Regulation of Ca2+ channels by SNAP-25 via recruitment of syntaxin-1 from plasma membrane clusters.
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.
Neurosecretion Group, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
Trine Lisberg
TL
Toft-Bertelsen
Neurosecretion Group, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
Iwona
I
Ziomkiewicz
Neurosecretion Group, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
Sébastien
S
Houy
Neurosecretion Group, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
Paulo S
PS
Pinheiro
Neurosecretion Group, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark jakobbs@sund.ku.dk.
Jakob B
JB
Sørensen
D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism* / Q000502:physiology; D002462:Cell Membrane; D005089:Exocytosis / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009419:Nerve Tissue Proteins / Q000378:metabolism; D018408:Patch-Clamp Techniques; D061251:Primary Cell Culture; D011485:Protein Binding; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism*; D050827:Syntaxin 1 / Q000378:metabolism*
D015220:Calcium Channels; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C494209:Snap25 protein, mouse; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D002118:Calcium