Cysteine-scanning mutagenesis of the fifth external loop of serotonin transporter.
External loop 5 (EL5) of serotonin transporter was analyzed by mutating each of the residues from Thr-480 to Ala-511, one at a time, with cysteine. Cysteine was well-tolerated at most positions, although G485C, Y495C, and E508C had low transport activities. Replacement with cysteine rendered mutants G484C-P499C sensitive to partial or complete inactivation by [2-(trimethylammonium)ethyl] methanethiosulfonate and (2-sulfonatoethyl) methanethiosulfonate. Within this sensitive region, the rates of reaction varied by over 2 orders of magnitude. Rates of inactivation were not significantly affected by removal of Na(+) or by addition of cocaine or serotonin. These results suggest that modification of EL5 interferes with the transport process but is not sensitive to substrate and ion binding.
Keller
Preston C
PC
Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520-8066, USA.
Stephan
Megan
M
Glomska
Hanna
H
Rudnick
Gary
G
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D003042:Cocaine / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D000939:Epitopes; D006367:HeLa Cells; D006801:Humans; D007477:Ions; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D026901:Membrane Transport Proteins; D008698:Mesylates / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D012701:Serotonin / Q000737:chemistry; D050486:Serotonin Plasma Membrane Transport Proteins; D012964:Sodium / Q000737:chemistry; D013912:Threonine / Q000737:chemistry
D002352:Carrier Proteins; D000939:Epitopes; D007477:Ions; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D008698:Mesylates; D009419:Nerve Tissue Proteins; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; C493232:Slc6a4 protein, rat; C109153:(2-sulfonatoethyl)methanethiosulfonate; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D013912:Threonine; D012701:Serotonin; D012964:Sodium; D003042:Cocaine; D003545:Cysteine; D000409:Alanine