Dataset Information

Allosteric control of syntaxin 1a by Munc18-1: characterization of the open and closed conformations of syntaxin.

ABSTRACT: Syntaxin 1a is a plasma membrane soluble N-ethylmaleimide-sensitive factor attachment receptor protein (SNARE) that contains an H3 domain (SNARE motif) and a regulatory Habc domain. These regions associate to produce a closed state, which is generally thought to suppress assembly of syntaxin into the SNARE complex. However, the molecular nature of the closed and open states of syntaxin is not well defined. Here, we use electron paramagnetic resonance spectroscopy to characterize conformational exchange in syntaxin. The data indicate that the H3 segment is in equilibrium between ordered and disordered states that have significant populations. In solution, the central region of the H3 segment is positioned close to the Habc domain and the configuration of syntaxin 1a is dominated by a closed state. However, an open state is enhanced in full-length membrane reconstituted syntaxin. Munc18-1 binding alters the equilibrium along H3 to favor the ordered, folded state. Munc18 also suppresses the minor open population and narrows the distance distributions between H3 and Habc. The allosteric control exhibited by Munc18 on the H3 segment and the suppression of the minor open component may both play a role in regulating membrane fusion by controlling the assembly of syntaxin into the SNARE complex.

SUBMITTER: Dawidowski D

PROVIDER: S-EPMC3617443 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Publications

Allosteric control of syntaxin 1a by Munc18-1: characterization of the open and closed conformations of syntaxin.

Dawidowski Damian D Cafiso David S DS

Biophysical journal 20130401 7

Syntaxin 1a is a plasma membrane soluble N-ethylmaleimide-sensitive factor attachment receptor protein (SNARE) that contains an H3 domain (SNARE motif) and a regulatory Habc domain. These regions associate to produce a closed state, which is generally thought to suppress assembly of syntaxin into the SNARE complex. However, the molecular nature of the closed and open states of syntaxin is not well defined. Here, we use electron paramagnetic resonance spectroscopy to characterize conformational e ...[more]

PMID: 23561535

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Project description:We have shown that SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins not only participate directly in exocytosis, but also regulate the dominant membrane-repolarizing Kv channels (voltage-gated K+ channels), such as Kv2.1, in pancreatic beta-cells. In a recent report, we demonstrated that WT (wild-type) Syn-1A (syntaxin-1A) inhibits Kv2.1 channel trafficking and gating through binding to the cytoplasmic C-terminus of Kv2.1. During beta-cell exocytosis, Syn-1A converts from a closed form into an open form which reveals its active H3 domain to bind its SNARE partners SNAP-25 (synaptosome-associated protein of 25 kDa) and synaptobrevin. In the present study, we compared the effects of the WT Syn-1A and a mutant open form Syn-1A (L165A, E166A) on Kv2.1 channel trafficking and gating. When co-expressed in HEK-293 cells (human embryonic kidney-293 cells), the open form Syn-1A decreased Kv2.1 current density more than (P<0.05) the WT Syn-1A (166+/-35 and 371+/-93 pA/pF respectively; control=911+/-91 pA/pF). Confocal microscopy and biotinylation experiments showed that both the WT and open form Syn-1A inhibited Kv2.1 expression at the plasma membrane to a similar extent, suggesting that the stronger reduction of Kv2.1 current density by the open form compared with the WT Syn-1A is probably due to a stronger direct inhibition of channel activity. Consistently, dialysis of the recombinant open form Syn-1A protein into Kv2.1-expressing HEK-293 cells caused stronger inhibition of Kv2.1 current amplitude (P<0.05) than the WT Syn-1A protein (73+/-2 and 82+/-3% of the control respectively). We found that the H3 but not H(ABC) domain is the putative active domain of Syn-1A, which bound to and inhibited the Kv2.1 channel. When co-expressed in HEK-293 cells, the open-form Syn-1A slowed down Kv2.1 channel activation (tau=12.3+/-0.8 ms) much more than (P<0.05) WT Syn-1A (tau=7.9+/-0.8 ms; control tau=5.5+/-0.6 ms). In addition, only the open form Syn-1A, but not the WT Syn-1A, caused a significant (P<0.05) left-shift in the steady-state inactivation curve (V(1/2)=33.1+/-1.3 and -29.4+/-1.1 mV respectively; control V(1/2)=-24.8+/-2 mV). The present study therefore indicates that the open form of Syn-1A is more potent than the WT Syn-1A in inhibiting the Kv2.1 channel. Such stronger inhibition by the open form of Syn-1A may limit K+ efflux and thus decelerate membrane repolarization during exocytosis, leading to optimization of insulin release.

Dataset Information

Allosteric control of syntaxin 1a by Munc18-1: characterization of the open and closed conformations of syntaxin.

Publications

Allosteric control of syntaxin 1a by Munc18-1: characterization of the open and closed conformations of syntaxin.

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