TY - JOUR
T1 - Expression and function of the rat vesicular monoamine transporter 2
AU - Adam, Yoav
AU - Edwards, Robert H.
AU - Schuldiner, Shimon
PY - 2008/4
Y1 - 2008/4
N2 - 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 ΔμH+-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.
AB - 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 ΔμH+-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.
KW - Ion-coupled transporters
KW - Membrane protein
KW - Monoamines
KW - Neurotransmitter storage
UR - http://www.scopus.com/inward/record.url?scp=41949089632&partnerID=8YFLogxK
U2 - 10.1152/ajpcell.00348.2007
DO - 10.1152/ajpcell.00348.2007
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C2 - 18287335
AN - SCOPUS:41949089632
SN - 0363-6143
VL - 294
SP - C1004-C1011
JO - American Journal of Physiology - Cell Physiology
JF - American Journal of Physiology - Cell Physiology
IS - 4
ER -