Cysteine-scanning mutagenesis of the fifth external loop of serotonin transporter

Biochemistry
Preston C KellerGary Rudnick

Abstract

External loop 5 (EL5) of serotonin transporter was analyzed by mutating each of the residues from Thr-480 to Ala-511, one at a time, with cysteine. Cysteine was well-tolerated at most positions, although G485C, Y495C, and E508C had low transport activities. Replacement with cysteine rendered mutants G484C-P499C sensitive to partial or complete inactivation by [2-(trimethylammonium)ethyl] methanethiosulfonate and (2-sulfonatoethyl) methanethiosulfonate. Within this sensitive region, the rates of reaction varied by over 2 orders of magnitude. Rates of inactivation were not significantly affected by removal of Na(+) or by addition of cocaine or serotonin. These results suggest that modification of EL5 interferes with the transport process but is not sensitive to substrate and ion binding.

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Citations

Apr 10, 2007·The Journal of Membrane Biology·Gary Rudnick
Jun 27, 2009·Chemical Communications : Chem Comm·Jacob AndersenKristian Strømgaard
Jul 24, 2008·Proceedings of the National Academy of Sciences of the United States of America·Lucy R ForrestGary Rudnick
Apr 19, 2011·The Journal of Biological Chemistry·Padmanabhan MannangattiLankupalle D Jayanthi
Jul 17, 2008·Protein Science : a Publication of the Protein Society·Melissa I Torres-AltoroEric L Barker
Sep 30, 2006·Protein Science : a Publication of the Protein Society·Kellie J WhiteEric L Barker
Jul 16, 2010·Journal of Neurochemistry·Balasubramaniam AnnamalaiLankupalle D Jayanthi
Mar 1, 2008·Molecular Membrane Biology·Yuxin MaoGary Rudnick
Mar 23, 2010·The Journal of Biological Chemistry·Melissa I Torres-AltoroEric L Barker
Jul 5, 2005·The Journal of Biological Chemistry·Yuan-Wei Zhang, Gary Rudnick
Sep 30, 2006·The Journal of Biological Chemistry·Yuan-Wei Zhang, Gary Rudnick
Jul 15, 2011·Pharmacological Reviews·Anders S KristensenUlrik Gether
Jan 26, 2006·Molecular Pharmacology·Henrik Amtoft NeubauerOve Wiborg

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