A conformationally sensitive residue on the cytoplasmic surface of serotonin transporter

Serotonin transporter (SERT) contains a single reactive external cysteine r esidue at position 109 (Chen, J. G., Liu-Chen, S., and Rudnick, G. (1997) B iochemistry 36, 1479-1486) and seven predicted cytoplasmic cysteines. A mut ant of rat SERT (X8C) in which those eight cysteine residues were replaced by other amino acids retained similar to 32% of wild type transport activit y and similar to 56% of wild type binding activity. In contrast to wild-typ e SERT or the C109A mutant, X8C was resistant to inhibition of high affinit y cocaine analog binding by the cysteine reagent 2-(aminoethyl)methanethios ulfonate hydrobromide (MTSEA) in membrane preparations from transfected cel ls. Each predicted cytoplasmic cysteine residue was reintroduced, one at a time, into the X8C template. Reintroduction of Cys-357, located in the thir d intracellular loop, restored MTSEA sensitivity similar to that of C109A. Replacement of only Cys-109 and Cys-357 was sufficient to prevent MTSEA sen sitivity. Thus, Cys-357 was the sole cytoplasmic determinant of MTSEA sensi tivity in SERT. Both serotonin and cocaine protected SERT from inactivation by MTSEA at Cys-357. This protection was apparently mediated through a con formational change following ligand binding. Although both ligands bind in the absence of Na+ and at 4 degreesC, their ability to protect Cys-357 requ ired Na+ and was prevented at 4 degreesC. The accessibility of Cys-357 to M TSEA inactivation was increased by monovalent cations. The K+ ion, which is believed to serve as a countertransport substrate for SERT, was the most e ffective ion for increasing Cys-357 reactivity.