The rat serotonin transporter: identification of cysteine residues important for substrate transport

Reduction and alkylation of disulfide bonds are known to affect substrate translocation by and antidepressant binding to the serotonin transporter (SERT). To identify functionally relevant cysteine residues, we substituted 16 cysteins of the rat SERT by alanine or serine residues and analyzed the transport and binding properties of the respective mutant transporters after heterologous expression in a mammalian cell line. Replacement of cysteine 209 by serine resulted in a marked reduction of the maximal transport rate, loss of positive cooperativity, and insensitivity to treatment with disulfide reducing agents, indicating that cysteine 209 participates in a structurally important disulfide bridge. Replacement of cysteine residues 147, 200, 369, and 540 caused a complete loss of both substrate transport and antidepressant binding, a result that is likely to reflect impaired processing and/or cell surface expression of the mutated polypeptides.