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.     

alpha(1C) (Ca(V)1.2) L-type calcium channel mediates mechanosensitive calcium regulation

Smooth muscle exhibitsmechanosensitivity independent of neural input, suggesting thatmechanosensitive pathways reside within smooth muscle cells. The nativeL-type calcium current recorded from human intestinal smooth muscle ismodulated by stretch. To define mechanosensitive mechanisms involved inthe regulation of smooth muscle calcium entry, we cloned the_1C L-type calcium channel subunit (Ca_V1.2)from human intestinal smooth muscle and expressed the channel in aheterologous system. This channel subunit retained mechanosensitivitywhen expressed alone or coexpressed with a ₂ calciumchannel subunit in HEK-293 or Chinese hamster ovary cells. Theheterologously expressed human cardiac _1C splice formalso demonstrated mechanosensitivity. Inhibition of kinase signalingdid not affect mechanosensitivity of the native channel. Truncation of the _1C COOH terminus, which containsan inhibitory domain and a proline-rich domain thought to mediatemechanosensitive signaling from integrins, did not disruptmechanosensitivity of the expressed channel. These data demonstratemechanical regulation of calcium entry through molecularly identifiedL-type calcium channels in mammalian cells and suggest that themechanosensitivity resides within the pore forming_1C-subunit.

     

AKAP79 modulation of L-type channels involves disruption of intramolecular interactions in the CaV1.2 subunit

L-type voltage gated calcium channels (VGCCs) interact with a variety of proteins that modulate both their function and localization. A-Kinase Anchoring Proteins (AKAPs) facilitate L-type calcium channel phosphorylation through β adrenergic stimulation. Our previous work indicated a role of neuronal AKAP79/150 in the membrane targeting of Ca(V)1.2 L-type calcium channels, which involved a proline rich domain (PRD) in the intracellular II-III loop of the channel.(1) Here, we show that mutation of proline 857 to alanine (P857A) into the PRD does not disrupt the AKAP79-induced increase in Ca(v)1.2 membrane expression. Furthermore, deletion of two other PRDs into the carboxy terminal domain of Ca(V)1.2 did not alter the targeting role of AKAP79. In contrast, the distal carboxy terminus region of the channel directly interacts with AKAP79. This protein-protein interaction competes with a direct association of the channel II-III linker on the carboxy terminal tail and modulates membrane targeting of Ca(V)1.2. Thus, our results suggest that the effects of AKAP79 occur through relief of an autoinhibitory mechanism mediated by intramolecular interactions of Ca(v)1.2 intracellular regions.     

AKAP79 modulation of L-type channels involves disruption of intramolecular interactions in the CaV1.2 subunit

L-type voltage gated calcium channels (VGCCs) interact with a variety of proteins that modulate both their function and localization. A-Kinase Anchoring Proteins (AKAPs) facilitate L-type calcium channel phosphorylation through β adrenergic stimulation. Our previous work indicated a role of neuronal AKAP79/150 in the membrane targeting of Ca_V1.2 L-type calcium channels, which involved a proline rich domain (PRD) in the intracellular II-III loop of the channel.¹ Altier C, Dubel SJ, Barrère C, Jarvis SE, Stotz SC, Spaetgens RL, et al. Trafficking of L-type calcium channels mediated by the postsynaptic scaffolding protein AKAP79. J Biol Chem 2002; 277:33598 - 603; http://dx.doi.org/10.1074/jbc.M202476200; PMID: 12114507 [Crossref], [PubMed], [Web of Science ®] , [Google Scholar] Here, we show that mutation of proline 857 to alanine (P857A) into the PRD does not disrupt the AKAP79-induced increase in Cav1.2 membrane expression. Furthermore, deletion of two other PRDs into the carboxy terminal domain of Ca_V1.2 did not alter the targeting role of AKAP79. In contrast, the distal carboxy terminus region of the channel directly interacts with AKAP79. This protein-protein interaction competes with a direct association of the channel II-III linker on the carboxy terminal tail and modulates membrane targeting of Ca_V1.2. Thus, our results suggest that the effects of AKAP79 occur through relief of an autoinhibitory mechanism mediated by intramolecular interactions of Cav1.2 intracellular regions.     

Swapping the I-II intracellular linker between L-type CaV1.2 and R-type CaV2.3 high-voltage gated calcium channels exchanges activation attributes

Calcium entry through voltage-gated calcium channels (VGCC) initiates diverse cellular functions. VGCC pore-forming subunit (Ca_Vα1) contains four homology repeats, each encompassing a voltage sensor and a pore domain. Three main classes of Ca_Vα1 subunits have been described, Ca_V1, Ca_V2 and Ca_V3 that differ in their voltage-dependence of activation and in the extent in which this process is modulated by the auxiliary β-subunit (Ca_Vβ). Association of Ca_Vβ induces a coil-to-helix conformation of the I-II intracellular linker joining the first and second repeat of CaVα1 that is thought to be crucial for modulation of channel function. When expressed in Xenopus laevis oocytes in the absence of Ca_Vβ the voltage to reach 50% activation (V_0.5) for Ca_V1.2 and Ca_V2.3 differs by more than 60 mV and the channel current-carrying capacity by more than thirty-fold. Here we report that the difference in V_0.5 is reduced to about 30 mV and the current-carrying capacity becomes virtually identical when the I-II linkers of Ca_V1.2 and Ca_V2.3 are swapped. Co-expression with Ca_Vβ increases the current-carrying capacity of chimeric channels by the same extent, while the difference in V_0.5 with respect to their corresponding parental channels vanishes. Our findings indicate that Ca_Vβ modulatory potency is determined by both, the nature of the I-II linker and the pore-forming subunit background. Moreover, they demonstrate that the I-II linker encodes self-reliant molecular determinants for channel activation and suggest that besides to the secondary structure adopted by this segment upon Ca_Vβ association, its chemical nature is as well relevant.     

Proteolytic processing of the C terminus of the alpha(1C) subunit of L-type calcium channels and the role of a proline-rich domain in membrane tethering of proteolytic fragments

Although most L-type calcium channel alpha(1C) subunits isolated from heart or brain are approximately 190-kDa proteins that lack approximately 50 kDa of the C terminus, the C-terminal domain is present in intact cells. To test the hypothesis that the C terminus is processed but remains functionally associated with the channels, expressed, full-length alpha(1C) subunits were cleaved in vitro by chymotrypsin to generate a 190-kDa C-terminal truncated protein and C-terminal fragments of 30-56 kDa. These hydrophilic C-terminal fragments remained membrane-associated. A C-terminal proline-rich domain (PRD) was identified as the mediator of membrane association. The alpha(1C) PRD bound to SH3 domains in Src, Lyn, Hck, and the channel beta(2) subunit. Mutant alpha(1C) subunits lacking either approximately 50 kDa of the C terminus or the PRD produced increased barium currents through the channels, demonstrating that these domains participate in the previously described (Wei, X., Neely, a., Lacerda, A. E. Olcese, r., Stefani, E., Perez-Reyes, E., and Birnbaumer, L. (1994) J. Biol. Chem. 269, 1635-1640) inhibition of channel function by the C terminus.     

Expression of the alpha(2)delta subunit interferes with prepulse facilitation in cardiac L-type calcium channels

We investigated the role of the accessory alpha(2)delta subunit on the voltage-dependent facilitation of cardiac L-type Ca(2+) channels (alpha(1C)). alpha(1C) Channels were coexpressed in Xenopus oocytes with beta(3) and alpha(2)delta calcium channel subunits. In alpha(1C) + beta(3), the amplitude of the ionic current (measured during pulses to 10 mV) was in average approximately 1.9-fold larger after the application of a 200-ms prepulse to +80 mV. This phenomenon, commonly referred to as voltage-dependent facilitation, was not observed when alpha(2)delta was coexpressed with alpha(1C) + beta(3). In alpha(1C) + beta(3), the prepulse produced a left shift ( approximately 40 mV) of the activation curve. Instead, the activation curve for alpha(1C) + beta(3) + alpha(2)delta was minimally affected by the prepulse and had a voltage dependence very similar to the G-V curve of the alpha(1C) + beta(3) channel facilitated by the prepulse. Coexpression of alpha(2)delta with alpha(1C) + beta(3) seems to mimic the prepulse effect by shifting the activation curve toward more negative potentials, leaving little room for facilitation. The facilitation of alpha(1C) + beta(3) was associated with an increase of the charge movement. In the presence of alpha(2)delta, the charge remained unaffected after the prepulse. Coexpression of alpha(2)delta seems to set all the channels in a conformational state from where the open state can be easily reached, even without prepulse.     

Protein kinase C enhances plasma membrane expression of cardiac L-type calcium channel,CaV1.2

L-type-voltage-dependent Ca²⁺ channels (L-VDCCs; Ca_V1.2, α_1C), crucial in cardiovascular physiology and pathology, are modulated via activation of G-protein-coupled receptors and subsequently protein kinase C (PKC). Despite extensive study, key aspects of the mechanisms leading to PKC-induced Ca²⁺ current increase are unresolved. A notable residue, Ser1928, located in the distal C-terminus (dCT) of α_1C was shown to be phosphorylated by PKC. Ca_V1.2 undergoes posttranslational modifications yielding full-length and proteolytically cleaved CT-truncated forms. We have previously shown that, in Xenopus oocytes, activation of PKC enhances α_1C macroscopic currents. This increase depended on the isoform of α_1C expressed. Only isoforms containing the cardiac, long N-terminus (L-NT), were upregulated by PKC. Ser1928 was also crucial for the full effect of PKC. Here we report that, in Xenopus oocytes, following PKC activation the amount of α_1C protein expressed in the plasma membrane (PM) increases within minutes. The increase in PM content is greater with full-length α_1C than in dCT-truncated α_1C, and requires Ser1928. The same was observed in HL-1 cells, a mouse atrium cell line natively expressing cardiac α_1C, which undergoes the proteolytic cleavage of the dCT, thus providing a native setting for exploring the effects of PKC in cardiomyocytes. Interestingly, activation of PKC preferentially increased the PM levels of full-length, L-NT α_1C. Our findings suggest that part of PKC regulation of Ca_V1.2 in the heart involves changes in channel's cellular fate. The mechanism of this PKC regulation appears to involve the C-terminus of α_1C, possibly corroborating the previously proposed role of NT-CT interactions within α_1C.     

CaBP1 regulates voltage-dependent inactivation and activation of Ca(V)1.2 (L-type) calcium channels

CaBP1 is a Ca(2+)-binding protein that regulates the gating of voltage-gated (Ca(V)) Ca(2+) channels. In the Ca(V)1.2 channel α(1)-subunit (α(1C)), CaBP1 interacts with cytosolic N- and C-terminal domains and blunts Ca(2+)-dependent inactivation. To clarify the role of the α(1C) N-terminal domain in CaBP1 regulation, we compared the effects of CaBP1 on two alternatively spliced variants of α(1C) containing a long or short N-terminal domain. In both isoforms, CaBP1 inhibited Ca(2+)-dependent inactivation but also caused a depolarizing shift in voltage-dependent activation and enhanced voltage-dependent inactivation (VDI). In binding assays, CaBP1 interacted with the distal third of the N-terminal domain in a Ca(2+)-independent manner. This segment is distinct from the previously identified calmodulin-binding site in the N terminus. However, deletion of a segment in the proximal N-terminal domain of both α(1C) isoforms, which spared the CaBP1-binding site, inhibited the effect of CaBP1 on VDI. This result suggests a modular organization of the α(1C) N-terminal domain, with separate determinants for CaBP1 binding and transduction of the effect on VDI. Our findings expand the diversity and mechanisms of Ca(V) channel regulation by CaBP1 and define a novel modulatory function for the initial segment of the N terminus of α(1C).     

Localization of the calcium channel subunits Cav1.2 (alpha1C) and Cav2.3 (alpha1E) in the mouse organ of Corti

Voltage-activated Ca2+ channels play an important role in synaptic transmission, signal processing and development. The immunohistochemical localization of Cav1.2 (alpha1C) and Cav2.3 (alpha1E) Ca2+ channels was studied in the developing and adult mouse organ of Corti using subunit-specific antibodies and fluorescent secondary antibodies with cochlear cryosections. Cav1.2 immunoreactivity has been detected from postnatal day 14 (P14) onwards at the synapses between cholinergic medial efferents and outer hair cells as revealed by co-staining with anti-synaptophysin and anti-choline acetyltransferase. Most likely the Cav1.2 immunoreactivity was located presynaptically at the site of contact of the efferent bouton with the outer hair cell which suggests a role for class C L-type Ca2+ channels in synaptic transmission of the medial efferent system. The localization of the second Ca2+ channel tested, Cav2.3, showed a pronounced change during cochlear development. From P2 until P10, Cav2.3 immunoreactivity was found in the outer spiral bundle followed by the inner spiral bundle, efferent endings and by medial efferent fibers. Around P14, Cav2.3 immunoreactivity disappeared from these structures and from P19 onwards it was observed in the basal poles of the outer hair cell membranes.     

Ni2+ block of CaV3.1 (alpha1G) T-type calcium channels

Ni(2+) inhibits current through calcium channels, in part by blocking the pore, but Ni(2+) may also allosterically affect channel activity via sites outside the permeation pathway. As a test for pore blockade, we examined whether the effect of Ni(2+) on Ca(V)3.1 is affected by permeant ions. We find two components to block by Ni(2+), a rapid block with little voltage dependence, and a slow block most visible as accelerated tail currents. Rapid block is weaker for outward vs. inward currents (apparent K(d) = 3 vs. 1 mM Ni(2+), with 2 mM Ca(2+) or Ba(2+)) and is reduced at high permeant ion concentration (110 vs. 2 mM Ca(2+) or Ba(2+)). Slow block depends both on the concentration and on the identity of the permeant ion (Ca(2+) vs. Ba(2+) vs. Na(+)). Slow block is 2-3x faster in Ba(2+) than in Ca(2+) (2 or 110 mM), and is approximately 10x faster with 2 vs. 110 mM Ca(2+) or Ba(2+). Slow block is orders of magnitude slower than the diffusion limit, except in the nominal absence of divalent cations ( approximately 3 muM Ca(2+)). We conclude that both fast and slow block of Ca(V)3.1 by Ni(2+) are most consistent with occlusion of the pore. The exit rate of Ni(2+) for slow block is reduced at high Ni(2+) concentrations, suggesting that the site responsible for fast block can "lock in" slow block by Ni(2+), at a site located deeper within the pore. In contrast to the complex pore block observed for Ca(V)3.1, inhibition of Ca(V)3.2 by Ni(2+) was essentially independent of voltage, and was similar in 2 mM Ca(2+) vs. Ba(2+), consistent with inhibition by a different mechanism, at a site outside the pore.     

Functional embryonic cardiomyocytes after disruption of the L-type alpha1C (Cav1.2) calcium channel gene in the mouse

The L-type alpha(1C) (Ca(v)1.2) calcium channel is the major calcium entry pathway in cardiac and smooth muscle. We inactivated the Ca(v)1.2 gene in two independent mouse lines that had indistinguishable phenotypes. Homozygous knockout embryos (Ca(v)1. 2-/-) died before day 14.5 postcoitum (p.c.). At day 12.5 p.c., the embryonic heart contracted with identical frequency in wild type (+/+), heterozygous (+/-), and homozygous (-/-) Ca(v)1.2 embryos. Beating of isolated embryonic cardiomyocytes depended on extracellular calcium and was blocked by 1 microm nisoldipine. In (+/+), (+/-), and (-/-) cardiomyocytes, an L-type Ba(2+) inward current (I(Ba)) was present that was stimulated by Bay K 8644 in all genotypes. At a holding potential of -80 mV, nisoldipine blocked I(Ba) of day 12.5 p.c. (+/+) and (+/-) cells with two IC(50) values of approximately 0.1 and approximately 1 microm. Inhibition of I(Ba) of (-/-) cardiomyocytes was monophasic with an IC(50) of approximately 1 microm. The low affinity I(Ba) was also present in cardiomyocytes of homozygous alpha(1D) (Ca(v)1.3) knockout embryos at day 12.5 p.c. These results indicate that, up to day 14 p.c., contraction of murine embryonic hearts requires an unidentified, low affinity L-type like calcium channel.     

Phosphorylation sites on calcium channel alpha1 and beta subunits regulate ERK-dependent modulation of neuronal N-type calcium channels

Voltage-dependent calcium channels (VDCCs) in sensory neurones are tonically up-regulated via Ras/extracellular signal regulated kinase (ERK) signalling. The presence of putative ERK consensus sites within the intracellular loop linking domains I and II of neuronal N-type (Ca(v)2.2) calcium channels and all four neuronal calcium channel beta subunits (Ca(v)beta), suggests that Ca(v)2.2 and/or Ca(v)betas may be ERK-phosphorylated. Here we report that GST-Ca(v)2.2 I-II loop, and to a lesser extent Ca(v)beta1b-His(6), are substrates for ERK1/2 phosphorylation. Serine to alanine mutation of Ser-409 and/or Ser-447 on GST-Ca(v)2.2 I-II loop significantly reduced phosphorylation. Loss of Ser-447 reduced phosphorylation to a greater extent than mutation of Ser-409. Patch-clamp recordings from wild-type Ca(v)2.2,beta1b,alpha2delta1 versus mutant Ca(v)2.2(S447A) or Ca(v)2.2(S409A) channels revealed that mutation of either site significantly reduced current inhibition by UO126, a MEK (ERK kinase)-specific inhibitor that down-regulates ERK activity. However, no additive effect was observed by mutating both residues together, suggesting some functional redundancy between these sites. Mutation of both Ser-161 and Ser-348 on Ca(v)beta1b did not significantly reduce phosphorylation but did reduce UO126-induced current inhibition. Crucially, co-expression of Ca(v)2.2(S447A) with Ca(v)beta1b(S161,348A) had an additive effect, abolishing the action of UO126 on channel current, an effect not seen when Ca(v)beta1b(S161,348A) was co-expressed with Ca(v)2.2(S409A). Thus, Ser-447 on Ca(v)2.2 and Ser-161 and Ser-348 of Ca(v)beta1b appear to be both necessary and sufficient for ERK-dependent modulation of these channels. Together, our data strongly suggest that modulation of neuronal N-type VDCCs by ERK involves phosphorylation of Ca(v)2.2alpha1 and to a lesser extent possibly also Ca(v)beta subunits.     

Role of Orai1 and L-type CaV1.2 channels in Endothelin-1 mediated coronary contraction under ischemia and reperfusion

Ischemia and Reperfusion (I/R) injuries are associated with coronary artery hypercontracture. They are mainly originated by an exacerbated response to agonists released by endothelium such as Endothelin (ET-1), involving the alteration in intracellular calcium handling. Recent evidences have highlighted the implication of Store-Operated Calcium Channels (SOCC) in intracellular calcium homeostasis in coronary artery. However, little is known about the role of SOCC in the regulation of coronary vascular tone under I/R.The aim of this study was to evaluate the role of SOCC and l-type Ca²⁺ channels (LTCC) in coronary artery vasoconstriction originated by ET-1 in I/R. We used Left Anterior Descendent coronary artery (LAD) rings, isolated from Wistar rats, to study the contractility and intracellular Ca²⁺ concentration ([Ca²⁺]_i) under a simulated I/R protocol. We observed that responses to high-KCL induced depolarization and caffeine-induced Ca²⁺ release are attenuated in coronary artery under I/R. Furthermore, ET-1 addition in ischemia promotes transient and small rise of [Ca²⁺]_i and coronary vascular tone. Meanwhile, these effects are significantly potentiated during reperfusion. The resulting ET-1-induced vasoconstrictions and [Ca²⁺]_i increase were abolished by; GSK-7975A and gadolinium, inhibitors of SOCC; and nifedipine a widely used inhibitor of LTCC. Interestingly, using in situ Proximity Ligation Assay (PLA) in isolated coronary smooth muscle cells we found significant colocalization of LTCC Ca_V1.2 isoform with Orai1, the pore forming subunit of SOCC, and TRPC1 under I/R.Our data suggest that hypercontraction of coronary artery induced by ET-1 after I/R involves the co-activation of LTCC and SOCC, which colocalize significantly in the sarcolemma of coronary smooth muscle cells.     

Custom distinctions in the interaction of G-protein beta subunits with N-type (CaV2.2) versus P/Q-type (CaV2.1) calcium channels

Inhibition of N- (Cav2.2) and P/Q-type (Cav2.1) calcium channels by G-proteins contribute importantly to presynaptic inhibition as well as to the effects of opiates and cannabinoids. Accordingly, elucidating the molecular mechanisms underlying G-protein inhibition of voltage-gated calcium channels has been a major research focus. So far, inhibition is thought to result from the interaction of multiple proposed sites with the Gbetagamma complex (Gbetagamma). Far less is known about the important interaction sites on Gbetagamma itself. Here, we developed a novel electrophysiological paradigm, "compound-state willing-reluctant analysis," to describe Gbetagamma interaction with N- and P/Q-type channels, and to provide a sensitive and efficient screen for changes in modulatory behavior over a broad range of potentials. The analysis confirmed that the apparent (un)binding kinetics of Gbetagamma with N-type are twofold slower than with P/Q-type at the voltage extremes, and emphasized that the kinetic discrepancy increases up to ten-fold in the mid-voltage range. To further investigate apparent differences in modulatory behavior, we screened both channels for the effects of single point alanine mutations within four regions of Gbeta1, at residues known to interact with Galpha. These residues might thereby be expected to interact with channel effectors. Of eight mutations studied, six affected G-protein modulation of both N- and P/Q-type channels to varying degrees, and one had no appreciable effect on either channel. The remaining mutation was remarkable for selective attenuation of effects on P/Q-, but not N-type channels. Surprisingly, this mutation decreased the (un)binding rates without affecting its overall affinity. The latter mutation suggests that the binding surface on Gbetagamma for N- and P/Q-type channels are different. Also, the manner in which this last mutation affected P/Q-type channels suggests that some residues may be important for "steering" or guiding the protein into the binding pocket, whereas others are important for simply binding to the channel.     

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.     

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.     

Antagonism of 4-substituted 1,4-dihydropyridine-3,5-dicarboxylates toward voltage-dependent L-type Ca2+ channels CaV1.3 and CaV1.2

L-type Ca²⁺ channels in mammalian brain neurons have either a Ca_V1.2 or Ca_V1.3 pore-forming subunit. Recently, it was shown that Ca_V1.3 Ca²⁺ channels underlie autonomous pacemaking in adult dopaminergic neurons in the substantia nigra pars compacta, and this reliance renders them sensitive to toxins used to create animal models of Parkinson’s disease. Antagonism of these channels with the dihydropyridine antihypertensive drug isradipine diminishes the reliance on Ca²⁺ and the sensitivity of these neurons to toxins, pointing to a potential neuroprotective strategy. However, for neuroprotection without an antihypertensive side effect, selective Ca_V1.3 channel antagonists are required. In an attempt to identify potent and selective antagonists of Ca_V1.3 channels, 124 dihydropyridines (4-substituted-1,4-dihydropyridine-3,5-dicarboxylic diesters) were synthesized. The antagonism of heterologously expressed Ca_V1.2 and Ca_V1.3 channels was then tested using electrophysiological approaches and the FLIPR Calcium 4 assay. Despite the large diversity in substitution on the dihydropyridine scaffold, the most Ca_V1.3 selectivity was only about twofold. These results support a highly similar dihydropyridine binding site at both Ca_V1.2 and Ca_V1.3 channels and suggests that other classes of compounds need to be identified for Ca_V1.3 selectivity.