A Central Small Amino Acid in the VAMP2 Transmembrane Domain Regulates the Fusion Pore in Exocytosis
Sci Rep. 2017-06-06; 7(1):
DOI: 10.1038/s41598-017-03013-3
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1. Sci Rep. 2017 Jun 6;7(1):2835. doi: 10.1038/s41598-017-03013-3.
A Central Small Amino Acid in the VAMP2 Transmembrane Domain Regulates the Fusion
Pore in Exocytosis.
Hastoy B(1)(2)(3), Scotti PA(1)(2), Milochau A(1)(2), Fezoua-Boubegtiten Z(1)(2),
Rodas J(2)(4)(5), Megret R(2)(4)(5), Desbat B(1)(2), Laguerre M(1)(2), Castano
S(1)(2), Perrais D(2)(6), Rorsman P(3), Oda R(1)(2), Lang J(7)(8).
Author information:
(1)Laboratory of Membrane Chemistry and Biology (CBMN), UMR CNRS 5248, Université
de Bordeaux, Allée de Geoffroy St Hilaire, 33600, Pessac, France.
(2)Université de Bordeaux, 351 Cours de la Libération, 33400, Talence, France.
(3)Oxford Centre for Diabetes, Endocrinology and Metabolism, University of
Oxford, Churchill Hospital, Oxford, OX3 7LJ, UK.
(4)Laboratoire de l’Intégration du Matériau au Système, UMR CNRS 5218, 351 Cours
de la Libération, 33400 Talence, France.
(5)Institut Polytechnique de Bordeaux, Avernue des Facultés, 33405, Talence,
France.
(6)Interdisciplinary Institute for Neuroscience, UMR CNRS 5287, 146, rue
Léo-Saignat, 33077, Bordeaux, France.
(7)Laboratory of Membrane Chemistry and Biology (CBMN), UMR CNRS 5248, Université
de Bordeaux, Allée de Geoffroy St Hilaire, 33600, Pessac, France.
.
(8)Université de Bordeaux, 351 Cours de la Libération, 33400, Talence, France.
.
Exocytosis depends on cytosolic domains of SNARE proteins but the function of the
transmembrane domains (TMDs) in membrane fusion remains controversial. The TMD of
the SNARE protein synaptobrevin2/VAMP2 contains two highly conserved small amino
acids, G100 and C103, in its central portion. Substituting G100 and/or C103 with
the β-branched amino acid valine impairs the structural flexibility of the TMD in
terms of α-helix/β-sheet transitions in model membranes (measured by infrared
reflection-absorption or evanescent wave spectroscopy) during increase in
protein/lipid ratios, a parameter expected to be altered by recruitment of SNAREs
at fusion sites. This structural change is accompanied by reduced membrane
fluidity (measured by infrared ellipsometry). The G100V/C103V mutation nearly
abolishes depolarization-evoked exocytosis (measured by membrane capacitance) and
hormone secretion (measured biochemically). Single-vesicle optical (by TIRF
microscopy) and biophysical measurements of ATP release indicate that G100V/C103V
retards initial fusion-pore opening, hinders its expansion and leads to premature
closure in most instances. We conclude that the TMD of VAMP2 plays a critical
role in membrane fusion and that the structural mobility provided by the central
small amino acids is crucial for exocytosis by influencing the molecular
re-arrangements of the lipid membrane that are necessary for fusion pore opening
and expansion.
DOI: 10.1038/s41598-017-03013-3
PMCID: PMC5460238
PMID: 28588281