A site-directed anti-peptide antibody (anti-CNA1) directed against the alpha 1 subunit of class A calcium channels (alpha 1A) recognizes a protein of approximately 190-200 kDa in immunoblot and immunoprecipitation analyses of rat brain glycoproteins. Calcium channels recognized by anti-CNA1 are distributed throughout the brain with a high concentration in the cerebellum. Calcium channels having alpha 1A subunits are concentrated in presynaptic terminals making synapses on cell bodies and on dendritic shafts and spines of many classes of neurons and are especially prominent in the synapses of the parallel fibers of cerebellar granule cells on Purkinje neurons where their localization in presynaptic terminals was confirmed by double labeling with the synaptic membrane protein syntaxin or the microinjected postsynaptic marker Neurobiotin. Calcium channels are present in lower density in the surface membrane of dendrites of most major classes of neurons. There was substantial labeling of Purkinje cell bodies, but less intense staining of the cell bodies of hippocampal pyramidal neurons, layer V pyramidal neurons in the dorsal cortex, and most other classes of neurons in the forebrain and cerebellum. Scattered cell bodies elsewhere in the brain were labeled at low levels. These results define a unique pattern of localization of class A calcium channels in the cell bodies, dendrites, and presynaptic terminals of most central neurons. Compared to class B N-type calcium channels, class A calcium channels are concentrated in a larger number of presynaptic nerve terminals implying a more prominent role in neurotransmitter release at many central synapses (Westenbroek, 1995).
The N channel is critical for regulating release of neurotransmitter at many synapses, and even subtle differences in its activity would be expected to influence the efficacy of synaptic transmission. Although several splice variants of the N channel are expressed in the mammalian nervous system, their biological importance is presently unclear. Variants of the alpha1B subunit of the N channel are expressed in sympathetic ganglia and alternative splicing within IIIS3-S4 and IVS3-S4 generate kinetically distinct channels. A striking difference is shown between the expression pattern of the S3-S4 variants in brain and peripheral ganglia; it is concluded that the brain-dominant form of the N channel gates 2-to-4-fold more rapidly than that predominant in ganglia (Lin, 1997).
P-type and Q-type calcium channels mediate neurotransmitter release at many synapses in the mammalian nervous system. The alpha 1A calcium channel has been implicated in the etiologies of conditions such as episodic ataxia, epilepsy and familial migraine, and shares several properties with native P- and Q-type channels. However, the exact relationship between alpha 1A and P- and Q-type channels is unknown. Alternative splicing of the alpha 1A subunit gene results in channels with distinct kinetic, pharmacological and modulatory properties. Overall, the results indicate that alternative splicing of the alpha 1A gene generates P-type and Q-type channels as well as multiple phenotypic variants (Bourinet, 1999).
The N-type Ca channel alpha1B subunit is localized to synapses throughout the nervous system and couples excitation to release of neurotransmitters. Two functionally distinct variants of the alpha1B subunit have been identified, rnalpha1B-b and rnalpha1B-d, that differ at two loci; four amino acids [SerPheMetGly (SFMG)] in IIIS3-S4 and two amino acids [GluThr (ET)] in IVS3-S4. These variants are reciprocally expressed in rat brain and sympathetic ganglia. The slower activation kinetics of rnalpha1B-b (DeltaSFMG/+ET) compared with rnalpha1B-d (+SFMG/DeltaET) channels are fully accounted for by the insertion of ET in IVS3-S4 and not by the lack of SFMG in IIIS3-S4. The inactivation kinetics of these two variants are indistinguishable. Through genomic analysis a six-base cassette exon is identified that encodes the ET site. With ribonuclease protection assays it has been demonstrated that the expression of this mini-exon is essentially restricted to alpha1B RNAs of peripheral neurons. Evidence is shown for regulated alternative splicing of a six-base exon encoding NP in the IVS3-S4 linker of the closely related alpha1A gene; residues NP can functionally substitute for ET in domain IVS3-S4 of alpha1B. The selective expression of functionally distinct Ca channel splice variants of alpha1B and alpha1A subunits in different regions of the nervous system adds a new dimension of diversity to voltage-dependent Ca signaling in neurons that may be important for optimizing action potential-dependent transmitter release at different synapses (Lin, 1999).
Two splice variants of the human homolog of the alpha1A subunit of voltage-gated Ca2+ channels have been cloned. The sequences of human alpha1A-1 and alpha1A-2 code for proteins of 2510 and 2662 amino acids, respectively. Human alpha1A-2alpha2bdeltabeta1b Ca2+ channels expressed in HEK293 cells activate rapidly (tau+10mV = 2.2 ms), deactivate rapidly (tau-90mV = 148 micros), inactivate slowly (tau+10mV = 690 ms), and have peak currents at a potential of +10 mV with 15 mM Ba2+ as charge carrier. In HEK293 cellsm transient expression of Ca2+ channels containing alpha1A/B(f), an alpha1A subunit containing a 112 amino acid segment of alpha1B-1 sequence in the IVS3-IVSS1 region, resulted in Ba2+ currents that were 30-fold larger compared to wild-type (wt) alpha1A-2-containing Ca2+ channels, and had inactivation kinetics similar to those of alpha1B-1-containing Ca2+ channels. Cells transiently transfected with alpha1A/B(f)alpha2bdeltabeta1b expressed higher levels of the alpha1, alpha2bdelta, and beta1b subunit polypeptides as detected by immunoblot analysis. By mutation analysis two locations were identified in domain IV within the extracellular loops S3-S4 (N1655P1656) and S5-SS1 (E1740) that influence the biophysical properties of alpha1A. alpha1AE1740R resulted in a threefold increase in current magnitude, a -10 mV shift in steady-state inactivation, and an altered Ba2+ current inactivation, but did not affect ion selectivity. The deletion mutant alpha1ADeltaNP shifted steady-state inactivation by -20 mV and increased the fast component of current inactivation twofold. The potency and rate of block by omega-Aga IVA was increased with alpha1ADeltaNP. These results demonstrate that the IVS3-S4 and IVS5-SS1 linkers play an essential role in determining multiple biophysical and pharmacological properties of alpha1A-containing Ca2+ channels (Hans, 1999).
The calcium channel alpha(1A) subunit gene codes for proteins with diverse structure and function. This diversity may be important for fine tuning neurotransmitter release at central and peripheral synapses. The alpha(1A) C terminus, which serves a critical role in processing information from intracellular signaling molecules, is capable of undergoing extensive alternative splicing. The purpose of this study was to determine the extent to which C-terminal alternative splicing affects some of the fundamental biophysical properties of alpha(1A) subunits. Specifically, the biophysical properties of two alternatively spliced alpha(1A) subunits were compared. One variant was identical to an isoform identified previously in human brain, and the other was a novel isoform isolated from human spinal cord. The variants differed by two amino acids (NP) in the extracellular linker between transmembrane segments IVS3 and IVS4 and in two C-terminal regions encoded by exons 37 and 44. Expression in Xenopus oocytes demonstrated that the two variants were similar with respect to current-voltage relationships and the voltage dependence of steady-state activation and inactivation. However, the rates of activation, inactivation, deactivation, and recovery from inactivation were all significantly slower for the spinal cord variant. A chimeric strategy demonstrated that the inclusion of the sequence encoded by exon 44 specifically affects the rate of inactivation. These findings demonstrate that C-terminal structural changes alone can influence the way in which alpha(1A) subunits respond to a depolarizing stimulus and add to the developing picture of the C terminus as a critical domain in the regulation of Ca2+ channel function (Krovetz, 2000).
Structural diversity of voltage-gated Ca channels underlies much of the functional diversity in Ca signaling in neurons. Alternative splicing is an important mechanism for generating structural variants within a single gene family. In vivo expression pattern of an alternatively spliced 21 amino acid encoding exon in the II-III cytoplasmic loop region of the N-type Ca channel alpha(1B) subunit is shown and its functional impact is assessed. Exon-containing alpha(1B) mRNA dominates in sympathetic ganglia and is present in approximately 50% of alpha(1B) mRNA in spinal cord and caudal regions of the brain and in the minority of alpha(1B) mRNA in neocortex, hippocampus, and cerebellum (<20%). The II-III loop exon affects voltage-dependent inactivation of the N-type Ca channel. Steady-state inactivation curves are shifted to more depolarized potentials without affects on either the rate or voltage dependence of channel opening. Differences in voltage-dependent inactivation between alpha(1B) splice variants are most clearly manifested in the presence of Ca channel beta(1b) or beta(4), rather than beta(2a) or beta(3), subunits. These results suggest that exon-lacking alpha(1B) splice variants that associate with beta(1b) and beta(4) subunits will be susceptible to voltage-dependent inactivation at voltages in the range of neuronal resting membrane potentials (-60 to -80 mV). In contrast, alpha(1B) splice variants that associate with either beta(2a) or beta(3) subunits will be relatively resistant to inactivation at these voltages. The potential to mix and match multiple alpha(1B) splice variants and beta subunits probably represents a mechanism for controlling the plasticity of excitation-secretion coupling at different synapses (Pan, 2000).
The localization of voltage-gated calcium channel (VGCC) alpha(1) subunits in cultured GABAergic mouse cortical neurons was examined by immunocytochemical methods. Ca(v)1.2 and Ca(v)1.3 subunits of L-type VGCCs were found in cell bodies and dendrites of GABA-immunopositive neurons. Likewise, the Ca(v)2.3 subunit of R-type VGCCs is expressed in a somatodendritic pattern. Ca(v)2.2 subunits of N-type channels are found exclusively in small varicosities that were identified as presynaptic nerve terminals based on their expression of synaptic marker proteins. Two splice variants of the Ca(v)2.1 subunit of P/Q-type VGCCs show widely differing expression patterns. The rbA isoform displays a purely somatodendritic staining pattern, whereas the BI isoform is confined to axon-like fibers and nerve terminals. The nerve terminals of these cultured GABAergic neurons express Ca(v)2.2 either alone or in combination with Ca(v)2.1 (BI isoform) but never express Ca(v)2.1 alone. The functional association between VGCCs and the neurotransmitter release machinery was probed using the FM1-43 dye-labeling technique. N-type VGCCs are found to be tightly coupled to exocytosis in these cultured cortical neurons, and P-type VGCCs are also important in a fraction of the cells. The predominant role of N-type VGCCs in neurotransmitter release and the specific localization of the BI isoform of Ca(v)2.1 in the nerve terminals of these neurons distinguish them from previously studied central neurons. The complementary localization patterns observed for two different isoforms of the Ca(v)2.1 subunits provide direct evidence for alternative splicing as a means of generating functional diversity among neuronal calcium channels (Timmermann, 2002).
The Ca(V)2 family of voltage-gated calcium channels, present in presynaptic nerve terminals, regulates exocytosis and synaptic transmission. Cumulative inactivation of these channels occurs during trains of action potentials, and this may control short-term dynamics at the synapse. Inactivation during brief, repetitive stimulation is primarily attributed to closed-state inactivation, and several factors modulate the susceptibility of voltage-gated calcium channels to this form of inactivation. Alternative splicing of an exon in a cytoplasmic region of the Ca(V)2.2 channel modulates its sensitivity to inactivation during trains of action potential waveforms. The presence of this exon, exon 18a, protects the Ca(V)2.2 channel from entry into closed-state inactivation specifically during short (10 ms to 3 s) and small depolarizations of the membrane potential (-60 mV to -50 mV). The reduced sensitivity to closed-state inactivation within this dynamic range likely underlies the differential responsiveness of Ca(V)2.2 splice isoforms to trains of action potential waveforms. Regulated alternative splicing of Ca(V)2.2 represents a possible mechanism for modulating short-term dynamics of synaptic efficacy in different regions of the nervous system (Thaler, 2004).
The CaV2.2 gene encodes the functional core of the N-type calcium channel. This gene has the potential to generate thousands of CaV2.2 splice isoforms with different properties. However, the functional significance of most sites of alternative splicing is not established. The IVS3-IVS4 region contains an alternative splice site that is conserved evolutionarily among CaValpha1 genes from Drosophila to human. In CaV2.2, inclusion of exon 31a in the IVS3-IVS4 region is restricted to the peripheral nervous system, and its inclusion slows the speed of channel activation. To investigate the effects of exon 31a in more detail, four tsA201 cell lines were generated that stably express CaV2.2 splice isoforms. Coexpression of auxiliary CaVbeta and CaValpha2delta subunits is required to reconstitute currents with the kinetics of N-type channels from neurons. Channels including exon 31a activate and deactivate more slowly at all voltages. Current densities were high enough in the stable cell lines co-expressing CaValpha2delta to resolve gating currents. The steady-state voltage dependence of charge movement is not consistently different between splice isoforms, but on gating currents from the exon 31a-containing CaV2.2 isoform decay with a slower time course, corresponding to slower movement of the charge sensor. Exon 31a-containing CaV2.2 is restricted to peripheral ganglia; and the slower gating kinetics of CaV2.2 splice isoforms containing exon 31a correlate reasonably well with the properties of native N-type currents in sympathetic neurons. These results suggest that alternative splicing in the S3-S4 linker influences the kinetics but not the voltage dependence of N-type channel gating (Lin, 2004).
The inhibition of presynaptic calcium channels via G-protein-dependent second messenger pathways is a key mechanism of transmitter release modulation. The calyx-type nerve terminal of the chick ciliary ganglion was used to examine which G-proteins are involved in the voltage-sensitive inhibition of presynaptic N-type calcium channels. Adenosine causes a prominent inhibition of the calcium current that is totally blocked by pretreatment with pertussis toxin (PTX), consistent with an exclusive involvement of Go/Gi in the G-protein pathway. Immunocytochemistry was used to localize these G-protein types to the nerve terminal and its transmitter release face. Two approaches were used to test for modulation by other G-protein types. (1) The terminals were treated with ligands for a variety of G-protein-linked neurotransmitter receptor types that have been associated with different G-protein families. Although small inhibitory effects are observed, these can all be eliminated by PTX, indicating that in this terminal the Gi family is the sole transmitter-induced G-protein inhibitory pathway. (2) The kinetics of calcium channel inhibition was examined by uncaging the nonselective and irreversible G-protein activator GTPgammaS, bypassing the receptors. A large fraction of the rapid GTPgammaS-induced inhibition persists, consistent with a Go/Gi-independent pathway. Immunocytochemistry identified Gq, G11, G12, and G13 as potential PTX-insensitive second messengers at this terminal. Thus, these results suggest that whereas neurotransmitter-mediated calcium channel inhibition is mainly, and possibly exclusively, via Go/Gi, other rapid PTX-insensitive G-protein pathways exist that may involve novel, and perhaps transmitter-independent, activating mechanisms (Mirotznik, 2000).
Voltage-dependent G-protein inhibition of presynaptic Ca2+ channels is a key mechanism for regulating synaptic efficacy. G-protein betagamma subunits produce such inhibition by binding to and shifting channel opening patterns from high to low open probability regimes, known respectively as 'willing' and 'reluctant' modes of gating. Recent macroscopic electrophysiological data hint that only N-type, but not P/Q-type channels can open in the reluctant mode, a distinction that could enrich the dimensions of synaptic modulation arising from channel inhibition. Here, using high-resolution single-channel recording of recombinant channels, this core contrast in the prevalence of reluctant openings is directly distinguished. Single, inhibited N-type channels manifest relatively infrequent openings of submillisecond duration (reluctant openings), which differ sharply from the high-frequency, millisecond gating events characteristic of uninhibited channels. By contrast, inhibited P/Q-type channels are electrically silent at the single-channel level. The functional impact of the differing inhibitory mechanisms is revealed in macroscopic Ca2+ currents evoked with neuronal action potential waveforms (APWs). Fitting with a change in the manner of opening, inhibition of such N-type currents produces both decreased current amplitude and temporally advanced waveform, effects that would not only reduce synaptic efficacy, but also influence the timing of synaptic transmission. However, inhibition of P/Q-type currents evoked by APWs show diminished amplitude without shape alteration, as expected from a simple reduction in the number of functional channels. Variable expression of N- and P/Q-type channels at spatially distinct synapses therefore offers the potential for custom regulation of both synaptic efficacy and synchrony, by G-protein inhibition (Colecraft, 2001).
N-type Ca2+ channels can be inhibited by neurotransmitter-induced release of G protein betagamma subunits. Two isoforms of Ca(v)2.2 alpha1 subunits of N-type calcium channels from rat brain [Ca(v)2.2a and Ca(v)2.2b; initially termed rbB-I and rbB-II] have different functional properties. Unmodulated Ca(v)2.2b channels are in an easily activated 'willing' (W) state with fast activation kinetics and no prepulse facilitation. Activating G proteins, shifts Ca(v)2.2b channels to a difficult to activate 'reluctant' (R) state with slow activation kinetics; they can be returned to the W state by strong depolarization resulting in prepulse facilitation. This contrasts with Ca(v)2.2a channels, which are tonically in the R state and exhibit strong prepulse facilitation. Activating or inhibiting G proteins has no effect. Thus, the R state of Ca(v)2.2a and its reversal by prepulse facilitation are intrinsic to the channel and independent of G protein modulation. Mutating G177 in segment IS3 of Ca(v)2.2b to E as in Ca(v)2.2a converts Ca(v)2.2b tonically to the R state, insensitive to further G protein modulation. The converse substitution in Ca(v)2.2a, E177G, converts it to the W state and restores G protein modulation. It is proposed that negatively charged E177 in IS3 interacts with a positive charge in the IS4 voltage sensor when the channel is closed and produces the R state of Ca(v)2.2a by a voltage sensor-trapping mechanism. G protein betagamma subunits may produce reluctant channels by a similar molecular mechanism (Zhong, 2001).
Presynaptic Ca2+ channels are inhibited by neurotransmitters acting through G protein-coupled receptors via a membrane-delimited pathway. Inhibition is reversed by strong depolarization, resulting in prepulse facilitation. Activated G protein betagamma subunits (Gbetagamma) are required for maximal prepulse facilitation. Gbetagamma binds to multiple sites on Ca(v)2.1, Ca(v)2.2, and Ca(v)2.3 alpha1 subunits. The functional relevance of a C-terminal binding site for Gbetagamma on Ca(v)2.2b channels, which mediate N-type Ca2+ currents, were examined. In vitro binding studies showed that Gbetagamma subunits bind to the intracellular loop connecting domains I and II and the C-terminal domain of Ca(v)2.2b but not the intracellular loops connecting domains II and III or III and IV. Deletion analysis revealed that the binding site is located near the C terminus, within amino acid residues 2257 to 2336. Directed yeast two-hybrid analysis confirmed this specific binding interaction in vivo in yeast cells. Ca(v)2.2b channels with this site deleted have normal function properties, and they are inhibited essentially normally by strong activation of G proteins with guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) and are facilitated nearly normally by depolarizing prepulses. Similarly deletion of this site has small, statistically insignificant effects on inhibition of Ca2+ current and on prepulse facilitation in the presence of somatostatin to stimulate receptor-mediated activation of G proteins. In contrast, deletion of the C-terminal Gbetagamma site substantially reduces the low level of intrinsic prepulse facilitation present at the basal level of G protein activation in tsA-201 cells. Thus, this C-terminal Gbetagamma binding site contributes to the affinity or efficacy of Gbetagamma regulation at basal levels of G protein activation. The simplest interpretation of these results is that the C-terminal binding site increases the affinity of Gbetagamma for the channel but is not required for Gbetagamma action. C-terminal binding of Gbetagamma may influence the physiological responsiveness of Ca2+ channels to low-level G protein activation (Li, 2004).
Presynaptic calcium influx at most excitatory central synapses is carried by both Cav2.1 and Cav2.2 channels. The kinetics and modulation of Cav2.1 and Cav2.2 channels differ and may affect presynaptic calcium influx. Release dynamics at CA3/CA1 synapses in rat hippocampus after selective blockade of either channel subtype and subsequent quantal content restoration were compared. Selective blockade of Cav2.1 channels enhanced paired-pulse facilitation, whereas blockade of Cav2.2 channels decreased it. This effect was observed at short (50 msec) but not longer (500 msec) intervals and was maintained during prolonged bursts of presynaptic activity. It did not reflect differences in the distance of the channels from the calcium sensor. The suppression of this effect by preincubation with the Go/i-protein inhibitor pertussis toxin suggests instead that high-frequency stimulation relieves inhibition of Cav2.2 by Go/i, thereby increasing the number of available channels (Scheuber, 2004).
L-type dihydropyridine-sensitive voltage dependent Ca2+ channels [L-VDCCs; alpha(1C)] are crucial in cardiovascular physiology. Currents via L-VDCCs are enhanced by hormones and transmitters operating via G(q), such as angiotensin II (AngII) and acetylcholine (ACh). It has been proposed that these modulations are mediated by protein kinase C (PKC). However, reports on effects of PKC activators on L-type channels are contradictory: inhibitory and/or enhancing effects have been observed. Attempts to reproduce the enhancing effect of AngII in heterologous expression systems failed. PKC modulation of the channel depends on alpha(1C) isoform used; only a long N-terminal (NT) isoform is up-regulated. This study reports the reconstitution of the AngII- and ACh-induced enhancement of the long-NT isoform of L-VDCC expressed in Xenopus oocytes. The current initially increases over several minutes but later declines to below baseline levels. Using different NT deletion mutants and human short- and long-NT isoforms of the channel, it was found the the initial segment of the NT is crucial for the enhancing, but not for the inhibitory, effect. Using blockers of PKC and of phospholipase C (PLC) and a mutated AngII receptor lacking G(q) coupling, it was demonstrated that the signaling pathway of the enhancing effect includes the activation of G(q), PLC, and PKC. The inhibitory modulation, present in both alpha(1C) isoforms, is G(q)- and PLC-independent and Ca2+-dependent, but not Ca2+-mediated, because only basal levels of Ca2+ are essential. Reconstitution of AngII and ACh effects in Xenopus oocytes will advance the study of molecular mechanisms of these physiologically important modulations (Weiss, 2004).
Neurotransmitter release at many central synapses is initiated by an influx of calcium ions through P/Q-type calcium channels, which are densely localized in nerve terminals. Because neurotransmitter release is proportional to the fourth power of calcium concentration, regulation of its entry can profoundly influence neurotransmission. N- and P/Q-type calcium channels are inhibited by G proteins, and recent evidence indicates feedback regulation of P/Q-type channels by calcium. Although calcium-dependent inactivation of L-type channels is well documented, little is known about how calcium modulates P/Q-type channels. A calcium-dependent interaction between calmodulin and a novel site in the carboxy-terminal domain of the alpha1A subunit of P/Q-type channels is reported. In the presence of low concentrations of intracellular calcium chelators, calcium influx through P/Q-type channels enhances channel inactivation, increases recovery from inactivation and produces a long-lasting facilitation of the calcium current. These effects are prevented by overexpression of a calmodulin-binding inhibitor peptide and by deletion of the calmodulin-binding domain. These results reveal an unexpected association of Ca2+/calmodulin with P/Q-type calcium channels that may contribute to calcium-dependent synaptic plasticity (Lee, 1999).
L-type Ca2+ channels are unusual in displaying two opposing forms of autoregulatory feedback, Ca2+-dependent inactivation and facilitation. Previous studies suggest that both involve direct interactions between calmodulin (CaM) and a consensus CaM-binding sequence (IQ motif) in the C terminus of the channel's alpha(1C) subunit. This study reports the functional effects of an extensive series of modifications of the IQ motif aimed at dissecting the structural determinants of the different forms of modulation. Although the combined substitution by alanine at five key positions [Ile(1624), Gln(1625), Phe(1628), Arg(1629), and Lys(1630)] abolishes all Ca2+ dependence, corresponding single alanine replacements behaves similarly to the wild-type channel (77wt) in four of five cases. The mutant I1624A stands out in displaying little or no Ca2+-dependent inactivation, but clear Ca2+- and frequency-dependent facilitation. An even more pronounced tilt in favor of facilitation is seen with the double mutant I1624A/Q1625A: overt facilitation is observed even during a single depolarizing pulse, as confirmed by two-pulse experiments. Replacement of Ile(1624) by 13 other amino acids produces graded and distinct patterns of change in the two forms of modulation. The extent of Ca2+-dependent facilitation is monotonically correlated with the affinity of CaM for the mutant IQ motif, determined in peptide binding experiments in vitro. Ca2+-dependent inactivation also depends on strong CaM binding to the IQ motif, but shows an additional requirement for a bulky, hydrophobic side chain at position 1624. Abolition of Ca2+-dependent modulation by IQ motif modifications mimics and occludes the effects of overexpressing a dominant-negative CaM mutant (Zuhlke, 2000).
Acute modulation of P/Q-type (alpha1A) calcium channels by neuronal activity-dependent changes in intracellular Ca2+ concentration may contribute to short-term synaptic plasticity, potentially enriching the neurocomputational capabilities of the brain. An unconventional mechanism for such channel modulation has been proposed in which calmodulin (CaM) may exert two opposing effects on individual channels, initially promoting ('facilitation') and then inhibiting ('inactivation') channel opening. Such dual regulation arises from three surprising Ca2+-transduction capabilities of CaM. (1) Although facilitation and inactivation are two competing processes, both require Ca2+-CaM binding to a single 'IQ-like' domain on the carboxy tail of alpha1A; a previously identified 'CBD' CaM-binding site has no detectable role. (2) Expression of a CaM mutant with impairment of all four of its Ca2+-binding sites (CaM1234) eliminates both forms of modulation. This result confirms that CaM is the Ca2+ sensor for channel regulation, and indicates that CaM may associate with the channel even before local Ca2+ concentration rises. (3) The bifunctional capability of CaM arises from bifurcation of Ca2+ signaling by the lobes of CaM: Ca2+ binding to the amino-terminal lobe selectively initiates channel inactivation, whereas Ca2+ sensing by the carboxy-terminal lobe induces facilitation. Such lobe-specific detection provides a compact means to decode local Ca2+ signals in two ways, and to separately initiate distinct actions on a single molecular complex (DeMaria, 2001).
Among the most intriguing forms of Ca2+ channel modulation is the regulation of L-type and P/Q-type channels by intracellular Ca2+, acting via unconventional channel-calmodulin (CaM) interactions. In particular, overexpressing Ca2+-insensitive mutant CaM abolishes Ca2+-dependent modulation, hinting that Ca2+-free CaM may 'preassociate' with these channels to enhance detection of local Ca2+. Despite the far-reaching consequences of this proposal, in vitro experiments testing for preassociation provide conflicting results. A three filter-cube fluorescence resonance energy transfer method (three-cube FRET) has been developed to directly probe for constitutive associations between channel subunits and CaM in single living cells. This FRET assay detects Ca2+-independent associations between CaM and the pore-forming alpha(1) subunit of L-type, P/Q-type, and, surprisingly, R-type channels. These results now definitively demonstrate channel-CaM preassociation in resting cells and underscore the potential of three-cube FRET for probing protein-protein interactions (Erickson, 2001).
Ca(v)2.1 channels, which mediate P/Q-type Ca2+ currents, undergo Ca2+/calmodulin (CaM)-dependent inactivation and facilitation that can significantly alter synaptic efficacy. The neuronal Ca2+-binding protein 1 (CaBP1) modulates Ca(v)2.1 channels in a manner that is markedly different from modulation by CaM. CaBP1 enhances inactivation, causes a depolarizing shift in the voltage dependence of activation, and does not support Ca2+-dependent facilitation of Ca(v)2.1 channels. These inhibitory effects of CaBP1 do not require Ca2+, but depend on the CaM-binding domain in the alpha1 subunit of Ca(v)2.1 channels (alpha12.1). CaBP1 binds to the CaM-binding domain, co-immunoprecipitates with alpha12.1 from transfected cells and brain extracts, and colocalizes with alpha12.1 in discrete microdomains of neurons in the hippocampus and cerebellum. These results identify an interaction between Ca2+ channels and CaBP1 that may regulate Ca2+-dependent forms of synaptic plasticity by inhibiting Ca2+ influx into neurons (Lee, 2002).
L-type (CaV1.2) and P/Q-type (CaV2.1) calcium channels possess lobe-specific CaM regulation, where Ca2+ binding to one or the other lobe of CaM triggers regulation, even with inverted polarity of modulation between channels. Other major members of the CaV1-2 channel family, R-type (CaV2.3) and N-type (CaV2.2), have appeared to lack such CaM regulation. R- and N-type channels undergo Ca2+-dependent inactivation, which is mediated by the CaM N-terminal lobe and present only with mild Ca2+ buffering (0.5 mM EGTA) characteristic of many neurons. These features, together with the CaM regulatory profiles of L- and P/Q-type channels, are consistent with a simplifying principle for CaM signal detection in CaV1-2 channels -- independent of channel context, the N- and C-terminal lobes of CaM appear invariably specialized for decoding local versus global Ca2+ activity, respectively (Liang, 2003).
N-type Ca2+ channels bind directly to the synaptic core complex of VAMP/synaptobrevin, syntaxin, and SNAP-25. Peptides containing the synaptic protein interaction ('synprint') site cause dissociation of N-type Ca2+ channels from the synaptic core complex. Introduction of synprint peptides into presynaptic superior cervical ganglion neurons reversibly inhibits synaptic transmission. Fast EPSPs due to synchronous transmitter release are inhibited, while late EPSPs arising from asynchronous release following a train of action potentials are increased and paired-pulse facilitation is increased. The corresponding peptides from L-type Ca2+ channels have no effect, and the N-type peptides have no effect on Ca2+ currents through N-type Ca2+ channels. These results are consistent with the hypothesis that binding of the synaptic core complex to presynaptic N-type Ca2+ channels is required for Ca2+ influx to elicit rapid, synchronous neurotransmitter release (Mochida, 1996).
Presynaptic N-type calcium channels interact with syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) through a binding site in the intracellular loop connecting domains II and III of the alpha1 subunit. This binding region was loaded into embryonic spinal neurons of Xenopus by early blastomere injection. After culturing, synaptic transmission of peptide-loaded and control cells was compared by measuring postsynaptic responses under different external Ca2+ concentrations. The relative transmitter release of injected neurons was reduced by approximately 25% at physiological Ca2+ concentration, whereas injection of the corresponding region of the L-type Ca2+ channel had virtually no effect. When applied to a theoretical model, these results imply that 70% of the formerly linked vesicles have been uncoupled after action of the peptide. These data suggest that severing the physical interaction between presynaptic calcium channels and synaptic proteins will not prevent synaptic transmission at this synapse but will make it less efficient by shifting its Ca2+ dependence to higher values (Rettig, 1997).
Syntaxin is a key presynaptic protein that binds to N- and P/Q-type Ca2+ channels in biochemical studies and affects gating of these Ca2+ channels in expression systems and in synaptosomes. The present study was aimed at understanding the molecular basis of syntaxin modulation of N-type channel gating. Mutagenesis of either syntaxin 1A or the pore-forming alpha(1B) subunit of N-type Ca2+ channels was combined with functional assays of N-type channel gating in a Xenopus oocyte coexpression system and in biochemical binding experiments in vitro. This analysis showed that the transmembrane region of syntaxin and a short region within the H3 helical cytoplasmic domain of syntaxin, containing residues Ala-240 and Val-244, appears critical for the channel modulation but not for biochemical association with the 'synprint site' in the II/III loop of alpha(1B). These results suggest that syntaxin and the alpha(1B) subunit engage in two kinds of interactions: an anchoring interaction via the II/III loop synprint site and a modulatory interaction via another site located elsewhere in the channel sequence. The segment of syntaxin H3 found to be involved in the modulatory interaction would lie hidden within the four-helix structure of the SNARE complex, supporting the hypothesis that syntaxin's ability to regulate N-type Ca2+ channels would be enabled after SNARE complex disassembly after synaptic vesicle exocytosis (Bezprovzvanny, 2000).
Syntaxin, a membrane protein vital in triggering vesicle fusion, interacts with voltage-gated N- and P/Q-type Ca2+ channels. This biochemical association is proposed to colocalize Ca2+ channels and presynaptic release sites, thus supporting rapid and efficient initiation of neurotransmitter release. The syntaxin channel interaction may also support a novel signaling function, to modulate Ca2+ channels according to the state of the associated release machinery. Syntaxin 1A (syn1A) coexpressed with N-type channels in Xenopus oocytes greatly promotes slow inactivation gating, but has little or no effect on the onset of and recovery from fast inactivation. Accordingly, the effectiveness of syntaxin depends strongly on voltage protocol. Slow inactivation is found for N-type channels even in the absence of syntaxin and can be distinguished from fast inactivation on the basis of its slow kinetics, distinct voltage dependence (voltage-independent at potentials higher than the level of half-inactivation), and temperature independence [Q(10), approximately 0.8]. Trains of action potential-like stimuli are more effective than steady depolarizations in stabilizing the slowly inactivated condition. Agents that stimulate protein kinase C decrease the inhibitory effect of syntaxin on N-type channels. Application of BoNtC1 to cleave syntaxin sharply attenuates the modulatory effects on Ca2+ channel gating, consistent with structural analysis of syntaxin modulation, supporting use of this toxin to test for the impact of syntaxin on Ca2+ influx in nerve terminals (Degtiar, 2000)
N-type Ca2+ channels are modulated by a variety of G-protein-coupled pathways. Some pathways produce a transient, voltage-dependent (VD) inhibition of N channel function and involve direct binding of G-protein subunits; others require the activation of intermediate enzymes and produce a longer-lasting, voltage-independent (VI) form of inhibition. The ratio of VD:VI inhibition differs significantly among cell types, suggesting that the two forms of inhibition play unique physiological roles in the nervous system. Mechanisms capable of altering the balance of VD and VI inhibition in chick dorsal root ganglion neurons have been explored. VD:VI inhibition is critically dependent on the Gbetagamma concentration, with VI inhibition dominant at low Gbetagamma concentrations. Syntaxin-1A (but not syntaxin-1B) shifts the ratio in favor of VD inhibition by potentiating the VD effects of Gbetagamma. Variations in expression levels of G-proteins and/or syntaxin provide the means to alter over a wide range both the extent and the rate of Ca2+ influx through N channels (Lü, 2001).
Syntaxin 1A, a component of the presynaptic SNARE complex, directly modulates N-type calcium channel gating in addition to promoting tonic G-protein inhibition of the channels, whereas syntaxin 1B affects channel gating but does not support G-protein modulation. The molecular determinants that govern the action of syntaxin 1 isoforms on N-type calcium channel function have been investigated. In vitro evidence shows that both syntaxin 1 isoforms physically interact with the G-protein beta subunit and the synaptic protein interaction (synprint) site contained within the N-type calcium channel domain II-III linker region. Moreover, in vitro evidence suggests that distinct domains of syntaxin participate in each interaction, with the COOH-terminal SNARE domain (residues 183-230) binding to Gbeta and the N-terminal (residues 1-69) binding to the synprint motif of the channel. Electrophysiological analysis of chimeric syntaxin 1A/1B constructs reveals that the variable NH(2)-terminal domains of syntaxin 1 are responsible for the differential effects of syntaxin 1A and 1B on N-type calcium channel function. Because syntaxin 1 exists in both 'open' and 'closed' conformations during exocytosis, a constitutively open form of syntaxin 1A was produced; it still promoted G-protein inhibition of the channels, but it did not affect N-type channel availability. This state dependence of the ability of syntaxin 1 to mediate N-type calcium channel availability suggests that syntaxin 1 dynamically regulates N-type channel function during various steps of exocytosis. Finally, syntaxin 1A appears to compete with Ggamma for the Gbeta subunit both in vitro and under physiological conditions, suggesting that syntaxin 1A may contain a G-protein gamma subunit-like domain (Jarvis, 2002).
When the presynaptic membrane protein syntaxin is coexpressed in Xenopus oocytes with N- or P/Q-type Ca2+ channels, it promotes their inactivation. These findings led to the hypothesis that syntaxin influences Ca2+ channel function in presynaptic endings, in a reversal of the conventional flow of information from Ca2+ channels to the release machinery. This effect was examined in isolated mammalian nerve terminals (synaptosomes). Botulinum neurotoxin type C1 (BoNtC1), which cleaves syntaxin, was applied to rat neocortical synaptosomes at concentrations that completely block neurotransmitter release. This treatment alters the pattern of Ca2+ entry monitored with fura-2. Whereas the initial Ca2+ rise induced by depolarization with K(+)-rich solution is unchanged, late Ca2+ entry is strongly augmented by syntaxin cleavage. Similar results were obtained when Ca2+ influx arose from repetitive firing induced by the K(+)-channel blocker 4-aminopyridine. Cleavage of vesicle-associated membrane protein with BoNtD or SNAP-25 with BoNtE failed to produce a significant change in Ca2+ entry. The BoNtC1-induced alteration in Ca2+ signaling is specific to voltage-gated Ca2+ channels, not Ca2+ extrusion or buffering, and it involves N-, P/Q- and R-type channels, the high voltage-activated channels most intimately associated with presynaptic release machinery. The modulatory effect of syntaxin is not immediately manifest when synaptosomes have been K(+)-predepolarized in the absence of external Ca2+, but develop with a delay after admission of Ca2+, suggesting that vesicular turnover may be necessary to make syntaxin available for its stabilizing effect on Ca2+ channel inactivation (Bergsman, 2000).
Activation of GABAB receptors in chick DRG neurons inhibits the Cav2.2 calcium channel in both a voltage-dependent and voltage-independent manner. The voltage-independent inhibition requires activation of a tyrosine kinase which phosphorylates the alpha 1 subunit of the channel and thereby recruits RGS12, a member of the "regulator of G protein signaling" (RGS) proteins. RGS12 binds to the SNARE-binding or synprint region (amino acids 726-985) in loop II-III of the calcium channel alpha 1 subunit. Recombinant protein encompassing the N-terminal PTB domain of RGS12 binds to the synprint region in protein overlay and surface plasmon resonance binding assays; this interaction is dependent on tyrosine phosphorylation yet within a sequence that differs from the canonical N-P-x-Y motif targeted by other PTB domains. In electrophysiological experiments, microinjection of DRG neurons with synprint-derived peptides containing the tyrosine residue Y804 alters the rate of desensitization of neurotransmitter-mediated inhibition of the Cav2.2 calcium channel while peptides centered about a second tyrosine residue, Y815, are without effect. RGS12 from DRG neuron lysate was precipitated using synprint peptides containing phosphorylated Y804. The high degree of conservation of Y804 in the SNARE binding region of Cav2.1 and Cav2.2 calcium channels suggests that this region, in addition to the binding of SNARE proteins, is also important for determining the time course of modulation of calcium current via tyrosine phosphorylation (Richman, 2004).
Calcium channel beta subunits are key modulators of calcium channel function and membrane targeting of the pore-forming alpha1 subunit. An invertebrate (Lymnaea stagnalis) homolog of P/Q- and N-type calcium channels (LCav2), although colocalized with beta subunits in synapses of mature neurons, is physically uncoupled from the beta subunits in the leading edge of growth cones of outgrowing neurons. Moreover, LCav2 channels that mediate transmitter release in mature synapses also participate in neuronal outgrowth in growth cones. The differential association of beta subunits with synaptic calcium channels and those expressed in emergent neuronal growth suggests that beta subunits may play a role in the transformation of Cav2 calcium channel function in immature neurons and mature synapses (Spafford, 2004).
The omega-conotoxins from fish-hunting cone snails are potent inhibitors of voltage-gated calcium channels. The omega-conotoxins MVIIA and CVID are selective N-type calcium channel inhibitors with potential in the treatment of chronic pain. The beta and alpha(2)delta-1 auxiliary subunits influence the expression and characteristics of the alpha(1B) subunit of N-type channels and are differentially regulated in disease states, including pain. This study examined the influence of these auxiliary subunits on the ability of the omega-conotoxins GVIA, MVIIA, CVID and their analogues to inhibit peripheral and central forms of the rat N-type channels. Although the beta3 subunit has little influence on the on- and off-rates of omega-conotoxins, coexpression of alpha(2)delta with alpha(1B) significantly reduces on-rates and equilibrium inhibition at both the central and peripheral isoforms of the N-type channels. The alpha(2)delta also enhances the selectivity of MVIIA, but not CVID, for the central isoform. Similar but less pronounced trends were also observed for N-type channels expressed in human embryonic kidney cells. The influence of alpha(2)delta is not affected by oocyte deglycosylation. The extent of recovery from the omega-conotoxin block is least for GVIA, intermediate for MVIIA, and almost complete for CVID. Application of a hyperpolarizing holding potential (-120 mV) does not significantly enhance the extent of CVID recovery. Interestingly, [R10K]MVIIA and [O10K]GVIA have greater recovery from the block, whereas [K10R]CVID has reduced recovery from the block, indicating that position 10 has an important influence on the extent of omega-conotoxin reversibility. Recovery from CVID block is reduced in the presence of alpha(2)delta in human embryonic kidney cells and in oocytes expressing alpha(1B-b). These results may have implications for the antinociceptive properties of omega-conotoxins, given that the alpha(2)delta subunit is up-regulated in certain pain states (Mould, 2004).
N-type and P/Q-type Ca2+ channels are inhibited by neurotransmitters acting through G protein-coupled receptors in a membrane-delimited pathway involving Gbetagamma subunits. Inhibition is caused by a shift from an easily activated 'willing' (W) state to a more-difficult-to-activate 'reluctant' (R) state. This inhibition can be reversed by strong depolarization, resulting in prepulse facilitation, or by protein kinase C (PKC) phosphorylation. Comparison of regulation of N-type Ca2+ channels containing Cav2.2a alpha(1) subunits and P/Q-type Ca2+ channels containing Ca(v)2.1 alpha(1) subunits revealed substantial differences. In the absence of G protein modulation, Ca(v)2.1 channels containing Ca(v)beta subunits are tonically in the W state, whereas Ca(v)2.1 channels without beta subunits and Ca(v)2.2a channels with beta subunits are tonically in the R state. Both Ca(v)2.1 and Ca(v)2.2a channels can be shifted back toward the W state by strong depolarization or PKC phosphorylation. These results show that the R state and its modulation by prepulse facilitation, PKC phosphorylation, and Ca(v)beta subunits are intrinsic properties of the Ca2+ channel itself in the absence of G protein modulation. A common allosteric model of G protein modulation of Ca2+-channel activity is presented incorporating an intrinsic equilibrium between the W and R states of the alpha(1) subunits and modulation of that equilibrium by G proteins, Ca(v)beta subunits, membrane depolarization, and phosphorylation by PKC accommodates these findings. Such regulation will modulate transmission at synapses that use N-type and P/Q-type Ca2+ channels to initiate neurotransmitter release (Herlitze, 2001).
Syntaxin 1A mediates two effects on N-type channels transiently expressed in tsA-201 cells: a hyperpolarizing shift in the steady-state inactivation curve as well as a tonic inhibition of the channel by G-protein betagamma subunits. Some of the molecular determinants and factors that modulate the action of syntaxin 1A on N-type calcium channels have been examined. With the additional coexpression of SNAP25, the syntaxin 1A-induced G-protein modulation of the channel becomes reduced in magnitude by approximately 50% but nonetheless remains significantly higher than the low levels of background inhibition seen with N-type channels alone. In contrast, coexpression of nSec-1 does not reduce the syntaxin 1A-mediated G-protein inhibition; however, interestingly, nSec-1 is able to induce tonic G-protein inhibition even in the absence of syntaxin 1A. Both SNAP25 and nSec-1 block the negative shift in half-inactivation potential that is induced by syntaxin 1A. Activation of protein kinase C via phorbol esters or site-directed mutagenesis of three putative PKC consensus sites in the syntaxin 1A binding region of the channel (S802, S896, S898) to glutamic acid (to mimic a permanently phosphorylated state) did not affect the syntaxin 1A-mediated G-protein modulation of the channel. However, in the S896E and S898E mutants, or after PKC-dependent phosphorylation of the wild-type channels, the susceptibility of the channel to undergo shifts in half-inactivation potential was removed. Thus, separate molecular determinants govern the ability of syntaxin 1A to affect N-type channel gating and its modulation by G-proteins (Jarvis, 2001).
L-type [alpha(1C)] calcium channels inactivate rapidly in response to localized elevation of intracellular Ca2+, providing negative Ca2+ feedback in a diverse array of biological contexts. The dominant Ca2+ sensor for such Ca2+-dependent inactivation is calmodulin, which appears to be constitutively tethered to the channel complex. This Ca2+ sensor induces channel inactivation by Ca2+-dependent CaM binding to an IQ-like motif situated on the carboxyl tail of alpha(1C). Apart from the IQ region, another crucial site for Ca2+ inactivation appears to be a consensus Ca2+-binding, EF-hand motif, located approximately 100 amino acids upstream on the carboxyl terminus. However, the importance of this EF-hand motif for channel inactivation has become controversial. This study demonstrates not only that the consensus EF hand is essential for Ca2+ inactivation, but that a four-amino acid cluster (VVTL) within the F helix of the EF-hand motif is itself essential for Ca2+ inactivation. Mutating these amino acids to their counterparts in non-inactivating alpha(1E) calcium channels (MYEM) almost completely ablates Ca2+ inactivation. In fact, only a single amino acid change of the second valine within this cluster to tyrosine (V1548Y) supports much of the functional knockout. However, mutations of presumed Ca2+-coordinating residues in the consensus EF hand reduce Ca2+ inactivation by only approximately 2-fold, fitting poorly with the EF hand serving as a contributory inactivation Ca2+ sensor, in which Ca2+ binds according to a classic mechanism. It is therefore suggested that while CaM serves as Ca2+ sensor for inactivation, the EF-hand motif of alpha(1C) may support the transduction of Ca2+-CaM binding into channel inactivation. The proposed transduction role for the consensus EF hand is compatible with the detailed Ca2+-inactivation properties of wild-type and mutant V1548Y channels, as gauged by a novel inactivation model incorporating multivalent Ca2+ binding of CaM (Peterson, 2000).
The leaner (tgla) mutation in mice results in severe ataxia and an overt neurodegeneration of the cerebellum. Positional cloning has revealed that the tgla mutation occurs in a gene encoding the voltage-activated calcium channel alpha1A subunit. The alpha1A subunit is highly expressed in the cerebellum and is thought to be the pore-forming subunit of P- and Q-type calcium channels. In this study both whole-cell and single-channel patch-clamp recordings were used to examine the functional consequences of the tgla mutation on P-type calcium currents. High-voltage-activated (HVA) calcium currents were recorded from acutely dissociated cerebellar Purkinje cells of homozygous leaner (tgla/tgla) and age-matched wild-type (+/+) mice. In whole cell recordings, a marked reduction of peak current density is observed in tgla/tgla Purkinje cells (-35.0 +/- 1.8 pA/pF) relative to that in +/+ (-103.1 +/- 5.9 pA/pF). The reduced whole-cell current in tgla/tgla cells is accompanied by little to no alteration in the voltage dependence of channel gating. In both genotypes, HVA currents are predominantly of the omega-agatoxin-IVA-sensitive P-type. Cell-attached patch-clamp recordings revealed no differences in single-channel conductance between the two genotypes and confirmed the presence of three distinct conductance levels (9, 13-14, and 17-18 pS) in cerebellar Purkinje cells. Analysis of patch open-probability (NPo) revealed a threefold reduction in the open-probability of channels in tgla/tgla patches (0.04 +/- 0.01) relative to that in +/+ (0.13 +/- 0.02), which may account for the reduced whole-cell current in tgla/tgla Purkinje cells. These results suggest that the tgla mutation can alter native P-type calcium channels at the single-channel level and that these alterations may contribute to the neuropathology of the leaner phenotype (Dove, 1998).
The Ca2+ channel alpha(1A)-subunit is a voltage-gated, pore-forming membrane protein positioned at the intersection of two important lines of research: one exploring the diversity of Ca2+ channels and their physiological roles, and the other pursuing mechanisms of ataxia, dystonia, epilepsy, and migraine. Alpha(1A)-Subunits are thought to support both P- and Q-type Ca2+ channel currents, but the most direct test, a null mutant, has not been described, nor is it known which changes in neurotransmission might arise from elimination of the predominant Ca2+ delivery system at excitatory nerve terminals. Alpha(1A)-deficient mice [alpha(1A)(-/-)] have been generated; they developed a rapidly progressive neurological deficit with specific characteristics of ataxia and dystonia before dying approximately 3-4 weeks after birth. P-type currents in Purkinje neurons and P- and Q-type currents in cerebellar granule cells are eliminated completely whereas other Ca2+ channel types, including those involved in triggering transmitter release, also undergo concomitant changes in density. Synaptic transmission in alpha(1A)(-/-) hippocampal slices persists despite the lack of P/Q-type channels but shows enhanced reliance on N-type and R-type Ca2+ entry. The alpha(1A)(-/-) mice provide a starting point for unraveling neuropathological mechanisms of human diseases generated by mutations in alpha(1A) (Jun, 1999).
Mutations of the alpha1A calcium channel subunit have been shown to cause such human neurological diseases as familial hemiplegic migraine, episodic ataxia-2, and spinocerebellar ataxia 6 and also to cause the murine neurological phenotypes of tottering and leaner. The leaner phenotype is recessive and characterized by ataxia with cortical spike and wave discharges (similar to absence epilepsy in humans) and a gradual degeneration of cerebellar Purkinje and granule cells. The mutation responsible is a single-base substitution that produces truncation of the normal open reading frame beyond repeat IV and expression of a novel C-terminal sequence. Whole-cell recordings have been used to determine whether the leaner mutation alters calcium channel currents in cerebellar Purkinje cells, both because these cells are profoundly affected in leaner mice and because they normally express high levels of alpha1A. In Purkinje cells from normal mice, 82% of the whole-cell current was blocked by 100 nM omega-agatoxin-IVA. In Purkinje cells from homozygous leaner mice, this omega-agatoxin-IVA-sensitive current was 65% smaller than in control cells. Although attenuated, the omega-agatoxin-IVA-sensitive current in homozygous leaner cells has properties indistinguishable from that of normal Purkinje neurons. Additionally, the omega-agatoxin-IVA-insensitive current is unaffected in homozygous leaner mice. Thus, the leaner mutation selectively reduces P-type currents in Purkinje cells, and the alpha1A subunit and P-type current appear to be essential for normal cerebellar function (Lorenzon, 1999).
alpha(1) subunit of the voltage-dependent Ca2+ channel is essential for channel function and determines the functional specificity of various channel types. alpha(1E) subunit was originally identified as a neuron-specific one, but the physiological function of the Ca2+ channel containing this subunit [alpha(1E) Ca2+ channel] was not clear compared with other types of Ca2+ channels because of the limited availability of specific blockers. To clarify the physiological roles of the alpha(1E) Ca2+ channel, alpha(1E) mutant [alpha(1E)-/-] mice were generated by gene targeting. The lacZ gene was inserted in-frame and used as a marker for alpha(1E) subunit expression. alpha(1E)-/- mice show reduced spontaneous locomotor activities and signs of timidness, but other general behaviors are apparently normal. As involvement of alpha(1E) in pain transmission is suggested by localization analyses with 5-bromo-4-chloro-3-indolyl beta-d-galactopyranoside staining, several pain-related behavioral tests were conducted using the mutant mice. Although alpha(1E)+/- and alpha(1E)-/- mice exhibit normal pain behaviors against acute mechanical, thermal, and chemical stimuli, they both show reduced responses to somatic inflammatory pain. alpha(1E)+/- mice show reduced response to visceral inflammatory pain, whereas alpha(1E)-/- mice showed apparently normal response compared with that of wild-type mice. Furthermore, alpha(1E)-/- mice that had been presensitized with a visceral noxious conditioning stimulus showed increased responses to a somatic inflammatory pain, in marked contrast with the wild-type mice in which long-lasting effects of descending antinociceptive pathway were predominant. These results suggest that the alpha(1E) Ca(2 +) channel controls pain behaviors by both spinal and supraspinal mechanisms (Saegusa, 2000).
N-type voltage-dependent Ca2+ channels (VDCCs), predominantly localized in the nervous system, have been considered to play an essential role in a variety of neuronal functions, including neurotransmitter release at sympathetic nerve terminals. As a direct approach to elucidating the physiological significance of N-type VDCCs, mice genetically deficient in the alpha(1B) subunit [Ca(v) 2.2] have been generated. The alpha(1B)-deficient null mice, surprisingly, have a normal life span and are free from apparent behavioral defects. A complete and selective elimination of N-type currents, sensitive to omega-conotoxin GVIA, was observed without significant changes in the activity of other VDCC types in neuronal preparations of mutant mice. The baroreflex response, mediated by the sympathetic nervous system, is markedly reduced after bilateral carotid occlusion. In isolated left atria prepared from N-type-deficient mice, the positive inotropic responses to electrical sympathetic neuronal stimulation are dramatically decreased compared with those of normal mice. In contrast, parasympathetic nervous activity in the mutant mice is nearly identical to that of wild-type mice. Interestingly, the mutant mice showed sustained elevation of heart rate and blood pressure. These results provide direct evidence that N-type VDCCs are indispensable for the function of the sympathetic nervous system in circulatory regulation and indicate that N-type VDCC-deficient mice will be a useful model for studying disorders attributable to sympathetic nerve dysfunction (Ino, 2001).
The expansion of polyglutamine tracts encoded by CAG trinucleotide repeats is a common mutational mechanism in inherited neurodegenerative diseases. Spinocerebellar ataxia type 6 (SCA6), an autosomal dominant, progressive disease, arises from trinucleotide repeat expansions present in the coding region of CACNA1A (chromosome 19p13). This gene encodes alpha(1A), the principal subunit of P/Q-type Ca2+ channels, which are abundant in the CNS, particularly in cerebellar Purkinje and granule neurons. Ion channel function was assayed by introduction of human alpha(1A) cDNAs in human embryonic kidney 293 cells that stably coexpressed beta(1) and alpha(2)delta subunits. Immunocytochemical analysis showed a rise in intracellular and surface expression of alpha(1A) protein when CAG repeat lengths reach or exceed the pathogenic range for SCA6. This gain at the protein level is not a consequence of changes in RNA stability. The electrophysiological behavior of alpha(1A) subunits containing expanded (EXP) numbers of CAG repeats (23, 27, and 72) was compared against that of wild-type subunits (WT) (4 and 11 repeats) using standard whole-cell patch-clamp recording conditions. The EXP alpha(1A) subunits yield functional ion channels that support inward Ca2+ channel currents, with a sharp increase in P/Q Ca2+ channel current density relative to WT. These results show that Ca2+ channels from SCA6 patients display near-normal biophysical properties but increased current density attributable to elevated protein expression at the cell surface (Piedras-Renteria, 2001).
Ca2+-dependent inactivation (CDI) of L-type Ca2+ channels plays a critical role in controlling Ca2+ entry and downstream signal transduction in excitable cells. Ca2+-insensitive forms of calmodulin (CaM) act as dominant negatives to prevent CDI, suggesting that CaM acts as a resident Ca2+ sensor. However, it is not known how the Ca2+ sensor is constitutively tethered. The tethering of Ca2+-insensitive CaM has been localized to the C-terminal tail of alpha(1C), close to the CDI effector motif, and it depends on nanomolar Ca2+ concentrations, likely attained in quiescent cells. Two stretches of amino acids are found to support the tethering and to contain putative CaM-binding sequences close to or overlapping residues previously shown to affect CDI and Ca2+-independent inactivation. Synthetic peptides containing these sequences display differences in CaM-binding properties, both in affinity and Ca2+ dependence, leading to the proposal of a novel mechanism for CDI. In contrast to a traditional disinhibitory scenario, it is suggested that apoCaM is tethered at two sites and signals actively to slow inactivation. When the C-terminal lobe of CaM binds to the nearby CaM effector sequence (IQ motif), the braking effect is relieved, and CDI is accelerated (Pitt, 2001).
Rocker (gene symbol rkr), a new neurological mutant phenotype, was found in descendents of a chemically mutagenized male mouse. Mutant mice display an ataxic, unstable gait accompanied by an intention tremor, typical of cerebellar dysfunction. These mice are fertile and appear to have a normal life span. Segregation analysis reveals rocker to be an autosomal recessive trait. The overall cytoarchitecture of the young adult brain appears normal, including its gross cerebellar morphology. Golgi-Cox staining, however, reveals dendritic abnormalities in the mature cerebellar cortex characterized by a reduction of branching in the Purkinje cell dendritic arbor and a 'weeping willow' appearance of the secondary branches. Using simple sequence length polymorphism markers, the rocker locus was mapped to mouse chromosome 8 within 2 centimorgans of the calcium channel alpha1a subunit (Cacna1a, formerly known as tottering) locus. Complementation tests with the leaner mutant allele (Cacna1ala) produced mutant animals, thus identifying rocker as a new allele of Cacna1a (Cacna1arkr). Sequence analysis of the cDNA revealed rocker to be a point mutation resulting in an amino acid exchange: T1310K between transmembrane regions 5 and 6 in the third homologous domain. Important distinctions between rocker and the previously characterized alleles of this locus include the absence of aberrant tyrosine hydroxylase expression in Purkinje cells and the separation of the absence seizures (spike/wave type discharges) from the paroxysmal dyskinesia phenotype. Overall these findings point to an important dissociation between the seizure phenotypes and the abnormalities in catecholamine metabolism, and they emphasize the value of allelic series in the study of gene function (Zwingman, 2001).
Differential properties of voltage-dependent Ca2+ channels have been primarily ascribed to the alpha1 subunit, of which 10 different subtypes are currently known. For example, channels that conduct the N-type Ca2+ current possess the alpha1B subunit (Cav2.2), which has been localized, inter alia, to the piriform cortex, hippocampus, hypothalamus, locus coeruleus, dorsal raphe, thalamic nuclei, and granular layer of the cortex. Some of these regions have been implicated in metabolic and vigilance state control, and selective block of the N-type Ca2+ channel causes circadian rhythm disruption. In this study of Cav2.2-/- knock-out mice, potential differences in feeding behavior, spontaneous locomotion, and the sleep-wake cycle were examined. Cav2.2-/- mice do not display an overt metabolic phenotype but are hyperactive, demonstrating a 20% increase in activity under novel conditions and a 95% increase in activity under habituated conditions during the dark phase, compared with wild-type littermates. Cav2.2-/- mice also display vigilance state differences during the light phase, including increased consolidation of rapid-eye movement (REM) sleep and increased intervals between non-REM (NREM) and wakefulness episodes. EEG spectral power is increased during wakefulness and REM sleep and is decreased during NREM sleep in Cav2.2-/- mice. These results indicate a role for the N-type Ca2+ channel in activity and vigilance state control, that is interpreted in terms of effects on neurotransmitter release (Beuckmann, 2003).
N-type calcium channels are modulated by acute and chronic ethanol exposure in vitro at concentrations known to affect humans, but it is not known whether N-type channels are important for behavioral responses to ethanol in vivo. In mice lacking functional N-type calcium channels, voluntary ethanol consumption is reduced and place preference is developed only at a low dose of ethanol. The hypnotic effects of ethanol are also substantially diminished, whereas ethanol-induced ataxia is mildly increased. These results demonstrate that N-type calcium channels modulate acute responses to ethanol and are important mediators of ethanol reward and preference (Newton, 2004).
Presynaptic N-type Ca2+ channels (CaV2.2, alpha1B) are thought to bind to SNARE (SNAP-25 receptor) complex proteins through a synaptic protein interaction (synprint) site on the intracellular loop between domains II and III of the alpha1B subunit. Whether binding of syntaxin to the N-type Ca2+ channels is required for coupling Ca2+ ion influx to rapid exocytosis has been the subject of considerable investigation. In this study, the synprint site was deleted from a recombinant alpha1B Ca2+ channel subunit and either the wild-type alpha1B or the synprint deletion mutant was transiently transfected into mouse pheochromocytoma (MPC) cell line 9/3L, a cell line that has the machinery required for rapid stimulated exocytosis but lacks endogenous voltage-dependent Ca2+ channels. Secretion was elicited by activation of exogenously transfected Ca2+ channel subunits. The current-voltage relationship was similar for the wild-type and mutant alpha1B-containing Ca2+ channels. Although total Ca2+ entry was slightly larger for the synprint deletion channel, compared with the wild-type channel, when Ca2+ entry was normalized to cell size and limited to cells with similar Ca2+ entry (approximately 150 x 106 Ca2+ ions/pF cell size), total secretion and the rate of secretion, determined by capacitance measurements, were significantly reduced in cells expressing the synprint deletion mutant channels, compared with wild-type channels. Furthermore, the amount of endocytosis was significantly reduced in cells with the alpha1B synprint deletion mutant, compared with the wild-type subunit. These results suggest that the synprint site is necessary for efficient coupling of Ca2+ influx through alpha1B-containing Ca2+ channels to exocytosis (Harkins, 2004).
Processing and storage of information by the nervous system requires the ability to modulate the response of excitable cells to neurotransmitter. A simple process of this type, known as adaptation or desensitization, occurs when prolonged stimulation triggers processes that attenuate the response to neurotransmitter. The C. elegans gene unc-2 is required for adaptation to two neurotransmitters, dopamine and serotonin. A loss-of-function mutation in unc-2 results in failure to adapt either to paralysis by dopamine or to stimulation of egg laying by serotonin. In addition, unc-2 mutants display behaviors similar to those induced by serotonin treatment. unc-2 encodes a homolog of a voltage-sensitive calcium-channel alpha-1 subunit. Expression of unc-2 occurs in two types of neurons implicated in the control of egg laying, a behavior regulated by serotonin. Unc-2 appears to be required in modulatory neurons to downregulate the response of the egg-laying muscles to serotonin. It is proposed that adaptation to serotonin occurs through activation of an Unc-2-dependent calcium influx, which modulates the postsynaptic response to serotonin, perhaps by inhibiting the release of a potentiating neuropeptide (Schafer, 1995).
Calcium signaling is known to be important for regulating the guidance of migrating neurons, yet the molecular mechanisms underlying this process are not well understood. Two different voltage-gated calcium channels are important for the accurate guidance of postembryonic neuronal migrations in the nematode C. elegans. In mutants carrying loss-of-function alleles of the calcium channel gene unc-2, the touch receptor neuron AVM and the interneuron SDQR often migrate inappropriately, leading to misplacement of their cell bodies. However, the AVM neurons in unc-2 mutant animals extended axons in a wild-type pattern, suggesting that the UNC-2 calcium channel specifically directs migration of the neuronal cell body and is not required for axonal pathfinding. In contrast, mutations in egl-19, which affect a different voltage-gated calcium channel, affect the migration of the AVM and SDQR bodies, as well as the guidance of the AVM axon. Thus, cell migration and axonal pathfinding in the AVM neurons appear to involve distinct calcium channel subtypes. Mutants defective in the unc-43/CaM kinase gene show a defect in SDQR and AVM positioning that resembles that of unc-2 mutants; thus, CaM kinase may function as an effector of the UNC-2-mediated calcium influx in guiding cell migration (Tam, 2000).
In response to retinal disease and injury, the axon terminals of rod photoreceptors demonstrate dramatic structural plasticity, including axonal retraction, neurite extension, and the development of presynaptic varicosities. Cone cell terminals, however, are relatively inactive. Similar events are observed in primary cultures of salamander photoreceptors. To investigate the mechanisms underlying these disparate presynaptic responses, antagonists to voltage-gated L-type and cGMP-gated channels, known to be present on rod and cone cell terminals, respectively, were used to block calcium influx during critical periods of plasticity in vitro. In rod cells, L-type channel antagonists nicardipine and verapamil inhibited not only the outgrowth of processes and the formation of varicosities, but also the synthesis of vesicle proteins, SV2 and synaptophysin. In contrast, the synthesis of opsin in rod cells was unaffected. In cone cells, L-type channel antagonists caused only modest changes. However, cobalt bromide, which blocks all calcium channels, and l-cis-diltiazem, a potent antagonist of cGMP-gated channels, significantly inhibited varicosity formation and synthesis of SV2 in cone cells. Moreover, the cGMP-gated channel agonist 8-bromo-cGMP caused a significant increase in varicosity formation by cone but not rod cells. Thus voltage-gated L-type channels in rod cells and cGMP-gated channels in cone cells are the primary calcium channels required for structural plasticity and the accompanying upregulation of synaptic vesicle synthesis. The differing responses of rod and cone terminals to injury and disease may be determined by these differences in the regulation of Ca2+ influx (Zhang, 2002).
At many central synapses, endocannabinoids released by postsynaptic cells inhibit neurotransmitter release by activating presynaptic cannabinoid receptors. The mechanisms underlying this important means of synaptic regulation are not fully understood. It has been shown at several synapses that endocannabinoids inhibit neurotransmitter release by reducing calcium influx into presynaptic terminals. One hypothesis maintains that endocannabinoids indirectly reduce calcium influx by modulating potassium channels and narrowing the presynaptic action potential. An alternative hypothesis is that endocannabinoids directly and selectively inhibit N-type calcium channels in presynaptic terminals. These hypotheses were tested at the granule cell to Purkinje cell synapse in cerebellar brain slices. By monitoring optically the presynaptic calcium influx (Cainflux) and measuring the EPSC amplitudes, it was found that cannabinoid-mediated inhibition arises solely from reduced presynaptic Cainflux. Next it was found that cannabinoid receptor activation does not affect the time course of presynaptic calcium entry, indicating that the reduced Cainflux reflects inhibition of presynaptic calcium channels. Finally, the classes of presynaptic calcium channels inhibited by cannabinoid receptor activation was assessed via peptide calcium channel antagonists. Previous studies established that N-type, P/Q-type, and R-type calcium channels are all present in granule cell presynaptic boutons. It was found that cannabinoid activation reduced Cainflux through N-type, P/Q-type, and R-type calcium channels to 29%, 60%, and 55% of control, respectively. Thus, rather than narrowing the presynaptic action potential or exclusively modulating N-type calcium channels, CB1 receptor activation inhibits synaptic transmission by modulating all classes of calcium channels present in the presynaptic terminal of the granule cell to Purkinje cell synapse (Brown, 2004).
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