Ca_V2.1 (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 Ca(2+) 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 Ca(2+)-independent coupling of the human neuronal P/Q-type calcium channel (Ca(V)2.1) with Sx1A, SNAP-25, Syt1 and synaptobrevin (VAMP) in BAPTA-injected Xenopus oocytes. The co-expression of Ca(V)2.1 with Sx1A, SNAP-25 and Syt1, produced a multiprotein complex with distinctive kinetic properties analogous to the excitosome complexes generated by Ca(V)1.2, Ca(V)2.2 and Ca(V)2.3. The distinct kinetic properties of Ca(V)2.1 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 Ca(V)2.1 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 Ca(2+) entry, setting the stage for the fusion of channel-tethered-vesicles upon the arrival of an action potential.

Timothy syndrome is associated with activity-dependent dendritic retraction in rodent and human neurons

“…Finally, introducing point mutations in the TS channel that prevent Ca 2+ flux through the channel does not prevent the channel from triggering retraction. These results are consistent with the idea that Ca V 1.2 channels can activate signaling cascades by voltage-dependent conformational changes 48,49 , reminiscent of the physical mechanism by which a related channel Ca V 1.1 causes excitation-contraction coupling in skeletal muscle 50 .…”

PRRT2 mutations cause hemiplegic migraine

“…9 SNAP25 is a part of a multiprotein complex implicated in the regulation of channel activity through modulation of the transport and membrane localization of the channel and inhibition of channel activation. 10,11 In particular, SNAP25 is thought to modulate the kinetics of voltage-gated Ca(21) channels, including the Ca v 2.1 calcium channel linked to HM.…”

The genetic relationship between epilepsy and hemiplegic migraine

“…Results of a yeast two-hybrid assay indicated that PRRT2 and SNAP25 interact 62,63. SNAP25 affects neurotransmitter release from the synapse and can regulate calcium channel dynamics, including for the Cav2.1 calcium channel 64. Some investigators have hypothesized that PRRT2 mutation impairs SNAP25 function, which then changes CaV2.1 activity, causes neuronal hyperexcitability, and results in epilepsy, hemiplegic migraine, or PKD.…”