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.
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Department of Neurochemistry, Max-Planck Institute for Brain Research, Deutschordenstrasse, 46, Frankfurt am Main, 60528, Germany.
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D000409:Alanine; D019943:Amino Acid Substitution; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D015283:Citalopram / Q000378:metabolism; D003545:Cysteine; D006801:Humans; D007099:Imipramine / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine; D012701:Serotonin / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins; D014162:Transfection
D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; C493232:Slc6a4 protein, rat; D015283:Citalopram; D012701:Serotonin; D012694:Serine; D003545:Cysteine; D000409:Alanine; D007099:Imipramine