Identification of conformationally sensitive residues essential for inhibition of vesicular monoamine transport by the noncompetitive inhibitor tetrabenazine.
Vesicular monoamine transporter 2 (VMAT2) transports monoamines into storage vesicles in a process that involves exchange of the charged monoamine with two protons. VMAT2 is a member of the DHA12 family of multidrug transporters that belongs to the major facilitator superfamily of secondary transporters. Tetrabenazine (TBZ) is a non-competitive inhibitor of VMAT2 that is used in the treatment of hyperkinetic disorders associated with Huntington disease and Tourette syndrome. Previous biochemical studies suggested that the recognition site for TBZ and monoamines is different. However, the precise mechanism of TBZ interaction with VMAT2 remains unknown. Here we used a random mutagenesis approach and selected TBZ-resistant mutants. The mutations clustered around the lumenal opening of the transporter and mapped to either conserved proline or glycine, or to residues immediately adjacent to conserved proline and glycine. Directed mutagenesis provides further support for the essential role of the latter residues. Our data strongly suggest that the conserved α-helix breaking residues identified in this work play an important role in conformational rearrangements required for TBZ binding and substrate transport. Our results provide a novel insight into the mechanism of transport and TBZ binding by VMAT2.
From the Department of Biological Chemistry, Alexander A. Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
Yelena
Y
Ugolev
From the Department of Biological Chemistry, Alexander A. Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
Tali
T
Segal
From the Department of Biological Chemistry, Alexander A. Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
Dana
D
Yaffe
From the Department of Biological Chemistry, Alexander A. Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
Yael
Y
Gros
From the Department of Biological Chemistry, Alexander A. Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel. Electronic address: Shimon.Schuldiner@huji.ac.il.
Shimon
S
Schuldiner
D018759:Adrenergic Uptake Inhibitors / Q000493:pharmacokinetics / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D001693:Biological Transport, Active / Q000187:drug effects / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D006816:Huntington Disease / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D051381:Rats; D012441:Saccharomyces cerevisiae; D013747:Tetrabenazine / Q000493:pharmacokinetics / Q000494:pharmacology*; D005879:Tourette Syndrome / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D050493:Vesicular Monoamine Transport Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*
D018759:Adrenergic Uptake Inhibitors; C493454:Slc18a2 protein, rat; D050493:Vesicular Monoamine Transport Proteins; D013747:Tetrabenazine
Directed Evolution; Ion-coupled Transporters; MFS Superfamily; Membrane Proteins; Multidrug Transporters; Neurotransmitter Transport; Neurotransmitters; PXXP Motifs