Tracking Ca2+-dependent and Ca2+-independent conformational transitions in syntaxin 1A during exocytosis in neuroendocrine cells

Abstract

SummaryA 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.

GABA B receptor deficiency causes failure of neuronal homeostasis in hippocampal networks

“…3B). The engineered Synt 1a FRET reporter has been shown to assemble into endogenous SNARE complexes and was able to reconstitute dense-core vesicle exocytosis in PC12 cells (27). Furthermore, we show that neurons transfected with the light chain of BoNT-C1 are not capable of synaptic vesicle recycling, even during strong stimulation (600 pulses at 20 Hz; Fig.…”

“…To assess whether chronic inactivity promotes Synt 1 opening, we used a recently developed intramolecular Synt 1a FRET probe (CFP-Synt 1a -YFP) that reports the closed-to-open transition as a decrease in FRET efficiency (27). The Synt 1a sensor contains CFP fluorophore inserted at the N terminus and YFP inserted after the SNARE motif (Fig.…”

“…Synt 1a (CSYS-5RK), Synt 1a Open , and Synt 1a K253I are as described in ref. 27. W66A point mutation was introduced to silence YFP in the YFP-Synt 1a -CFP construct (Fig.…”

The Munc18-1 domain 3a hinge-loop controls syntaxin-1A nanodomain assembly and engagement with the SNARE complex during secretory vesicle priming

“…Munc18-1 is critically involved in delivering syntaxin-1A to the plasma membrane ( Rickman et al, 2007 ; Han et al, 2009 ; Malintan et al, 2009 ) and in controlling its opening and access to other SNARE proteins ( Fasshauer and Margittai, 2004 ; Rickman et al, 2007 ; Greitzer-Antes et al, 2013 ). We therefore investigated whether syntaxin-1A was also present in Munc18-1 nanodomains.…”

VAMP721 Conformations Unmask an Extended Motif for K+ Channel Binding and Gating Control

“…Because we suspected that Tyr-57 and the residues around it might affect the longin conformation and its interaction with the SNARE domain, we sought to explore this possibility using Förster resonance energy transfer (FRET). The efficiency of FRET energy transfer is high over molecular dimensions and falls off with the sixth power of distance between donor and acceptor fluorophores, making the FRET fluorescence signal ideal for structural studies of proteins and their complexes in vivo (Deniz et al, 2000;Kang et al, 2012;Greitzer-Antes et al, 2013).…”