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

TY - JOUR

T1 - Regulation of Ca2+ channels by SNAP-25 via recruitment of syntaxin-1 from plasma membrane clusters

AU - Toft-Bertelsen, Trine Lisberg

AU - Ziomkiewicz, Iwona

AU - Houy, Sébastien

AU - da Silva Pinheiro, Paulo César

AU - Sørensen, Jakob B

N1 - © 2016 Toft-Bertelsen et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

PY - 2016/11/1

Y1 - 2016/11/1

N2 - 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 chromaffn 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 fndings 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+ in?ux, 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+ in?ux by expanding or contracting syntaxin-1 clusters.

AB - 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 chromaffn 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 fndings 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+ in?ux, 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+ in?ux by expanding or contracting syntaxin-1 clusters.

U2 - 10.1091/mbc.e16-03-0184

DO - 10.1091/mbc.e16-03-0184

M3 - Journal article

C2 - 27605709

SN - 1059-1524

VL - 27

SP - 3329

EP - 3341

JO - Molecular Biology of the Cell

JF - Molecular Biology of the Cell

IS - 21

ER -