Poster Session 1 - B21
1,2,3H. K.-H. MAH, 1,2,3C. SNIDAL, 1,2,3R. H.-C. CHEN, 2,3Q. LI, 1,2,3E. F. STANLEY
1 Dept. of Physiology, University of Toronto; 2 Genetics and development, Krembil Research Institute, Toronto, ON, Canada, 3 Univ. Hlth. Network, Toronto, ON, Canada.
In fast transmitting synapses synaptic vesicles (SVs) fuse with the surface membrane at the active zone and discharge neurotransmitter into the synaptic cleft. SV fusion is gated by Ca2+ ions that enter through nearby voltage gated calcium channels (CaVs), typically Cav2.1 or Cav2.2. The finding that the fusion of an SV could be gated by a single CaV led to the prediction that the SVs are linked to the channels by a molecular tether (Stanley 1993). In an accompanying study (Snidal et al. Soc. Neurosci. Abstr. 2017; Identification of a second SV binding site in the middle region of the C-terminal of presynaptic Voltage Gated Calcium channels) we used an in vitro binding assay (SV-PD) to explore SV capture by the chick CaV2.1 channel C-terminal and identified an attachment site in its C2 (mid) region (SV-AT). Cloning of the chick CaV2.1 channel revealed a splice variant that lacked this site (SV-AT(-)) and, consistent with our earlier findings, a fusion protein of this variant exhibited much weaker SV capture. In this study we have generated a rabbit polyclonal antibody (PC2var) against the chicken SV-AT site to explore the properties and distribution of the splice-positive channel. PC2var was characterized by western blot and successfully identified a band corresponding to CaV2.1 in synaptosome ghost lysates. It also identified CaV2.1 SV-AT(+) but not SV-AT(-) C2 region fusion protein, confirming its specificity. Chick cerebellar frozen section immunocytochemistry exhibited positive staining on Purkinje neurons which are known to be rich in CaV2.1 channels, demonstrating that the antibody can be used to localize the SV-AT(+) channel variant. We also observed positive punctate staining of the transmitter release face of calyx-type presynaptic terminals in the chick brain stem MNTB and these puncta abutted clouds of SVs, as identified by SV2. These results suggest that SV-AT-containing CaV2.1 channels cluster at the active zone, consistent with a role in action potential-gated transmitter release.