Expression and function of the rat vesicular monoamine transporter 2.
The vesicular monoamine transporters (VMATs) are essential proteins, involved in the storage of monoamines in the central nervous system and in endocrine cells, in a process that involves exchange of 2H(+) with one substrate molecule. The VMATs interact with various native substrates and clinically relevant drugs and display the pharmacological profile of multidrug transporters. Vesicular transporters suffer from a lack of biochemical and structural data due to the difficulties in their expression. In this work we present the high-level expression of rat VMAT2 (rVMAT2) in a stable a human embryonic kidney cell line (HEK293), generated using the resistance to the neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) conferred by the protein. In addition, we describe novel procedures for the solubilization and purification of active protein, and its reconstitution into proteoliposomes. The partially purified protein in detergent binds the inhibitor tetrabenazine and, after reconstitution, displays high levels of Deltamu(H+)-driven electrogenic transport of serotonin. The reconstituted purified rVMAT2 has wild-type affinity for serotonin, and its turnover rate is approximately 0.4 substrate molecule/s.
Adam
Yoav
Y
Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, Israel.
Edwards
Robert H
RH
Schuldiner
Shimon
S
D015655:1-Methyl-4-phenylpyridinium; D000818:Animals; D002460:Cell Line; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D006801:Humans; D051381:Rats; D050493:Vesicular Monoamine Transport Proteins / Q000235:genetics* / Q000378:metabolism*
C493454:Slc18a2 protein, rat; D050493:Vesicular Monoamine Transport Proteins; D015655:1-Methyl-4-phenylpyridinium