was shifted to more positive membrane potentials. These results indicate that gamma1 decreases the amount of Ca(2+) entry during stimulation of skeletal muscle.     

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.     

Plasma membrane targeting is essential for Rem-mediated Ca2+ channel inhibition

The small GTPase Rem is a potent negative regulator of high voltage-activated Ca(2+) channels and a known interacting partner for Ca(2+) channel accessory beta subunits. The mechanism for Rem-mediated channel inhibition remains controversial, although it has been proposed that Ca(V)beta association is required. Previous work has shown that a C-terminal truncation of Rem (Rem-(1-265)) displays reduced in vivo binding to membrane-localized beta 2a and lacks channel regulatory function. In this paper, we describe a role for the Rem C terminus in plasma membrane localization through association with phosphatidylinositol lipids. Moreover, Rem-(1-265) can associate with beta 2a in vitro and beta 1b in vivo, suggesting that the C terminus does not directly participate in Ca(V)beta association. Despite demonstrated beta 1b binding, Rem-(1-265) was not capable of regulating a Ca(V)1.2-beta 1b channel complex, indicating that beta subunit binding is not sufficient for channel regulation. However, fusion of the CAAX domain from K-Ras4B or H-Ras to the Rem-(1-265) C terminus restored membrane localization and Ca(2+) channel regulation, suggesting that beta binding and membrane localization are independent events required for channel inhibition.     

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.     

L-type calcium channel alpha-subunit and protein kinase inhibitors modulate Rem-mediated regulation of current

Cardiac voltage-gated L-type Ca channels (Ca(V)) are multiprotein complexes, including accessory subunits such as Ca(V)beta2 that increase current expression. Recently, members of the Rad and Gem/Kir-related family of small GTPases have been shown to decrease current, although the mechanism remains poorly defined. In this study, we evaluated the contribution of the L-type Ca channel alpha-subunit (Ca(V)1.2) to Ca(V)beta2-Rem inhibition of Ca channel current. Specifically, we addressed whether protein kinase A (PKA) modulation of the Ca channel modifies Ca(V)beta2-Rem inhibition of Ca channel current. We first tested the effect of Rem on Ca(V)1.2 in human embryonic kidney 293 (HEK-293) cells using the whole cell patch-clamp configuration. Rem coexpression with Ca(V)1.2 reduces Ba current expression under basal conditions, and Ca(V)beta2a coexpression enhances Rem block of Ca(V)1.2 current. Surprisingly, PKA inhibition by 133 nM H-89 or 50 microM Rp-cAMP-S partially relieved the Rem-mediated inhibition of current activity both with and without Ca(V)beta2a. To test whether the H-89 action was a consequence of the phosphorylation status of Ca(V)1.2, we examined Rem regulation of the PKA-insensitive Ca(V)1.2 serine 1928 (S1928) to alanine mutation (Ca(V)1.2-S1928A). Ca(V)1.2-S1928A current was not inhibited by Rem and when coexpression with Ca(V)beta2a was not completely blocked by Rem coexpression, suggesting that the phosphorylation of S1928 contributes to Rem-mediated Ca channel modulation. As a model for native Ca channel complexes, we tested the ability of Rem overexpression in HIT-T15 cells and embryonic ventricular myocytes to interfere with native current. We find that native current is also sensitive to Rem block and that H-89 pretreatment relieves the ability of Rem to regulate Ca current. We conclude that Rem is capable of regulating L-type current, that release of Rem block is modulated by cellular kinase pathways, and that the Ca(V)1.2 COOH terminus contributes to Rem-dependent channel inhibition.     

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.     

S-nitrosation controls gating and conductance of the alpha 1 subunit of class C L-type Ca(2+) channels

Modulation of smooth muscle, L-type Ca(2+) channels (class C, Ca(V)1.2b) by thionitrite S-nitrosoglutathione (GSNO) was investigated in the human embryonic kidney 293 expression system at the level of whole-cell and single-channel currents. Extracellular administration of GSNO (2 mM) rapidly reduced whole-cell Ba(2+) currents through channels derived either by expression of alpha1C-b or by coexpression of alpha1C-b plus beta2a and alpha2-delta. The non-thiol nitric oxide (NO) donors 2,2-diethyl-1-nitroso-oxhydrazin (2 mM) and 3-morpholinosydnonimine-hydrochloride (2 mM), which elevated cellular cGMP levels to a similar extent as GSNO, failed to affect Ba(2+) currents significantly. Intracellular administration of copper ions, which promote decomposition of the thionitrite, antagonized its inhibitory effect, and loading of cells with high concentrations of dithiothreitol (2 mM) prevented the effect of GSNO on alpha1C-b channels. Intracellular loading of cells with oxidized glutathione (2 mM) affected neither alpha1C-b channel function nor their modulation by GSNO. Analysis of single-channel behavior revealed that GSNO inhibited Ca(2+) channels mainly by reducing open probability. The development of GSNO-induced inhibition was associated with the transient occurrence of a reduced conductance state of the channel. Our results demonstrate that GSNO modulates the alpha1 subunit of smooth muscle L-type Ca(2+) channels by an intracellular mechanism that is independent of NO release and stimulation of guanylyl cyclase. We suggest S-nitrosation of intracellularly located sulfhydryl groups as an important determinant of Ca(2+) channel gating and conductance.     

Rem2-targeted shRNAs reduce frequency of miniature excitatory postsynaptic currents without altering voltage-gated Ca²⁺ currents

Ca²⁺ influx through voltage-gated Ca²⁺ channels (VGCCs) plays important roles in neuronal cell development and function. Rem2 is a member of the RGK (Rad, Rem, Rem2, Gem/Kir) subfamily of small GTPases that confers potent inhibition upon VGCCs. The physiologic roles of RGK proteins, particularly in the brain, are poorly understood. Rem2 was implicated in synaptogenesis through an RNAi screen and proposed to regulate Ca²⁺ homeostasis in neurons. To test this hypothesis and uncover physiological roles for Rem2 in the brain, we investigated the molecular mechanisms by which Rem2 knockdown affected synaptogenesis and Ca²⁺ homeostasis in cultured rat hippocampal neurons. Expression of a cocktail of shRNAs targeting rat Rem2 (rRem2) reduced the frequency of miniature excitatory postsynaptic currents (mEPSCs) measured 10 d after transfection (14 d in vitro), but did not affect mEPSC amplitude. VGCC current amplitude after rRem2-targeted knockdown was not different from that in control cells, however, at either 4 or 10 d post transfection. Co-expression of a human Rem2 that was insensitive to the shRNAs targeting rRem2 was unable to prevent the reduction in mEPSC frequency after rRem2-targeted knockdown. Over-expression of rRem2 resulted in 50% reduction in VGCC current, but neither the mEPSC frequency nor amplitude was affected. Taken together, the observed effects upon synaptogenesis after shRNA treatment are more likely due to mechanisms other than modulation of VGCCs and Ca²⁺ homeostasis, and may be independent of Rem2. In addition, our results reveal a surprising lack of contribution of VGCCs to synaptogenesis during early development in cultured hippocampal neurons.     

Biochemical and biophysical evidence for gamma 2 subunit association with neuronal voltage-activated Ca2+ channels.

A novel gene (Cacng2; gamma(2)) encoding a protein similar to the voltage-activated Ca(2+) channel gamma(1) subunit was identified as the defective gene in the epileptic and ataxic mouse, stargazer. In this study, we analyzed the association of this novel neuronal gamma(2) subunit with Ca(2+) channels of rabbit brain, and the function of the gamma(2) subunit in recombinant neuronal Ca(2+) channels expressed in Xenopus oocytes. Our results showed that the gamma(2) subunit and a closely related protein (called gamma(3)) co-sedimented and co-immunoprecipitated with neuronal Ca(2+) channel subunits in vivo. Electrophysiological analyses showed that gamma(2) co-expression caused a significant decrease in the current amplitude of both alpha(1B)(alpha(1)2.2)-class (36.8%) and alpha(1A)(alpha(1)2.1)-class (39.7%) Ca(2+) channels (alpha(1)beta(3)alpha(2)delta). Interestingly, the inhibitory effects of the gamma(2) subunit on current amplitude were dependent on the co-expression of the alpha(2)delta subunit. In addition, co-expression of gamma(2) or gamma(1) also significantly decelerates the activation kinetics of alpha(1B)-class Ca(2+) channels. Taken together, these results suggest that the gamma(2) subunit is an important constituent of the neuronal Ca(2+) channel complex and that it down-regulates neuronal Ca(2+) channel activity. Furthermore, the gamma(2) subunit likely contributes to the fine-tuning of neuronal Ca(2+) channels by counterbalancing the effects of the alpha(2)delta subunit.     

Nuclear sequestration of beta-subunits by Rad and Rem is controlled by 14-3-3 and calmodulin and reveals a novel mechanism for Ca2+ channel regulation

Voltage-gated Ca2+ channels (VDCCs) are heteromultimeric proteins that mediate Ca2+ influx into cells upon membrane depolarization. These channels are involved in various cellular events, including gene expression, regulation of hormone secretion and synaptic transmission. Kir/Gem, Rad, Rem, and Rem2 belong to the RGK family of Ras-related small G proteins. RGK proteins interact with the beta-subunits and downregulate VDCC activity. Kir/Gem was proposed to prevent surface expression of functional Ca2+ channels, while for Rem2 the mechanism remains controversial. Here, we have analyzed the mechanism by which Rad and Rem regulate VDCC activity. We show that, similar to Kir/Gem and Rem2, 14-3-3 and CaM binding regulate the subcellular distribution of Rad and Rem, which both inhibit Ca2+ channel activity by preventing its expression on the cell surface. This function is regulated by calmodulin and 14-3-3 binding only for Rad and not for Rem. Interestingly, nuclear targeting of Rad and Rem can relocalize and sequester the beta-subunit to the nucleus, thus providing a novel mechanism for Ca2+ channel downregulation.     

Modulation of the conductance of unitary cardiac L-type Ca(2+) channels by conditioning voltage and divalent ions

The accompanying paper (Josephson, I. R., A. Guia, E. G. Lakatta, and M. D. Stern. 2002. Biophys. J. 83:2575-2586) examined the effects of conditioning prepulses on the kinetics of unitary L-type Ca(2+) channel currents using Ca(2+) and Ba(2+) ions to determine the ionic-dependence of gating mechanisms responsible for channel inactivation and facilitation. Here we demonstrate that in addition to alterations in gating kinetics, the conductance of single L-type Ca(2+) channels was also dependent on the prior conditioning voltage and permeant ions. All recordings were made in the absence of any Ca(2+) channel agonists. Strongly depolarizing prepulses produced an increased frequency of long-duration (mode 2) openings during the test voltage steps. Mode 2 openings also displayed >25% larger single channel current amplitude (at 0 mV) than briefer (but well-resolved) mode 1 openings. The conductance of mode 2 openings was 26 pS for 105 mM Ba(2+), 18 pS for 5 mM Ba(2+), and 6 pS for 5 mM Ca(2+) ions; these values were 70% greater than the conductance of Ca(2+) channel openings of all durations (mode 1 and mode 2). Thus, the prepulse-driven shift into mode 2 gating results in a longer-lived Ca(2+) channel conformation that, in addition, displays altered permeation properties. These results, and those in the accompanying paper, support the hypothesis that multiple aspects of single L-type Ca(2+) channel behavior (gating kinetics, modal transitions, and ion permeation) are interrelated and are modulated by the magnitude of the conditioning depolarization and the nature and concentration of the ions permeating the channel.     

Ca2+ channel currents and contraction in CaVbeta3-deficient ileum smooth muscle from mouse

Voltage activated L-type Ca(2+) channels are the principal Ca(2+) channels in intestinal smooth muscle cells. They comprise the ion conducting Ca(V)1 pore and the ancillary subunits alpha(2)delta and beta. Of the four Ca(V)beta subunits Ca(V)beta(3) is assumed to be the relevant Ca(V)beta protein in smooth muscle. In protein lysates isolated from mouse ileum longitudinal smooth muscle we could identify the Ca(V)1.2, Ca(V)alpha(2), Ca(V)beta(2) and Ca(V)beta(3) proteins, but not the Ca(V)beta(1) and Ca(V)beta(4) proteins. Protein levels of Ca(V)1.2, Ca(V)alpha(2) and Ca(V)beta(2) are not altered in ileum smooth muscle obtained from Ca(V)beta(3)-deficient mice indicating that there is no compensatory increase of the expression of these channel proteins. Neither the Ca(V)beta(2) nor the other Ca(V)beta proteins appear to substitute for the lacking Ca(V)beta(3). L-type Ca(2+) channel properties including current density, inactivation kinetics as well as Cd(2+)- and dihydropyridine sensitivity were identical in cells of both genotypes suggesting that they do not require the presence of a Ca(V)beta(3) protein. However, a key hallmark of the Ca(V)beta modulation of Ca(2+) current, the hyperpolarisation of channel activation is slightly but significantly reduced by 4 mV. In addition to L-type Ca(2+) currents T-type Ca(2+) currents could be recorded in the murine ileum smooth muscle cells, but T-type currents were not affected by the lack of Ca(V)beta(3). Both proteins, Ca(V)beta(2) and Ca(V)beta(3) are localized near the plasma membrane and the localization of Ca(V)beta(2) is not altered in Ca(V)beta(3) deficient cells. Spontaneous contractions and potassium and carbachol induced contractions are not significantly different between ileum longitudinal smooth muscle strips from mice of both genotypes. In summary the data show that in ileum smooth muscle cells, Ca(V)beta(3) has only subtle effects on L-type Ca(2+) currents, appears not to be required for spontaneous and potassium induced contraction but might have a function beyond being a Ca(2+) channel subunit.     

Calcineurin Aalpha but not Abeta augments ICl(Ca) in rabbit pulmonary artery smooth muscle cells

Activation of Ca(2+)-dependent Cl(-) currents (I(Cl(Ca))) increases membrane excitability in vascular smooth muscle cells. Previous studies showed that Ca(2+)-dependent phosphorylation suppresses I(Cl(Ca)) in pulmonary artery myocytes, and the aim of the present study was to determine the role of the Ca(2+)-dependent phosphatase calcineurin on chloride channel activity. Immunocytochemical and Western blot studies with isoform-specific antibodies revealed that the alpha and beta forms of the CaN catalytic subunit are expressed in PA cells but that only the alpha variant translocated to the cell periphery upon a rise in intracellular [Ca(2+)]. I(Cl(Ca)) evoked by pipette solutions containing a [Ca(2+)] set at 500 nm was considerably larger when the pipette solution included constitutively active CaN containing the alpha catalytic isoform. This stimulatory effect was lost by boiling the enzyme or by the inclusion of a specific CaN inhibitory peptide and was not shared by the inclusion of the beta form of the catalytic subunit. In the absence of constitutively active CaN, cyclosporin A, an inhibitor of CaN, suppressed I(Cl(Ca)) evoked by 500 nm Ca(2+) when the current amplitude was relatively large but was ineffective in cells with smaller currents. In perforated patch recordings, cyclosporin A consistently inhibited I(Cl(Ca)) evoked as a consequence of Ca(2+) influx through voltage-dependent calcium channels. These novel data show that in PA myocytes activation of I(Cl(Ca)) is enhanced by Ca(2+)-dependent dephosphorylation and that the regulation of this conductance is highly isoform-specific.