Neuronal CaV2 Research Articles

Depolarization-Evoked Secretion Requires Two Vicinal Transmembrane Cysteines of Syntaxin 1A

BackgroundThe interactions of the voltage-gated Ca2+ channel (VGCC) with syntaxin 1A (Sx 1A), Synaptosome-associated protein of 25 kD (SNAP-25), and synaptotagmin, couple electrical excitation to evoked secretion. Two vicinal Cys residues, Cys 271 and Cys 272 in the Sx 1A transmembrane domain, are highly conserved and participate in modulating channel kinetics. Each of the Sx1A Cys mutants, differently modify the kinetics of Cav1.2, and neuronal Cav2.2 calcium channel.Methodology/Principle FindingsWe examined the effects of various Sx1A Cys mutants and the syntaxin isoforms 2, 3, and 4 each of which lack vicinal Cys residues, on evoked secretion, monitoring capacitance transients in a functional release assay. Membrane capacitance in Xenopus oocytes co-expressing Cav1.2, Sx1A, SNAP-25 and synaptotagmin, which is Bot C- and Bot A-sensitive, was elicited by a double 500 ms depolarizing pulse to 0 mV. The evoked-release was obliterated when a single Cys Sx1A mutant or either one of the Sx isoforms were substituted for Sx 1A, demonstrating the essential role of vicinal Cys residues in the depolarization mediated process. Protein expression and confocal imaging established the level of the mutated proteins in the cell and their targeting to the plasma membrane.Conclusions/SignificanceWe propose a model whereby the two adjacent transmembranal Cys residues of Sx 1A, lash two calcium channels. Consistent with the necessity of a minimal fusion complex termed the excitosome, each Sx1A is in a complex with SNAP-25, Syt1, and the Ca2+ channel. A Hill coefficient >2 imply that at least three excitosome complexes are required for generating a secreting hetero-oligomer protein complex. This working model suggests that a fusion pore that opens during membrane depolarization could be lined by alternating transmembrane segments of Sx1A and VGCC. The functional coupling of distinct amino acids of Sx 1A with VGCC appears to be essential for depolarization-evoked secretion.

Functional Interaction of Neuronal Cav1.3 L-type Calcium Channel with Ryanodine Receptor Type 2 in the Rat Hippocampus

Neuronal L-type Ca(2+) channels do not support synaptic transmission, but they play an essential role in synaptic activity-dependent gene expression. Ca(v)1.2 and Ca(v)1.3 are the two most widely expressed L-type Ca(2+) channels in neurons and have different biophysical and subcellular distributions. The function of the Ca(v) 1.3 L-type Ca(2+) channel and its cellular mechanisms in the central nervous system are poorly understood. In this study, using a yeast two-hybrid assay, we found that the N terminus of the rat Ca(v)1.3 alpha(1) subunit interacts with a partial N-terminal amino acid sequence of ryanodine receptor type 2 (RyR2). Reverse transcription-PCR and Western blot assays revealed high expression of both Ca(v)1.3 and RyR2 in the rat hippocampus. We also demonstrate a physical association of Ca(v)1.3 with RyR2 using co-immunoprecipitation assays. Moreover, immunocytochemistry revealed prominent co-localization between Ca(v)1.3 and RyR2 in hippocampal neurons. Depolarizing cells by an acute treatment of a high concentration of KCl (high-K, 60 mm) showed that the activation of L-type Ca(2+) channels induced RyR opening and led to RyR-dependent Ca(2+) release, even in the absence of extracellular Ca(2+). Furthermore, we found that RyR2 mRNA itself is increased by long term treatment of high-K via activation of L-type Ca(2+) channels. These acute and long term effects of high-K on RyRs were selectively blocked by small interfering RNA-mediated silencing of Ca(v)1.3. These results suggest a physical and functional interaction between Ca(v)1.3 and RyR2 and important implications of Ca(v)1.3/RyR2 clusters in translating synaptic activity into alterations in gene expression.

Abstract 355: Modulation of Cardiac L-type Calcium Channels by Phospholemman

Ca 2+ entry through L-type Ca 2+ channels plays a critical role in shaping the action potential and is the initial trigger for EC-coupling. The gating, expression and targeting Ca 2+ channels are tightly regulated by auxiliary subunits and second messenger signaling mechanisms. Here, we report that cardiac Ca 2+ channels are directly modulated by phospholemman (PLM), a single transmembrane protein that is important for regulating ion homeostasis in the heart through its interactions with the Na,K-ATPase and Na/Ca Exchanger (NCX). Experiments using confocal immunofluorescence microscopy indiate that PLM and the Ca 2+ channel alpha-1 subunit, Ca V 1.2, co-localize to the plasma membranes of HEK 293 and COS-7 cells. Recipricol co-immunoprecipitation studies demonstrate that PLM and Ca V 1.2 are specifically associated in the mouse heart (see Figure ) and HEK 293 cells expressing the two proteins (not shown). Whole-cell patch-clamp was used to assess the functional consequences of the interaction between PLM and the Ca V 1.2 subunit using HEK 293 cells transfected with PLM ((+)PLM) or empty PLM vector ((−)PLM). These studies demonstrate that PLM substantially slows the activation kinetics of Ca V 1.2 channels (see Figure ), but has no effect on neuronal Ca V 2.1 Ca 2+ channels (not shown). As a result, the level of Ca 2+ entry during the first 50 msec of channel activation is decreased by up to 32%. Since PLM is upregulated in post-ischemic rat hearts and due to the tight functional coupling between the cardiac Ca 2+ channel and NCX, we propose that PLM-induced slowing channel activation and PLM-dependent inhibition of NCX combine synergistically to reduce peak [Ca 2+ ]i in infacted myocytes.

Spatial Association between L‐type Calcium Channels and Integrins

Current through the L-type calcium channel (Cav1.2) is enhanced by α 5β1 integrin activation in vascular smooth muscle cells and neurons. We examined the spatial association between α5β1 integrin and Cav1.2 using Cav1.2 mutants either with truncation of the Cav1.2 C-terminal region just proximal to the PKA phosphorylation site at Ser1901(stop5) or missing the two C-terminal proline rich domains between amino acids 1955–1973 (dp1/p2). The effects on Cav1.2 current and integrin interaction were determined using co-immunoprecipitation (IP) and patch clamp recording of Cav1.2 current in HEK293 cells transfected with wild type (WT) or mutant neuronal Cav1.2 cDNA. After integrin activation with α5β1 integrin antibody, current was potentiated by 107% in WT Cav1.2, by 10% in the stop5 mutant, and by 88% in the dp1/p2 mutant. Co-association of Cav1.2 with β1 integrin was detected by IP in cells plated on fibronectin (FN), but not on poly-l-lysine. On FN, notably less co-association of Cav1.2 with β1 integrin was seen in the dp1/p2 mutant (59% of WT, p<0.05), but only slightly less co-association with β1 integrin was seen in the stop5 mutant (79% of WT). Lack of correlation between the patch clamp and IP data may result either from the differences in the timing of integrin activation in the two protocols and/or by a requirement for other regulatory regions in Cav1.2 that mediate acute signaling events upon integrin activation. Overall, our data suggest that regions in the C-terminal of Cav1.2 are crucial for association with β1 integrin, but their roles in modulating the effects of integrin activation on Cav1.2 need further investigation.

The Molecular Chaperone hsp70 Interacts with the Cytosolic II-III Loop of the Cav2.3 E-type Voltagegated Ca2+ Channel

Multiple types of voltage-activated Ca2+ channels(T, L, N, P, Q, R type) coexist in excitable cells andparticipate in synaptic differentiation, secretion,transmitter release, and neuronal plasticity. Ca2+ ionsentering cells trigger these events through theirinteraction with the ion channel itself or through Ca2+binding to target proteins initiating signallingcascades at cytosolic loops of the ion conductingsubunit (Cava1). These loops interact with targetproteins in a Ca2+-dependent or independent manner.In Cav2.3-containing channels the cytosolic linkerbetween domains II and III confers a novel Ca2+sensitivity to E-type Ca2+ channels including phorbolester sensitive signalling via protein kinase C (PKC)in Cav2.3 transfected HEK-293 cells. To understand Ca2+ and phorbol ester mediated activation of Cav2.3Ca2+ channels, protein interaction partners of the II-IIIloop were identified. FLAG-tagged II-III - loop of humanCav2.3 was over-expressed in HEK 293 cells, and themolecular chaperone hsp70, which is known tointeract with PKC, was identified as a novel functionalinteraction partner. Immunopurified II-III loop-proteinof neuronal and endocrine Cav2.3 splice variantsstimulate autophosphorylation of PKCa, leading to thesuggestion that hsp70 - binding to the II-III loop - mayact as an adaptor for Ca2+ dependent targeting ofPKC to E-type Ca2+ channels.

Neuronal L-Type Calcium Channels Open Quickly and Are Inhibited Slowly

Neuronal L-type calcium channels are essential for regulating activity-dependent gene expression, but they are thought to open too slowly to contribute to action potential-dependent calcium entry. A complication of studying native L-type channels is that they represent a minor fraction of the whole-cell calcium current in most neurons. Dihydropyridine antagonists are therefore widely used to establish the contribution of L-type channels to various neuronal processes and to study their underlying biophysical properties. The effectiveness of these antagonists on L-type channels, however, varies with stimulus and channel subtype. Here, we study recombinant neuronal L-type calcium channels, CaV1.2 and CaV1.3. We show that these channels open with fast kinetics and carry substantial calcium entry in response to individual action potential waveforms, contrary to most studies of native L-type currents. Neuronal CaV1.3 L-type channels were as efficient as CaV2.2 N-type channels at supporting calcium entry during action potential-like stimuli. We conclude that the apparent slow activation of native L-type currents and their lack of contribution to single action potentials reflect the state-dependent nature of the dihydropyridine antagonists used to study them, not the underlying properties of L-type channels.

Specific Kinetic Alterations of Human CaV2.1 Calcium Channels Produced by Mutation S218L Causing Familial Hemiplegic Migraine and Delayed Cerebral Edema and Coma after Minor Head Trauma

Mutation S218L in the Ca(V)2.1 alpha(1) subunit of P/Q-type Ca(2+) channels produces a severe clinical phenotype in which typical attacks of familial hemiplegic migraine (FHM) triggered by minor head trauma are followed, after a lucid interval, by deep (even fatal) coma and long lasting severe cerebral edema. We investigated the functional consequences of this mutation on human Ca(V)2.1 channels expressed in human embryonic kidney 293 cells and in neurons from Ca(V)2.1 alpha(1)(-/-) mice by combining single channel and whole cell patch clamp recordings. Mutation S218L produced a shift to lower voltages of the single channel activation curve and a consequent increase of both single channel and whole cell Ba(2+) influx in both neurons and human embryonic kidney 293 cells. Compared with the other FHM-1 mutants, the S218L shows one of the largest gains of function, especially for small depolarizations, which are insufficient to open the wild-type channel. S218L channels open at voltages close to the resting potential of many neurons. Moreover, the S218L mutation has unique effects on the kinetics of inactivation of the channel because it introduces a large component of current that inactivates very slowly, and it increases the rate of recovery from inactivation. During long depolarizations at voltages that are attained during cortical spreading depression, the extent of inactivation of the S218L channel is considerably smaller than that of the wild-type channel. We discuss how the unique combination of a particularly slow inactivation during cortical spreading depression and a particularly low threshold of channel activation might lead to delayed severe cerebral edema and coma after minor head trauma.

A Cacna1a Knockin Migraine Mouse Model with Increased Susceptibility to Cortical Spreading Depression

Migraine is a common, disabling, multifactorial, episodic neurovascular disorder of unknown etiology. Familial hemiplegic migraine type 1 (FHM-1) is a Mendelian subtype of migraine with aura that is caused by missense mutations in the CACNA1A gene that encodes the α 1 subunit of neuronal Ca v2.1 Ca 2+ channels. We generated a knockin mouse model carrying the human pure FHM-1 R192Q mutation and found multiple gain-of-function effects. These include increased Ca v2.1 current density in cerebellar neurons, enhanced neurotransmission at the neuromuscular junction, and, in the intact animal, a reduced threshold and increased velocity of cortical spreading depression (CSD; the likely mechanism for the migraine aura). Our data show that the increased susceptibility for CSD and aura in migraine may be due to cortical hyperexcitability. The R192Q FHM-1 mouse is a promising animal model to study migraine mechanisms and treatments.

R-type voltage-gated ca 2+ channel interacts with synaptic proteins and recruits synaptotagmin to the plasma membrane of xenopus oocytes

It is well established that syntaxin 1A, synaptosomal-associated protein of 25 kDa (SNAP-25) and synaptotagmin either alone or in combination, modulate the kinetic properties of voltage-gated Ca 2+ channels Ca v1.2 (Lc-channel) Ca v2.2 (N-type) and Ca v2.1 (P/Q-type). The interaction interface was found to reside at the cytosolic II–III domain of the α1 subunit of the channels. In this study, we demonstrated a functional coupling of human neuronal Ca v2.3 (R-type channel) with syntaxin 1A, SNAP-25 and synaptotagmin in BAPTA injected Xenopus oocytes. The kinetic properties of Ca v2.3 assembled with syntaxin 1A, SNAP-25 or synaptotagmin individually differed from Ca v2.3 associated with binary complexes syntaxin 1A/SNAP-25, syntaxin 1A/synaptotagmin or SNAP-25/synaptotagmin. Co-expression of Ca v2.3 with syntaxin 1A, SNAP-25 and synaptotagmin together, produced a channel with distinctive kinetic properties analogous to excitosome multiprotein complex generated by Ca v1.2 and Ca v2.2. Exchanging the current-carrying ions altered the kinetics of channel/synaptic proteins interaction, indicating a tight crosstalk formed between the permeation pathway of Ca v2.3 and the fusion apparatus during membrane depolarization. This putative coupling could predict how the release site might be organized to allow a rapid communication between the channel and the release machinery. In vivo confocal imaging of oocytes revealed GFP-synaptotagmin at the plasma membrane when the channel was present, as opposed to random distribution in its absence, consistent with Ca 2+-independent molecular link of synaptotagmin and the channel. Synaptotagmin was detected at the membrane also in oocytes co-expressing the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Both imaging studies and protein-protein interactions in Xenopus oocytes show that channel linkage to synaptotagmin precedes Ca 2+ influx. Altogether, the R-type channel appears to associate with synaptic proteins to generate a multiprotein excitosome complex prior to Ca 2+-entry. We propose that the distinct kinetics of the Ca 2+-channel acquired by the close association with the vesicle and the t-SNAREs within the excitosome complex may be essential for depolarization evoked transmitter release.

The C2A Domain of Synaptotagmin Alters the Kinetics of Voltage-gated Ca2+ Channels Cav1.2 (Lc-type) and Cav2.3 (R-type)

Biochemical and genetic studies implicate synaptotagmin (Syt 1) as a Ca2+ sensor for neuronal and neuroendocrine neurosecretion. Calcium binding to Syt 1 occurs through two cytoplasmic repeats termed the C2A and C2B domains. In addition, the C2A domain of Syt 1 has calcium-independent properties required for neurotransmitter release. For example, mutation of a polylysine motif (residues 189-192) reverses the inhibitory effect of injected recombinant Syt 1 C2A fragment on neurotransmitter release from PC12 cells. Here we examined the requirement of the C2A polylysine motif for Syt 1 interaction with the cardiac Cav1.2 (L-type) and the neuronal Cav2.3 (R-type) voltage-gated Ca2+ channels, two channels required for neurotransmission. We find that the C2A polylysine motif presents a critical interaction surface with Cav1.2 and Cav2.3 since truncated Syt 1 containing a mutated motif (Syt 1*1-264) was ineffective at modifying the channel kinetics. Mutating the polylysine motif also abolished C2A binding to Lc753-893, the cytosolic interacting domain of Syt 1 at Cav1.2 1 subunit. Syt 1 and Syt 1* harboring the mutation at the KKKK motif modified channel activation, while Syt 1* only partially reversed the syntaxin 1A effects on channel activity. This mutation would interfere with the assembly of Syt 1/channel/syntaxin into an exocytotic unit. The functional interaction of the C2A polylysine domain with Cav1.2 and Cav2.3 is consistent with tethering of the secretory vesicle to the Ca2+ channel. It indicates that calcium-independent properties of Syt 1 regulate voltage-gated Ca2+ channels and contribute to the molecular events underlying transmitter release.

Reconstituted Slow Muscarinic Inhibition of Neuronal (Ca V1.2c) L-Type Ca 2+ Channels

Ca 2+ influx through L-type channels is critical for numerous physiological functions. Relatively little is known about modulation of neuronal L-type Ca 2+ channels. We studied modulation of neuronal Ca V1.2c channels heterologously expressed in HEK293 cells with each of the known muscarinic acetylcholine receptor subtypes. G αq/11-coupled M1, M3, and M5 receptors each produced robust inhibition of Ca V1.2c, whereas G αi/o-coupled M2 and M4 receptors were ineffective. Channel inhibition through M1 receptors was studied in detail and was found to be kinetically slow, voltage-independent, and pertussis toxin-insensitive. Slow inhibition of Ca V1.2c was blocked by coexpressing RGS2 or RGS3T or by intracellular dialysis with antibodies directed against G αq/11. In contrast, inhibition was not reduced by coexpressing βARK1ct or G αt. These results indicate that slow inhibition required signaling by G αq/11, but not G βγ, subunits. Slow inhibition did not require Ca 2+ transients or Ca 2+ influx through Ca V1.2c channels. Additionally, slow inhibition was insensitive to pharmacological inhibitors of phospholipases, protein kinases, and protein phosphatases. Intracellular BAPTA prevented slow inhibition via a mechanism other than Ca 2+ chelation. The cardiac splice-variant of Ca V1.2 (Ca V1.2a) and a splice-variant of the neuronal/neuroendocrine Ca V1.3 channel also appeared to undergo slow muscarinic inhibition. Thus, slow muscarinic inhibition may be a general characteristic of L-type channels having widespread physiological significance.

Alternate Splicing in the Cytosolic II–III Loop and the Carboxy Terminus of Human E-type Voltage-Gated Ca 2+ Channels: Electrophysiological Characterization of Isoforms

There is growing evidence that Ca v2.3 (α1E, E-type) transcripts may encode the ion-conducting subunit of a subclass of R-type Ca 2+ channels, a heterogeneous group of channels by definition resistant to blockers of L-, N-, and P/Q-type Ca 2+ channels. To understand whether splice variation of Ca v2.3 contributes to the divergence of R-type channels, individual variants of Ca v2.3 were constructed and expressed in HEK-293 cells. With Ba 2+ as charge carrier, the tested biophysical properties were similar. In Ca 2+, the inactivation time course was slower and the recovery from short-term inactivation was faster; however, this occurred only in variants containing a 19-amino-acid-long insertion, which is typical for neuronal Ca v2.3 Ca 2+ channel subunits. This different Ca 2+ sensitivity is not responsible for the major differences between various R-type channels, and future studies might clarify its importance for in vivo synaptic or dendritic integration and the reasons for its loss in endocrine Ca v2.3 splice variants.

Functional expression and characterization of a voltage-gated CaV1.3 (alpha1D) calcium channel subunit from an insulin-secreting cell line.

L-type calcium channels mediate depolarization-induced calcium influx in insulin-secreting cells and are thought to be modulated by G protein-coupled receptors (GPCRs). The major fraction of L-type alpha1-subunits in pancreatic beta-cells is of the neuroendocrine subtype (CaV1.3 or alpha1D). Here we studied the biophysical properties and receptor regulation of a CaV1.3 subunit previously cloned from HIT-T15 cells. In doing so, we compared this neuroendocrine CaV1.3 channel with the cardiac L-type channel CaV1.2a (or alpha1C-a) after expression together with alpha2delta- and beta3-subunits in Xenopus oocytes. Both the current voltage relation and voltage dependence of inactivation for the neuroendocrine CaV1.3 channel were shifted to more negative potentials compared with the cardiac CaV1.2 channel. In addition, the CaV1.3 channel activated and inactivated more rapidly than the CaV1.2a channel. Both subtypes showed a similar sensitivity to the dihydropyridine (+)isradipine. More interestingly, the CaV1.3 channels were found to be stimulated by ligand-bound G(i)/G(o)-coupled GPCRs whereas a neuronal CaV2.2 (or alpha1B) channel was inhibited. The observed receptor-induced stimulation of CaV1.3 channels could be mimicked by phorbol-12-myristate-13-acetate and was sensitive to inhibitors of protein kinases, but not to the phosphoinositol-3-kinase-inhibitor wortmannin, pointing to serine/threonine kinase-dependent regulation. Taken together, we describe a neuroendocrine L-type CaV1.3 calcium channel that is stimulated by G(i)/G(o)-coupled GPCRs and differs significantly in distinct biophysical characteristics from the cardiac subtype (CaV1.2a), suggesting that the channels have different roles in native cells.