title: Tracking Ca2+-dependent and Ca2+-independent conformational transitions in syntaxin 1A during exocytosis in neuroendocrine cells.
abstract: A key issue for understanding exocytosis is elucidating the various protein interactions and the associated conformational transitions underlying soluble N-ethylmeleimide-sensitive factor attachment protein receptor (SNARE) protein assembly. To monitor dynamic changes in syntaxin 1A (Syx) conformation along exocytosis, we constructed a novel fluorescent Syx-based probe that can be efficiently incorporated within endogenous SNARE complexes, support exocytosis, and report shifts in Syx between 'closed' and 'open' conformations by fluorescence resonance energy transfer analysis. Using this probe we resolve two distinct Syx conformational transitions during membrane depolarization-induced exocytosis in PC12 cells: a partial 'opening' in the absence of Ca(2+) entry and an additional 'opening' upon Ca(2+) entry. The Ca(2+)-dependent transition is abolished upon neutralization of the basic charges in the juxtamembrane regions of Syx, which also impairs exocytosis. These novel findings provide evidence of two conformational transitions in Syx during exocytosis, which have not been reported before: one transition directly induced by depolarization and an additional transition that involves the juxtamembrane region of Syx. The superior sensitivity of our probe also enabled detection of subtle Syx conformational changes upon interaction with VAMP2, which were absolutely dependent on the basic charges of the juxtamembrane region. Hence, our results further suggest that the Ca(2+)-dependent transition in Syx involves zippering between the membrane-proximal juxtamembrane regions of Syx and VAMP2 and support the recently implied existence of this zippering in the final phase of SNARE assembly to catalyze exocytosis.
authors: Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Ramat Aviv 69978, Israel.
authors: Dafna
authors: D
authors: Greitzer-Antes
authors: Noa
authors: N
authors: Barak-Broner
authors: Shai
authors: S
authors: Berlin
authors: Yoram
authors: Y
authors: Oron
authors: Dodo
authors: D
authors: Chikvashvili
authors: Ilana
authors: I
authors: Lotan
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D005089:Exocytosis / Q000235:genetics*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D015870:Gene Expression; D057054:Molecular Imaging; D016716:PC12 Cells; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D055672:Static Electricity; D050827:Syntaxin 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D050988:Vesicle-Associated Membrane Protein 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; C494213:Stx1a protein, rat; D050827:Syntaxin 1; C494706:Vamp2 protein, rat; D050988:Vesicle-Associated Membrane Protein 2; D002118:Calcium
keywords: Exocytosis; FRET; PC12 cells; SNARE; Syntaxin 1A