文章基本信息

标题：Emulating proton-induced conformational changes in the vesicular monoamine transporter VMAT2 by mutagenesis
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作者：Dana Yaffe ; Ariela Vergara-Jaque ; Lucy R. Forrest 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2016
卷号：113
期号：47
页码：E7390-E7398
DOI：10.1073/pnas.1605162113
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceVesicular monoamine transporters (VMATs) are the targets of numerous psychoactive drugs, and play a critical role in the overall process of synaptic transmission by replenishing depleted monoamine stores in synaptic vesicles. VMATs transport monoamines in a process that involves exchange of two H+ per substrate. Here we show that two potent inhibitors of VMAT2, tetrabenazine and reserpine, bind to different conformations of the protein. The transition that generates a reserpine-binding site requires a proton gradient across the membrane. Here we emulate the effect of the proton gradient by tinkering with residues that form the cytoplasmic gate. These findings provide vital information about the conformational dynamics of a mammalian H+-coupled antiporter. Such conformational transitions constitute essential steps in all transport processes. Neurotransporters located in synaptic vesicles are essential for communication between nerve cells in a process mediated by neurotransmitters. Vesicular monoamine transporter (VMAT), a member of the largest superfamily of transporters, mediates transport of monoamines to synaptic vesicles and storage organelles in a process that involves exchange of two H+ per substrate. VMAT transport is inhibited by the competitive inhibitor reserpine, a second-line agent to treat hypertension, and by the noncompetitive inhibitor tetrabenazine, presently in use for symptomatic treatment of hyperkinetic disorders. During the transport cycle, VMAT is expected to occupy at least three different conformations: cytoplasm-facing, occluded, and lumen-facing. The lumen- to cytoplasm-facing transition, facilitated by protonation of at least one of the essential membrane-embedded carboxyls, generates a binding site for reserpine. Here we have identified residues in the cytoplasmic gate and show that mutations that disrupt the interactions in this gate also shift the equilibrium toward the cytoplasm-facing conformation, emulating the effect of protonation. These experiments provide significant insight into the role of proton translocation in the conformational dynamics of a mammalian H+-coupled antiporter, and also identify key aspects of the mode of action and binding of two potent inhibitors of VMAT2: reserpine binds the cytoplasm-facing conformation, and tetrabenazine binds the lumen-facing conformation.
关键词：neurotransmitter transporter ; ion coupling ; reserpine ; tetrabenazine