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.
['Ugolev|Yelena|Y|', 'Segal|Tali|T|', 'Yaffe|Dana|D|', 'Gros|Yael|Y|', 'Schuldiner|Shimon|S|']
D018759:Adrenergic Uptake Inhibitors D000818:Animals D001665:Binding Sites D001693:Biological Transport, Active D057809:HEK293 Cells D006801:Humans D006816:Huntington Disease D016297:Mutagenesis, Site-Directed D017433:Protein Structure, Secondary D051381:Rats D012441:Saccharomyces cerevisiae D013747:Tetrabenazine D005879:Tourette Syndrome D050493:Vesicular Monoamine Transport Proteins
Multidrug Transporters Directed Evolution Ion-coupled Transporters MFS Superfamily Membrane Proteins Neurotransmitter Transport Neurotransmitters PXXP Motifs