Cysteine-Scanning Mutagenesis of the Fifth External Loop of Serotonin Transporter

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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.