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

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.