Emulating proton-induced conformational changes in the vesicular monoamine transporter VMAT2 by mutagenesis.
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.
Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University, Jerusalem 91904, Israel.
Dana
D
Yaffe
Computational Structural Biology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.
Ariela
A
Vergara-Jaque
Computational Structural Biology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.
Lucy R
0000-0003-1855-7985
LR
Forrest
Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University, Jerusalem 91904, Israel; shimon.schuldiner@huji.ac.il.
Shimon
0000-0002-4874-6237
S
Schuldiner
D000818:Animals; D001665:Binding Sites; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D011522:Protons; D051381:Rats; D012110:Reserpine / Q000378:metabolism*; D013747:Tetrabenazine / Q000378:metabolism*; D050493:Vesicular Monoamine Transport Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D011522:Protons; C493454:Slc18a2 protein, rat; D050493:Vesicular Monoamine Transport Proteins; D012110:Reserpine; D013747:Tetrabenazine
ion coupling; neurotransmitter transporter; reserpine; tetrabenazine