The third transmembrane domain of the serotonin transporter contains residues associated with substrate and cocaine binding.
Twenty residues in the third transmembrane domain of the serotonin transporter (SERT) were mutated, one at a time, to cysteine. Almost all of these mutants were fully active for serotonin (5-HT) transport and insensitive to inactivation by the positively charged cysteine reagent [2-(trimethylammonium)ethyl]methanethiosul-fonate (MTSET). Two active mutants, I172C and I179C, were sensitive to rapid inactivation by MTSET but were relatively insensitive to the negatively charged reagent (2-sulfonatoethyl)methanethiosulfonate (MTSES). Inactivation of I172C was blocked by 5-HT and cocaine, but I179C was not similarly protected. Replacement of Tyr-175 with cysteine resulted in a mutant with low transport activity, and, at the neighboring Tyr-176, cysteine replacement completely blocked transport. The Y175C and Y176C mutants were expressed on the cell surface at levels 84% and 69%, respectively, that of wild type (C109A) SERT. Mutants Y175C and Y176C had lower cocaine affinity than C109A, as measured by displacement of the high affinity cocaine analog 2beta-carbomethoxy-3beta-(4-[125I]iodophenyl)tropane (beta-CIT). For Y176C, 5-HT affinity also was decreased. MTSET inactivated beta-CIT binding to I172C and Y176C, but only slightly inhibited binding to I179C and C109A. The MTSET sensitivity of cysteine replacements at positions 172, 176, and 179 was not observed when these positions were replaced with alanine, serine, or methionine. The results suggest that Ile-172, Tyr-176 and Ile-179 are on one face of an alpha-helical transmembrane element, and that Ile-172 and Tyr-176 are in proximity to the binding site for 5-HT and cocaine.
Chen
J G
JG
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510-8066, USA.
Sachpatzidis
A
A
Rudnick
G
G
D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003042:Cocaine / Q000031:analogs & derivatives / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology; D007532:Isoleucine / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008698:Mesylates / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins; D017433:Protein Structure, Secondary; D012701:Serotonin / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D007202:Indicators and Reagents; D008024:Ligands; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D008698:Mesylates; D009419:Nerve Tissue Proteins; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; C093171:methanethiosulfonate ethylammonium; D007532:Isoleucine; C109153:(2-sulfonatoethyl)methanethiosulfonate; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D012701:Serotonin; D014443:Tyrosine; C069723:2beta-carbomethoxy-3beta-(4-iodophenyl)tropane; D001216:Asparagine; D005020:Ethyl Methanesulfonate; D003042:Cocaine; D003545:Cysteine