TY - JOUR
T1 - Emulating proton-induced conformational changes in the Vesicular monoamine transporter VMAT2 by mutagenesis
AU - Yaffe, Dana
AU - Vergara-Jaque, Ariela
AU - Forrest, Lucy R.
AU - Schuldiner, Shimon
PY - 2016/11/22
Y1 - 2016/11/22
N2 - 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 ofmonoamines 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 lumento 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.
AB - 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 ofmonoamines 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 lumento 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.
KW - Ion coupling
KW - Neurotransmitter transporter
KW - Reserpine
KW - Tetrabenazine
UR - http://www.scopus.com/inward/record.url?scp=84996563942&partnerID=8YFLogxK
U2 - 10.1073/pnas.1605162113
DO - 10.1073/pnas.1605162113
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C2 - 27821772
AN - SCOPUS:84996563942
SN - 0027-8424
VL - 113
SP - E7390-E7398
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 47
ER -