The presynaptic CaV2.2 channel-transmitter release site core complex.
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.
Genetics and Development Division, Toronto Western Research Institute, 399 Bathurst Street, Toronto, ON, Canada M5T 2S8.
Rajesh
R
Khanna
Qi
Q
Li
Joerg
J
Bewersdorf
Elise F
EF
Stanley
D000818:Animals; D000831:Animals, Newborn; D001665:Binding Sites / Q000502:physiology; D020864:Calcium Channels, N-Type / Q000737:chemistry / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology; D002645:Chickens; D005726:Ganglia, Parasympathetic / Q000378:metabolism / Q000648:ultrastructure; D008561:Membrane Fusion / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000378:metabolism; D018377:Neurotransmitter Agents / Q000378:metabolism*; D017729:Presynaptic Terminals / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D017207:Rats, Sprague-Dawley; D013347:Subcellular Fractions / Q000737:chemistry; D013570:Synaptic Membranes / Q000378:metabolism* / Q000648:ultrastructure; D009435:Synaptic Transmission / Q000502:physiology*; D013572:Synaptic Vesicles / Q000378:metabolism* / Q000648:ultrastructure
C503279:Cacna1b protein, rat; D020864:Calcium Channels, N-Type; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents