The presynaptic CaV2.2 channel–transmitter release site core complex

Abstract

CaV2.2 channels play a key role in the gating of transmitter release sites (TRS) at presynaptic terminals. Physiological studies predict that the channels are linked directly to the TRS but the molecular composition of this complex remains poorly understood. We have used a high-affinity anti-CaV2.2 antibody, Ab571, to test a range of proteins known to contribute to TRS function for both an association in situ and a link in vitro. CaV2.2 clusters were isolated intact on immunoprecipitation beads and coprecipitated with a number of these proteins. Quantitative staining covariance analysis (ICA/ICQ method) was applied to the transmitter release face of the giant calyx terminal in the chick ciliary ganglion to test for TRS proteins with staining intensities that covary in situ with CaV2.2, resulting in a covariance sequence of NSF>RIM>spectrin>Munc18>VAMP>alpha-catenin, CASK>SV2>Na+-K+ approximately 0. A high-NaCl dissociation challenge applied to the immunoprecipitated complex, using the fractional recovery (FR) method [Khanna, R., Li, Q. & Stanley, E.F. (2006) PLoS.ONE., 1, e67], was used to test which proteins were most intimately associated with the channel, generating an FR sequence for CaV2.2 of: VAMP>or=actin>tubulin, NSF, Munc18, syntaxin 1>spectrin>CASK, SNAP25>RIM, Na+-K+ pump, v-ATPase, beta-catenin approximately 0. Proteins associated with endocytosis are considered in a companion paper [Khanna et al. (2007)Eur. J. Neurosci., 26, 560-574]. With the exception of VAMP and RIM, the ICQ and FR sequences were consistent, suggesting that proteins that covary the most strongly with CaV2.2 in situ are also the most intimately attached. Our findings suggest that the CaV2.2 cluster is an integral element of a multimolecular vesicle-fusion module that forms the core of a multifunctional TRS.

In Silico Docking and Electrophysiological Characterization of Lacosamide Binding Sites on Collapsin Response Mediator Protein-2 Identifies a Pocket Important in Modulating Sodium Channel Slow Inactivation

“…The supernatant was collected, and the protein concentration was determined by the BCA assay (Thermo Fisher Scientific, Shelbyville, IN). Twenty micrograms of protein was separated by SDS-PAGE (4 -12% polyacrylamide gradient gel) and electrophoretically transferred onto polyvinylidene difluoride membranes (Invitrogen) and assayed for the presence of CRMP-2 (1:1,000, Chemicon, Billerica, MA), GFP (1:10,000, Clontech, Mountain View, CA), NaV channels (1:200, Alomone Laboratories, Jerusalem, Israel), and ␤-tubulin (1:1000, Promega, Madison WI) proteins using specific antibodies (31). The membranes were blocked for 1 h in 5% skim milk powder in TBST (25 mM Tris-Cl, pH 8.0, 125 mM NaCl, 0.1% to 2% polyoxyethylene sorbitan monolaurate (Tween 20)) at room temperature.…”

Old proteins, developing roles: The regulation of calcium channels by synaptic proteins

“…It does not seem to target N-type to the presynaptic nerve terminal. 125,126 RIM possesses an N-terminal domain that binds to Rab3, 127 and two C2 domains C2A and C2B similar to those found in synaptotagmin I, indicating it may be a calcium sensor. 122 This C2 domain can selectively associate with SNAP-25 or synaptotagmin I in a calcium-dependent manner (favoring synaptotagmin I binding at concentrations higher than 75 μM).…”

“…An additional proposed role of modular adaptor proteins may be the physical anchoring of calcium channels to release sites 42,131 though recent evidence suggests otherwise. 125,126 …”

Quantitative proteomics of the Cav2 channel nano-environments in the mammalian brain

“…Finally, reliable identification of less-abundant or smaller proteins requires high-resolution quantitative MS (24). In the light of these concerns, previous efforts to identify proteins associated with Cav channels (25,26) might have missed important components or contained false-positive interactors.…”