Mechanisms of calcium independent but voltage dependent secretion(CiVDS) in somata of mammalian primary sensory neurons
【摘要】：The somata of primary sensory neurons, including dorsal root ganglion(DRG) neurons, release neurotransmitters and neuropeptides. Following physiological action potentials, in addition to Ca~(2+)-dependent secretion, we have discovered and studied Ca~(2+)-independent but voltage-dependent secretion(CiVDS) in somata of freshly isolated DRG neurons(Zhang et al, 2002, 2004; Zheng et al, 2009, Liu et al, 2011). Major open question of CiVDS is the molecular mechanism, including 3 components: fusion pore machinery(FP), voltage sensor(VS) and the FP-VS linker(LK). Here we report, by using exocytosis assays of patch-clamp recording of membrane capacitance, and single vesicle imaging(EM and TIRF),(1) FP is jointly contributed by 2 components of SNARE complex, SNAP25 and syntaxin;(2) VS is contributed by voltage-gated Ca channels(VGCCs), Cav2.2(N-type VGCC);(3) LK is the "synprint", Cav2.2 intracellular loop718-963 aa(Catterall, 1999);(4) following automatic knockdown of CiVDS by 3 d-culture of DRG neurons, CiVDS is rescued by overexpressing any component of FP(SNAPE25 or syntaxin) or VS(Cav2.2);(5) CiVDS is inhibited by blockers against FP(SNAP25 or sytaxin), VS(Cav2.2) and LK(synprint-truncated);(6) finally, CiVDS is blocked by Cav2.2-RNAi-KD in DRG pre-transfected in vivo.