期刊
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY
卷 294, 期 4, 页码 C1004-C1011出版社
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajpcell.00348.2007
关键词
membrane protein; ion-coupled transporters; neurotransmitter storage; monoamines
资金
- NIGMS NIH HHS [P50 GM73210] Funding Source: Medline
- NINDS NIH HHS [NS-16708] Funding Source: Medline
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 mu(H+) -driven electrogenic transport of serotonin. The reconstituted purified rVMAT2 has wild- type affinity for serotonin, and its turnover rate is similar to 0.4 substrate molecule/s.
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