CaV2.1 (P/Q channel) interaction with synaptic proteins is essential for depolarization-evoked release

Abstract

It is well established that syntaxin 1A (Sx1A), SNAp-25 and synaptotagmin (Syt1) either alone or in combination, modify the kinetic properties of voltage-gated Ca2+ channels (VGCCs). The interaction interface resides mainly at the cytosolic II-III domain of the alpha1 subunit of the channels, while sx1A interacts with the channel also via two highly conserved cysteine residues at the transmembrane domain. In the present study, we characterized Ca2+-independent coupling of the human neuronal p/Q-type calcium channel (CaV2.1) with sx1A, sNAp-25, syt1 and synaptobrevin (VAMp) in BApTA-injected Xenopus oocytes. The co-expression of CaV2.1 with sx1A, sNAp-25 and syt1, produced a multiprotein complex with distinctive kinetic properties analogous to the excitosome complexes generated by CaV1.2, CaV2.2 and CaV2.3. The distinct kinetic properties of CaV2.1 acquired by close association with syt1 and t-sNARes, suggests that the vesicle is tethered to the neuronal channel and to the exocytotic machinery independently of intracellular Ca 2+. To explore the relevance of these interactions to secretion we exploited a BotC1-and a BotA-sensitive secretion system developed for Xenopus oocytes not buffered by BApTA, in which depolarization-evoked secretion is monitored by a change in membrane capacitance. The reconstituted release mediated by CaV2.1 is consistent with the model in which the VGCC plays a signaling role in triggering release, acting from within the exocytotic complex. The relevance of these results to secretion posits the role of possible rearrangements within the excitosome subsequent to Ca2+ entry, setting the stage for the fusion of channel-tethered-vesicles upon the arrival of an action potential. © 2010 Landes Bioscience.