Mechanism of auxiliary subunit modulation of neuronal alpha1E calcium channels

Voltage-gated calcium channels are composed of a main pore-forming alpha1 moiety, and one or more auxiliary subunits (beta, alpha2 delta) that modulate channel properties. Because modulatory properties may vary greatly with different channels, expression systems, and protocols, it is advantageous to study subunit regulation with a uniform experimental strategy. Here, in HEK 293 cells, we examine the expression and activation gating of alpha1E calcium channels in combination with a beta (beta1-beta4) and/or the alpha2 delta subunit, exploiting both ionic- and gating-current measurements. Furthermore, to explore whether more than one auxiliary subunit can concomitantly specify gating properties, we investigate the effects of cotransfecting alpha2delta with beta subunits, of transfecting two different beta subunits simultaneously, and of COOH-terminal truncation of alpha1E to remove a second beta binding site. The main results are as follows. (a) The alpha2delta and beta subunits modulate alpha1E in fundamentally different ways. The sole effect of alpha2 delta is to increase current density by elevating channel density. By contrast, though beta subunits also increase functional channel number, they also enhance maximum open probability (Gmax/Qmax) and hyperpolarize the voltage dependence of ionic-current activation and gating-charge movement, all without discernible effect on activation kinetics. Different beta isoforms produce nearly indistinguishable effects on activation. However, beta subunits produced clear, isoform-specific effects on inactivation properties. (b) All the beta subunit effects can be explained by a gating model in which subunits act only on weakly voltage-dependent steps near the open state. (c) We find no clear evidence for simultaneous modulation by two different beta subunits. (d) The modulatory features found here for alpha1E do not generalize uniformly to other alpha1 channel types, as alpha1C activation gating shows marked beta isoform dependence that is absent for alpha1E. Together, these results help to establish a more comprehensive picture of auxiliary-subunit regulation of alpha1E calcium channels.     

The action of calcium channel blockers on recombinant L-type calcium channel alpha1-subunits

1. CHO cells expressing the alpha(1C-a) subunit (cardiac isoform) and the alpha(1C-b) subunit (vascular isoform) of the voltage-dependent L-type Ca2+ channel were used to investigate whether tissue selectivity of Ca2+ channel blockers could be related to different affinities for alpha1C isoforms. 2. Inward current evoked by the transfected alpha1 subunit was recorded by the patch-clamp technique in the whole-cell configuration. 3. Neutral dihydropyridines (nifedipine, nisoldipine, (+)-PN200-110) were more potent inhibitors of alpha(1C-)b-subunit than of alpha(1C-a)-subunit. This difference was more marked at a holding potential of -100 mV than at -50 mV. SDZ 207-180 (an ionized dihydropyridine) exhibited the same potency on the two isoforms. 4. Pinaverium (ionized non-dihydropyridine derivative) was 2 and 4 fold more potent on alpha(1C-a) than on alpha(1C-b) subunit at Vh of -100 mV and -50 mV, respectively. Effects of verapamil were identical on the two isoforms at both voltages. 5. [3H]-(+)-PN 200-110 binding experiments showed that neutral dihydropyridines had a higher affinity for the alpha(1C-b) than for the alpha(1C-a) subunit. SDZ 207-180 had the same affinity for the two isoforms and pinaverium had a higher affinity for the alpha(1C-a) subunit than for the alpha(1C-b) subunit. 6. These results indicate marked differences among Ca2+ channel blockers in their selectivity for the alpha(1C-a) and alpha(1C-b) subunits of the Ca2+ channel.     

Distribution of alpha 1A, alpha 1B and alpha 1E voltage-dependent calcium channel subunits in the human hippocampus and parahippocampal gyrus

The distribution of voltage-dependent calcium channel subunits in the central nervous system may provide information about the function of these channels. The present study examined the distribution of three alpha-1 subunits, alpha 1A, alpha 1B and alpha 1E, in the normal human hippocampal formation and parahippocampal gyrus using the techniques of in situ hybridization and immunocytochemistry. All three subunit mRNAs appeared to be similarly localized, with high levels of expression in the dentate granule and CA pyramidal layer. At the protein level, alpha 1A, alpha 1B and alpha 1E subunits were differentially localized. In general, alpha 1A-immunoreactivity was most intense in cell bodies and dendritic processes, including dentate granule cells, CA3 pyramidal cells and entorhinal cortex pre-alpha and pri-alpha cells. The alpha 1B antibody exhibited relatively weak staining of cell bodies but stronger staining of neuropil, especially in certain regions of high synaptic density such as the polymorphic layer of the dentate gyrus and the stratum lucidum and radiatum of the CA regions. The alpha 1E staining pattern shared features in common with both alpha 1A and alpha 1B, with strong immunoreactivity in dentate granule, CA3 pyramidal and entorhinal cortex pri-alpha cells, as well as staining of the CA3 stratum lucidum. These findings suggest regions in which particular subunits may be involved in synaptic communication. For example, comparison of alpha 1B and alpha 1E staining in the CA3 stratum lucidum with calbindin-immuno-reactivity suggested that these two calcium channels subunits may be localized presynaptically in mossy fibre terminals and therefore may be involved in neurotransmitter release from these terminals.     

G-Protein-dependent facilitation of neuronal alpha1A, alpha1B, and alpha1E Ca channels

Modulation of neuronal voltage-gated Ca channels has important implications for synaptic function. To investigate the mechanisms of Ca channel modulation, we compared the G-protein-dependent facilitation of three neuronal Ca channels. alpha1A, alpha1B, or alpha1E subunits were transiently coexpressed with alpha2-deltab and beta3 subunits in HEK293 cells, and whole-cell currents were recorded. After intracellular dialysis with GTPgammaS, strongly depolarized conditioning pulses facilitated currents mediated by each Ca channel type. The magnitude of facilitation depended on current density, with low-density currents being most strongly facilitated and high-density currents often lacking facilitation. Facilitating depolarizations speeded channel activation approximately 1.7-fold for alpha1A and alpha1B and increased current amplitudes by the same proportion, demonstrating equivalent facilitation of G-protein-inhibited alpha1A and alpha1B channels. Inactivation typically obscured facilitation of alpha1E current amplitudes, but the activation kinetics of alpha1E currents showed consistent and pronounced G-protein-dependent facilitation. The onset and decay of facilitation had the same kinetics for alpha1A, alpha1B, and alpha1E, suggesting that Gbeta gamma dimers dissociate from and reassociate with these Ca channels at very similar rates. To investigate the structural basis for N-type Ca channel modulation, we expressed a mutant of alpha1B missing large segments of the II-III loop and C terminus. This deletion mutant exhibited undiminished G-protein-dependent facilitation, demonstrating that a Gbeta gamma interaction site recently identified within the C terminus of alpha1E is not required for modulation of alpha1B.     

Abnormally expressed low-voltage-activated calcium channels in beta-cells from NOD mice and a related clonal cell line

A macroscopic low-voltage-activated (LVA) inward current was found in pancreatic beta-cells isolated from NOD mice. However, this current was not present in nondiabetic prone mouse (e.g., Swiss-Webster) pancreatic beta-cells. We performed pharmacological analyses on this current in NOD insulinoma tumor cells (NIT-1). This cell line was developed from pancreatic beta-cells of a transgenic NOD mouse. The sodium-channel blocker, tetrodotoxin (TTX; 2 micromol/l) had no effect on this LVA current. The amplitudes of currents elicited by a -20 mV test pulse retained similarity when the extracellular sodium concentration was increased from 0 to 115 mmol/l; when the extracellular calcium concentration was decreased from 10 to 2 mmol/l, there was an approximate 50% reduction of this current elicited by a -30 mV test pulse. Neither the L-type calcium-channel blocker, nifedipine (3 micromol/l), nor the N-type calcium-channel blocker, omega-CgTx-GVIA (1 micromol/l), at -30 mV produced an appreciable effect. The T-type calcium-channel blockers, nickel (3 micromol/l) and amiloride (250 micromol/l), effectively reduced the peak of this current. In 2 mmol/l calcium external solution, the threshold of voltage-dependent activation of this calcium current was approximately -65 mV, and the peak current occurred at -20 mV. Half-maximum steady-state inactivation was around -43 mV. The mean time constant of slow deactivating tail currents generated by a preceding 20 mV pulse was 2.53 ms. The intracellular free calcium concentration was two- to threefold higher in NOD mouse pancreatic beta-cells compared with Swiss-Webster pancreatic beta-cells. We concluded that there are LVA calcium channels abnormally expressed in NOD mouse beta-cells. This LVA calcium channel may be factorial to the high cytosolic free calcium concentration observed in these cells, and thereby may contribute to the pathogenesis of NOD mouse beta-cells.     

The Ca2+ channel beta3 subunit differentially modulates G-protein sensitivity of alpha1A and alpha1B Ca2+ channels

We have shown previously that the Ca2+ channel beta3 subunit is capable of modulating tonic G-protein inhibition of alpha1A and alpha1B Ca2+ channels expressed in oocytes. Here we determine the modulatory effect of the Ca2+ channel beta3 subunit on M2 muscarinic receptor-activated G-protein inhibition and whether the beta3 subunit modulates the G-protein sensitivity of alpha1A and alpha1B currents equivalently. To compare the relative inhibition by muscarinic activation, we have used successive ACh applications to remove the large tonic inhibition of these channels. We show that the resulting rebound potentiation results entirely from the loss of tonic G-protein inhibition; although the currents are temporarily relieved of tonic inhibition, they are still capable of undergoing inhibition through the muscarinic pathway. Using this rebound protocol, we demonstrate that the inhibition of peak current amplitude produced by M2 receptor activation is similar for alpha1A and alpha1B calcium currents. However, the contribution of the voltage-dependent component of inhibition, characterized by reduced inhibition at very depolarized voltage steps and the relief of inhibition by depolarizing prepulses, was slightly greater for the alpha1B current than for the alpha1A current. After co-expression of the beta3 subunit, the sensitivity to M2 receptor-induced G-protein inhibition was reduced for both alpha1A and alpha1B currents; however, the reduction was significantly greater for alpha1A currents. Additionally, the difference in the voltage dependence of inhibition of alpha1A and alpha1B currents was heightened after co-expression of the Ca2+ channel beta3 subunit. Such differential modulation of sensitivity to G-protein modulation may be important for fine tuning release in neurons that contain both of these Ca2+ channels.     

Activation kinetics and single channel properties of recombinant alpha1beta2gamma2L GABA(A) receptor channels

Recombinant alpha1beta2gamma2L GABA(A) receptor channels, transiently expressed in HEK 293 cells, were investigated using the patch-clamp technique in combination with a device for ultra-fast solution exchange. The dose-response relationship revealed an EC50 of 11.6 +/- 0.9 microM and saturated with 3 mM GABA. The slope between 0.001 and 0.01 mM GABA was 2.2 +/- 0.4, indicating at least three binding sites for GABA. The rise time decreased from about 120 ms at 0.001 mM GABA to about 0.8 ms at 10 mM GABA. Single channel openings were grouped in bursts with an average duration of 10.3 +/- 3.0 ms. More than 95% of the current was represented by a single channel slope conductance of about 29 pS.     

Ion permeation and glutamate residues linked by Poisson-Nernst-Planck theory in L-type calcium channels

L-type Ca channels contain a cluster of four charged glutamate residues (EEEE locus), which seem essential for high Ca specificity. To understand how this highly charged structure might produce the currents and selectivity observed in this channel, a theory is needed that relates charge to current. We use an extended Poisson-Nernst-Planck (PNP2) theory to compute (mean) Coulombic interactions and thus to examine the role of the mean field electrostatic interactions in producing current and selectivity. The pore was modeled as a central cylinder with tapered atria; the cylinder (i.e., "pore proper") contained a uniform volume density of fixed charge equivalent to that of one to four carboxyl groups. The pore proper was assigned ion-specific, but spatially uniform, diffusion coefficients and excess chemical potentials. Thus electrostatic selection by valency was computed self-consistently, and selection by other features was also allowed. The five external parameters needed for a system of four ionic species (Na, Ca, Cl, and H) were determined analytically from published measurements of thre limiting conductances and two critical ion concentrations, while treating the pore as a macroscopic ion-exchange system in equilibrium with a uniform bath solution. The extended PNP equations were solved with these parameters, and the predictions were compared to currents measured in a variety of solutions over a range of transmembrane voltages. The extended PNP theory accurately predicted current-voltage relations, anomalous mole fraction effects in the observed current, saturation effects of varied Ca and Na concentrations, and block by protons. Pore geometry, dielectric permittivity, and the number of carboxyl groups had only weak effects. The successful prediction of Ca fluxes in this paper demonstrates that ad hoc electrostatic parameters, multiple discrete binding sites, and logistic assumptions of single-file movement are all unnecessary for the prediction of permeation in Ca channels over a wide range of conditions. Further work is needed, however, to understand the atomic origin of the fixed charge, excess chemical potentials, and diffusion coefficients of the channel. The Appendix uses PNP2 theory to predict ionic currents for published "barrier-and-well" energy profiles of this channel.     

Cloning and functional expression of a voltage-gated calcium channel alpha1 subunit from jellyfish

Voltage-gated Ca2+ channels in vertebrates comprise at least seven molecular subtypes, each of which produces a current with distinct kinetics and pharmacology. Although several invertebrate Ca2+ channel alpha1 subunits have also been cloned, their functional characteristics remain unclear, as heterologous expression of a full-length invertebrate channel has not previously been reported. We have cloned a cDNA encoding the alpha1 subunit of a voltage-gated Ca2+ channel from the scyphozoan jellyfish Cyanea capillata, one of the earliest existing organisms to possess neural and muscle tissue. The deduced amino acid sequence of this subunit, named CyCaalpha1, is more similar to vertebrate L-type channels (alpha1S, alpha1C, and alpha1D) than to non-L-type channels (alpha1A, alpha1B, and alpha1E) or low voltage-activated channels (alpha1G). Expression of CyCaalpha1 in Xenopus oocytes produces a high voltage-activated Ca2+ current that, unlike vertebrate L-type currents, is only weakly sensitive to 1,4-dihydropyridine or phenylalkylamine Ca2+ channel blockers and is not potentiated by the agonist S(-)-BayK 8644. In addition, the channel is less permeable to Ba2+ than to Ca2+ and is more permeable to Sr2+. CyCaalpha1 thus represents an ancestral L-type alpha1 subunit with significant functional differences from mammalian L-type channels.     

Identification of benz(othi)azepine-binding regions within L-type calcium channel alpha1 subunits

To identify the binding domain for diltiazem-like Ca2+ antagonists on L-type Ca2+ channel alpha1 subunits we synthesized the benzazepine [3H]benziazem as a novel photoaffinity probe. [3H]Benziazem reversibly labeled the benzothiazepine (BTZ)-binding domain of partially purified skeletal muscle Ca2+ channels with high affinity (Kd = 12 nM) and photoincorporated into its binding domain with high yield (>66%). Antibody mapping of proteolytic labeled fragments revealed specific labeling of regions associated with transmembrane segments S6 in repeats III and IV. More than 50% of the labeling was found in the tryptic fragment alanine 1023-lysine 1077 containing IIIS6 together with extracellular and intracellular amino acid residues. The remaining labeling was identified in a second site comprising segment S6 in repeat IV and adjacent residues. Unlike for dihydropyridines, no labeling was observed in the connecting IIIS5-IIIS6 linker. The [3H]benziazem photolabeled regions must be in close contact to the drug molecule when bound to the channel. We propose that the determinants for high affinity BTZ binding are located within or in close proximity to segments IIIS6 and/or IVS6. Therefore the binding domain for BTZs, like for the other main classes of Ca2+ antagonists, must be located in close proximity to pore-forming regions of the channel.     

Antisense oligonucleotides against alpha1E reduce R-type calcium currents in cerebellar granule cells

Many neurons of the central nervous system display multiple high voltage-activated Ca2+ currents, pharmacologically classified as L-, N-, P-, Q-, and R-type. Of these current types, the R-type is the least understood. The leading candidate for the molecular correlate of R-type currents in cerebellar granule cells is the alpha1E subunit, which yields Ca2+ currents very similar to the R-type when expressed in heterologous systems. As a complementary approach, we tested whether antisense oligonucleotides against alpha1E could decrease the expression of R-type current in rat cerebellar granule neurons in culture. Cells were supplemented with either antisense or sense oligonucleotides and whole-cell patch clamp recordings were obtained after 6-8 days in vitro. Incubation with alpha1E antisense oligonucleotide caused a 52.5% decrease in the peak R-type current density, from -10 +/- 0.6 picoamperes/picofarad (pA/pF) (n = 6) in the untreated controls to -4.8 +/- 0.8 pA/pF (n = 11) (P < 0.01). In contrast, no significant changes in the current expression were seen in sense oligonucleotide-treated cells (-11.3 +/- 3.2 pA/pF). The specificity of the alpha1E antisense oligonucleotides was supported by the lack of change in estimates of the P/Q current amplitude. Furthermore, antisense and sense oligonucleotides against alpha1A did not affect R-type current expression (-11.5 +/- 1.7 and -11.7 +/- 1.7 pA/pF, respectively), whereas the alpha1A antisense oligonucleotide significantly reduced whole cell currents under conditions in which P/Q current is dominant. Our results support the hypothesis that members of the E class of alpha1 subunits support the high voltage-activated R-type current in cerebellar granule cells.     

Antibodies to synthetic peptides of the alpha1A subunit of the voltage-gated calcium channel in Lambert-Eaton myasthenic syndrome

STUDY OBJECTIVE: This study aimed to evaluate the effects of nasal continuous positive airway pressure (nCPAP) therapy on sleep and daytime symptoms of bed partners and patients with obstructive sleep apnea (OSA). DESIGN: A cross-sectional questionnaire survey. SETTING: The sleep laboratory of a university teaching hospital. PATIENTS: Ninety-one consecutive OSA patients within 2 to 12 months of being prescribed nCPAP. RESULTS: Eighty-five replies (93% of sample population) were received. Twelve patients (14% of replies) had discontinued nCPAP therapy; two patients had not yet been supplied with an nCPAP device. Seventy-one patients continued nightly nCPAP therapy. Bed partners of these patients (n = 55) answered a separate questionnaire assessing improvements in their own sleep quality, daytime alertness, mood and quality of life (questions 1 to 4), and evaluated the same parameters for the patients (questions 5 to 8). Possible scores ranged from -1 (worse) to +3 (marked improvement). Questions 1 to 4 yielded median scores of 2, 1, 1, and 2, respectively, and scores of 3, 3, 2, and 3 for questions 5 to 8. A ninth question addressing perceived changes in the quality of their relationship resulted in a median score of 2. Mean (SD) Epworth sleepiness scores improved from 14.3 (5.8) to 5.2 (4.3) in patients receiving therapy (p < 0.005). CONCLUSIONS: These data indicate that bed partners of OSA patients treated with nCPAP experience important improvements in symptoms and personal relationships. The findings are of practical clinical use when counseling patients with OSA and their partners on the likely impact of nCPAP therapy on their quality of life.     

Molecular diversity of the calcium channel alpha2delta subunit

Sequence database searches with the alpha2delta subunit as probe led to the identification of two new genes encoding proteins with the essential properties of this calcium channel subunit. Primary structure comparisons revealed that the novel alpha2delta-2 and alpha2delta-3 subunits share 55.6 and 30.3% identity with the alpha2delta-1 subunit, respectively. The number of putative glycosylation sites and cysteine residues, hydropathicity profiles, and electrophysiological character of the alpha2delta-3 subunit indicates that these proteins are functional calcium channel subunits. Coexpression of alpha2delta-3 with alpha1C and cardiac beta2a or alpha1E and beta3 subunits shifted the voltage dependence of channel activation and inactivation in a hyperpolarizing direction and accelerated the kinetics of current inactivation. The kinetics of current activation were altered only when alpha2delta-1 or alpha2delta-3 was expressed with alpha1C. The effects of alpha2delta-3 on alpha1C but not alpha1E are indistinguishable from the effects of alpha2delta-1. Using Northern blot analysis, it was shown that alpha2delta-3 is expressed exclusively in brain, whereas alpha2delta-2 is found in several tissues. In situ hybridization of mouse brain sections showed mRNA expression of alpha2delta-1 and alpha2delta-3 in the hippocampus, cerebellum, and cortex, with alpha2delta-1 strongly detected in the olfactory bulb and alpha2delta-3 in the caudate putamen.     

Identification of the amino terminus of neuronal Ca2+ channel alpha1 subunits alpha1B and alpha1E as an essential determinant of G-protein modulation

We have examined the basis for G-protein modulation of the neuronal voltage-dependent calcium channels (VDCCs) alpha1E and alpha1B. A novel PCR product of alpha1E was isolated from rat brain. This contained an extended 5' DNA sequence and was subcloned onto the previously cloned isoform rbEII, giving rise to alpha1Elong whose N terminus was extended by 50 amino acids. VDCC alpha1 subunit constructs were co-expressed with the accessory alpha2-delta and beta2a subunits in Xenopus oocytes and mammalian (COS-7) cells. The alpha1Elong showed biophysical properties similar to those of rbEII; however, when G-protein modulation of expressed alpha1 subunits was induced by activation of co-expressed dopamine (D2) receptors with quinpirole (100 nM) in oocytes, or by co-transfection of Gbeta1gamma2 subunits in COS-7 cells, alpha1Elong, unlike alpha1E(rbEII), was found to be G-protein-modulated, in terms of both a slowing of activation kinetics and a reduction in current amplitude. However, alpha1Elong showed less modulation than alpha1B, and substitution of the alpha1E1-50 with the corresponding region of alpha1B1-55 produced a chimera alpha1bEEEE, with G-protein modulation intermediate between alpha1Elong and alpha1B. Furthermore, deletion of the N-terminal 1-55 sequence from alpha1B produced alpha1BDeltaN1-55, which could not be modulated, thus identifying the N-terminal domain as essential for G-protein modulation. Taken together with previous studies, these results indicate that the intracellular N terminus of alpha1E1-50 and alpha1B1-55 is likely to contribute to a multicomponent site, together with the intracellular I-II loop and/or the C-terminal tail, which are involved in Gbetagamma binding and/or in subsequent modulation of channel gating.     

Genetic and developmental characterization of Dmca1D, a calcium channel alpha1 subunit gene in Drosophila melanogaster

To begin unraveling the functional significance of calcium channel diversity, we identified mutations in Dmca1D, a Drosophila calcium channel alpha1 subunit cDNA that we recently cloned. These mutations constitute the l(2)35Fa lethal locus, which we rename Dmca1D. A severe allele, Dmca1D(X10), truncates the channel after the IV-S4 transmembrane domain. These mutants die as late embryos because they lack vigorous hatching movements. In the weaker allele, Dmca1D(AR66), a cysteine in transmembrane domain I-S1 is changed to tyrosine. Dmca1D(AR66) embryos hatch but pharate adults have difficulty eclosing. Those that do eclose have difficulty in fluid-filling of the wings. These studies show that this member of the calcium channel alpha1 subunit gene family plays a nonredundant, vital role in larvae and adults.     

The calcium channel blocker debate

What, finally can we conclude from the work to date regarding the use of CCB's? The large clinical trials here give a number of clear messages to the prescriber. Short-acting dihydropyridine CCB's are unproven as prophylactic agents in ischaemic heart disease. In patients with poor left ventricular function post-infarct, CCB's are associated with an unchanged or increased mortality. Use of medications in the treatment of hypertension should be with proven first-line therapeutic agents; beta-blockers, diuretics; and the long-acting dihydropyridine CCB's nifedipine GITS and nitrendipine. The final argument in the discussion over the safety or otherwise of calcium channel blockers will rest in the completion in the future of a number of prospective, randomised, place-controlled clinical drug trials. These trials are currently ongoing, and their results may not be available until after the year 2000.     

Single-channel properties of L-type calcium channels from failing human ventricle

OBJECTIVE: The aim of our study was to analyse the single-channel properties of L-type calcium channels from failing human heart and to compare them to the respective animal data. Furthermore, we intended to evaluate the feasibility of future single-channel studies on the role of calcium channels in the pathophysiology of heart failure. METHODS: Single L-type calcium channels were recorded in ventricular myocytes from explanted failing human heart, using the cell-attached configuration of the patch-clamp technique. RESULTS: One or more successful registrations of calcium channels could be obtained in 11 of 19 cell isolations. Determination of single-channel conductance yielded a mean value of 16.6 +/- 1.2 pS (70 mM Ba2+ as the charge carrier) under control conditions and 23.7 +/- 2.8 pS in presence of the calcium-channel agonist FPL 64176. The rapid gating process could be described by a C<-->C<-->O gating scheme. Slow gating analysis revealed a highly significant clustering of active and non-active sweeps. CONCLUSION: Single-channel measurements of L-type calcium channels in human failing ventricle are feasible and reproducible despite the varying patient characteristics. Their channel properties are qualitatively comparable to those found in other mammals. Whether there are quantitative differences due to the underlying heart failure can be elucidated in further studies.     

Interaction of cysteine string proteins with the alpha1A subunit of the P/Q-type calcium channel

Cysteine string proteins (Csps) are J-domain chaperone proteins anchored at the surface of synaptic vesicles. Csps are involved in neurotransmitter release and may modulate presynaptic calcium channel activity, although the molecular mechanisms are unknown. Interactions between Csps, proteins of the synaptic core (SNARE) complex, and P/Q-type calcium channels were therefore explored. Co-immunoprecipitation suggested that Csps occur in complexes containing synaptobrevin (VAMP), but not syntaxin 1, SNAP-25, nor P/Q-type calcium channels labeled with 125I-omega-conotoxin MVIIC. However binding experiments with 35S-labeled Csp1 demonstrated an interaction (apparent KD = 700 nM at pH 7.4 and 4 degreesC) with a fusion protein containing a segment of the cytoplasmic loop linking homologous domains II-III of the alpha1A calcium channel subunit (BI isoform, residues 780-969). Binding was specific as it was displaced by unlabeled Csp1, and no interactions were detected with fusion proteins containing other calcium channel domains, VAMP, or syntaxin 1A. A Csp binding site on the P/Q-type calcium channel is thus located within the 200 residue synaptic protein interaction site that can also bind syntaxin I, SNAP-25, and synaptotagmin I. Csp may act as a molecular chaperone to direct assembly or disassembly of exocytotic complexes at the calcium channel.