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Am J Physiol Cell Physiol 294: C1004-C1011, 2008. First published February 20, 2008; doi:10.1152/ajpcell.00348.2007
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Expression and function of the rat vesicular monoamine transporter 2

Yoav Adam,1 Robert H. Edwards,2 and Shimon Schuldiner1

1Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, Israel; and 2Departments of Neurology and Physiology, University of California School of Medicine, San Francisco, California

Submitted 5 August 2007 ; accepted in final form 15 February 2008

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 {Delta}µH+-driven electrogenic transport of serotonin. The reconstituted purified rVMAT2 has wild-type affinity for serotonin, and its turnover rate is ~0.4 substrate molecule/s.

membrane protein; ion-coupled transporters; neurotransmitter storage; monoamines


Address for reprint requests and other correspondence: S. Schuldiner, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, Israel (e-mail: Shimon.Schuldiner{at}huji.ac.il)






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