Mutational scanning of the human serotonin transporter reveals fast translocating serotonin transporter mutants.
The serotonin transporter (SERT) belongs to a family of sodium-chloride-dependent transporters responsible for uptake of amino acids and biogenic amines from the extracellular space. SERT represents a major pharmacological target in the treatment of several clinical conditions, including depression and anxiety. In the present study we have undertaken a mutational scanning of human SERT in order to identify residues that are responsible for individual differences among related monoamine transporters. One mutant, G100A, was inactive in transport. However, ligand binding affinity was similar to wild-type, suggesting that G100A amongst different possible SERT conformations is restrained to a binding conformation. We suggest that the main role of glycine-100 is to confer structural flexibility during substrate translocation. For the two single mutants, T178A and F263C, uptake rates and K(m) values were both several-fold higher than wild-type while binding affinities and inhibitory potencies decreased considerably for several drugs. Ion dependency increased and only at hyperosmotic concentrations were K(m) values partly restored. For the double mutant, T178A/F263C, shifts in uptake kinetics and ligand affinities, as well as ion dependencies, were drastic. Effects were synergistic compared to the corresponding single mutants. In conclusion, we suggest that mutating threonine-178 to an alanine and phenylalanine-263 to a cysteine mainly alter the overall uptake kinetics of SERT by affecting the conformational equilibrium of different transporter conformations.
Kristensen
Anders S
AS
Laboratory of Molecular Neurobiology, Department of Biological Psychiatry, Aarhus Psychiatric University Hospital, Skovagervej 2, 8240 Risskov, Denmark.
Larsen
Mads B
MB
Johnsen
Laust B
LB
Wiborg
Ove
O
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D001692:Biological Transport; D015153:Blotting, Western / Q000379:methods; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002678:Chimera; D002522:Chlorocebus aethiops; D002794:Choline / Q000494:pharmacology; D003001:Cloning, Molecular; D003042:Cocaine / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D026901:Membrane Transport Proteins; D008698:Mesylates / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009419:Nerve Tissue Proteins; D019275:Radiopharmaceuticals / Q000493:pharmacokinetics; D012701:Serotonin / Q000031:analogs & derivatives / Q000378:metabolism; D050486:Serotonin Plasma Membrane Transport Proteins; D012964:Sodium / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014162:Transfection / Q000379:methods
D002352:Carrier Proteins; D007202:Indicators and Reagents; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D008698:Mesylates; D009419:Nerve Tissue Proteins; D019275:Radiopharmaceuticals; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; C109153:(2-sulfonatoethyl)methanethiosulfonate; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D012701:Serotonin; C069723:2beta-carbomethoxy-3beta-(4-iodophenyl)tropane; D012964:Sodium; D003042:Cocaine; D002794:Choline