Critical amino acid residues in transmembrane span 7 of the serotonin transporter identified by random mutagenesis.
Transmembrane span 7 of the rat brain serotonin transporter was subjected to random mutagenesis. Of the 27 amino acid residues mutated, six were identified as functionally important by their sensitivity to nonconservative mutations. These residues were Asn-368 and Tyr-385, where substitutions that retained hydrogen-bonding ability were preferred; Gly-376 and Gly-384, where only glycine was accepted; Phe-380, where a phenyl ring was preferred; and Met-386, where hydrophobic substitutions were preferred. Mutations that did not preserve these structural characteristics were highly detrimental to serotonin transport activity. These six residues form a stripe that runs at an angle down the side of the putative alpha-helix, lending support to this structural prediction. Mutations at some of these positions also specifically impaired transport activity under low Na+ conditions. Other mutations at nearby positions in transmembrane span 7 also impaired activity in low Na+, although the activity of the mutants in high Na+ was similar to wild type. These results suggest that at least some of the six critical residues play a role in Na+ binding or perhaps in the coupling of Na+ binding to later steps in the transport cycle. These residues may be important in other aspects of the transporter's function as well.
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
K M
KM
Penado
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G
Rudnick
M M
MM
Stephan
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D012701:Serotonin / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins; D013329:Structure-Activity Relationship
D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D050486:Serotonin Plasma Membrane Transport Proteins; C493232:Slc6a4 protein, rat; D012701:Serotonin