External cysteine residues in the serotonin transporter.
Hydropathy analysis predicts three cysteines (C109, C200, and C209) in extracellular loops of the rat serotonin transporter (SERT). We mutated these residues, singly and in combination, to either alanine or serine and expressed the mutant transporters in HeLa cells using the vaccinia-T7 transient expression system. Mutation of C109 to alanine had no effect on transport activity or surface expression of the transporter. In Na+-containing solutions, methanethiosulfonate (MTS) reagents had little effect on transport activity in the wild type or in the C109A mutant. When Na+ was replaced with Li+, inactivation of wild type by MTS reagents increased dramatically, but C109A was still resistant. The results suggest that C109 is exposed to the external medium in a manner dependent on cation binding. Replacing either C200 or C209 with serine resulted in either a partial (C200S) or almost total (C209S) loss of transport activity. MTS reagents rapidly inactivated transport activity in mutant C200S, suggesting increased accessibility of a previously unreactive cysteine residue. The double mutants C200S-C109A and C200S-C209S each retained partial activity. C200S-C109A was very sensitive to MTS reagents, but the C200S-C209S mutant was much less sensitive, similar to the wild type transporter. Replacement of C200 or C209 with serine dramatically decreased surface expression of the fully glycosylated transporter. Expression was normal, however, in the C200S-C209S double mutant. The Na+ dependence of transport and ligand binding was abnormal in both C200S and C200S-C209S mutants. Replacing C200 or C209 had similar effects on Na+ dependence and surface expression. Together with the increased MTS reactivity of C200S, these results support the possibility that C200 and C209 may be linked by a disulfide bond in the second external loop of SERT.
Chen
J G
JG
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Liu-Chen
S
S
Rudnick
G
G
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000032:analysis*; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D012701:Serotonin / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins; D012964:Sodium / Q000378:metabolism; D014162:Transfection
D002352:Carrier Proteins; D007202:Indicators and Reagents; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; C493232:Slc6a4 protein, rat; C093171:methanethiosulfonate ethylammonium; D012701:Serotonin; D005020:Ethyl Methanesulfonate; D012964:Sodium; D003545:Cysteine