Abstract
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.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Alanine / chemistry
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Amino Acid Sequence
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Animals
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Binding Sites
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Biological Transport
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Carrier Proteins / chemistry*
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Carrier Proteins / metabolism
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Cocaine / chemistry
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Cysteine / chemistry*
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Dose-Response Relationship, Drug
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Epitopes
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HeLa Cells
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Humans
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Ions
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Membrane Glycoproteins / chemistry*
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Membrane Glycoproteins / metabolism
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Membrane Transport Proteins*
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Mesylates / pharmacology
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Molecular Sequence Data
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Mutagenesis, Site-Directed*
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Mutation
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Nerve Tissue Proteins / chemistry*
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Nerve Tissue Proteins / metabolism
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Protein Structure, Secondary
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Protein Structure, Tertiary
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Rats
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Sequence Homology, Amino Acid
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Serotonin / chemistry
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Serotonin Plasma Membrane Transport Proteins
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Sodium / chemistry
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Threonine / chemistry
Substances
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Carrier Proteins
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Epitopes
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Ions
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Membrane Glycoproteins
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Membrane Transport Proteins
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Mesylates
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Nerve Tissue Proteins
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SLC6A4 protein, human
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Serotonin Plasma Membrane Transport Proteins
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Slc6a4 protein, rat
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(2-sulfonatoethyl)methanethiosulfonate
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(2-(trimethylammonium)ethyl)methanethiosulfonate
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Threonine
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Serotonin
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Sodium
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Cocaine
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Cysteine
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Alanine