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Emulating proton-induced conformational changes in the vesicular monoamine transporter VMAT2 by mutagenesis

  1. Shimon Schuldinera,1
  1. aDepartment of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University, Jerusalem 91904, Israel;
  2. bComputational Structural Biology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892

  1. Edited by H. Ronald Kaback, University of California, Los Angeles, CA, and approved October 7, 2016 (received for review March 31, 2016)

Significance

Vesicular 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.

Abstract

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.

Footnotes

  • ↵1To whom correspondence should be addressed. Email: shimon.schuldiner{at}huji.ac.il.

  • Author contributions: D.Y., L.R.F., and S.S. designed research; D.Y. and A.V.-J. performed research; D.Y., A.V.-J., L.R.F., and S.S. analyzed data; and D.Y., L.R.F., and S.S. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • Data deposition: The rVMAT2 model reported in this paper has been deposited in the Protein Model DataBase, https://bioinformatics.cineca.it/PMDB/ (accession no. PM0080553).

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1605162113/-/DCSupplemental.

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