Regulation of Ca²⁺channels by SNAP-25 via recruitment of syntaxin-1 from plasma membrane clusters

Abstract

SNAP-25 regulates Ca²⁺ channels, with potentially important consequences for diseases involving an aberrant SNAP-25 expression level. How this regulation is executed mechanistically remains unknown. We investigated this question in mouse adrenal chromaffin cells and found that SNAP-25 inhibits Ca²⁺ currents, with the B-isoform being more potent than the A-isoform, but not when syntaxin-1 is cleaved by botulinum neurotoxin C. In contrast, syntaxin-1 inhibits Ca²⁺ currents independently of SNAP-25. Further experiments using immunostaining showed that endogenous or exogenous SNAP-25 expression recruits syntaxin-1 from clusters on the plasma membrane, thereby increasing the immunoavailability of syntaxin-1 and leading indirectly to Ca²⁺ current inhibition. Expression of Munc18-1, which recruits syntaxin-1 within the exocytotic pathway, does not modulate Ca²⁺ channels, whereas overexpression of the syntaxin-binding protein Doc2B or ubMunc13-2 increases syntaxin-1 immunoavailability and concomitantly down-regulates Ca²⁺ currents. Similar findings were obtained upon chemical cholesterol depletion, leading directly to syntaxin-1 cluster dispersal and Ca²⁺ current inhibition. We conclude that clustering of syntaxin-1 allows the cell to maintain a high syntaxin-1 expression level without compromising Ca²⁺ influx, and recruitment of syntaxin-1 from clusters by SNAP-25 expression makes it available for regulating Ca²⁺ channels. This mechanism potentially allows the cell to regulate Ca²⁺ influx by expanding or contracting syntaxin-1 clusters.