TY - JOUR
T1 - The dual function of the polybasic juxtamembrane region of syntaxin 1a in clamping spontaneous release and stimulating Ca 2+ -triggered release in neuroendocrine cells
AU - Singer-Lahat, Dafna
AU - Barak-Broner, Noa
AU - Sheinin, Anton
AU - Greitzer-Antes, Dafna
AU - Michaelevski, Izhak
AU - Lotan, Ilana
N1 - Publisher Copyright:
© 2018 the authors.
PY - 2018/1/3
Y1 - 2018/1/3
N2 - The exact function of the polybasic juxtamembrane region (5RK) of the plasma membrane neuronal SNARE, syntaxin 1A (Syx), in vesicle exocytosis, although widely studied, is currently not clear. Here, we addressed the role of 5RK in Ca 2+ -triggered release, using our Syx-based intramolecular fluorescence resonance energy transfer (FRET) probe, which previously allowed us to resolve a depolarization-induced Ca 2+ -dependent close-to-open transition (CDO) of Syx that occurs concomitant with evoked release, both in PC12 cells and hippocampal neurons and was abolished upon charge neutralization of 5RK. First, using dynamic FRET analysis in PC12 cells, we show that CDO occurs following assembly of SNARE complexes that include the vesicular SNARE, synaptobrevin 2, and that the participation of 5RK in CDO goes beyond its participation in the final zippering of the complex, because mutations of residues adjacent to 5RK, believed to be crucial for final zippering, do not abolish this transition. In addition, we show that CDO is contingent on membrane phosphatidylinositol 4,5-bisphosphate (PIP2), which is fundamental for maintaining regulated exocytosis, as depletion of membranal PIP2 abolishes CDO. Prompted by these results, which underscore a potentially significant role of 5RK in exocytosis, we next amperometrically analyzed catecholamine release from PC12 cells, revealing that charge neutralization of 5RK promotes spontaneous and inhibits Ca 2+ -triggered release events. Namely, 5RK acts as a fusion clamp, making release dependent on stimulation by Ca 2+ .
AB - The exact function of the polybasic juxtamembrane region (5RK) of the plasma membrane neuronal SNARE, syntaxin 1A (Syx), in vesicle exocytosis, although widely studied, is currently not clear. Here, we addressed the role of 5RK in Ca 2+ -triggered release, using our Syx-based intramolecular fluorescence resonance energy transfer (FRET) probe, which previously allowed us to resolve a depolarization-induced Ca 2+ -dependent close-to-open transition (CDO) of Syx that occurs concomitant with evoked release, both in PC12 cells and hippocampal neurons and was abolished upon charge neutralization of 5RK. First, using dynamic FRET analysis in PC12 cells, we show that CDO occurs following assembly of SNARE complexes that include the vesicular SNARE, synaptobrevin 2, and that the participation of 5RK in CDO goes beyond its participation in the final zippering of the complex, because mutations of residues adjacent to 5RK, believed to be crucial for final zippering, do not abolish this transition. In addition, we show that CDO is contingent on membrane phosphatidylinositol 4,5-bisphosphate (PIP2), which is fundamental for maintaining regulated exocytosis, as depletion of membranal PIP2 abolishes CDO. Prompted by these results, which underscore a potentially significant role of 5RK in exocytosis, we next amperometrically analyzed catecholamine release from PC12 cells, revealing that charge neutralization of 5RK promotes spontaneous and inhibits Ca 2+ -triggered release events. Namely, 5RK acts as a fusion clamp, making release dependent on stimulation by Ca 2+ .
KW - Amperometry
KW - Exocytosis
KW - FRET
KW - Fusion clamp
KW - Neuroendocrine cells
KW - Syntaxin-1A
UR - http://www.scopus.com/inward/record.url?scp=85040083964&partnerID=8YFLogxK
U2 - 10.1523/JNEUROSCI.1541-17.2017
DO - 10.1523/JNEUROSCI.1541-17.2017
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AN - SCOPUS:85040083964
SN - 0270-6474
VL - 38
SP - 220
EP - 231
JO - Journal of Neuroscience
JF - Journal of Neuroscience
IS - 1
ER -