Amino acid residues that control pH modulation of transport-associated current in mammalian serotonin transporters.
The rat and human serotonin transporters (rSERT and hSERT, respectively) were expressed in Xenopus oocytes and studied using site-directed mutagenesis, electrophysiological recordings, and [3H]5-HT uptake measurements. rSERT, but not hSERT, displayed increased transport-associated current at low pH. Chimeras and point mutations showed that, of the 52 nonidentical residues, a single residue at position 490 (threonine in rSERT and lysine in hSERT) governs this difference. Furthermore, potentiation required the glutamate residue at position 493. Cysteine substitution and alkylation experiments showed that residue 493 is extracellular. Cysteine at 493 increased, whereas aspartate decreased, the net charge movement per transported 5-HT molecule. The mutations at this region did not significantly affect other aspects of SERT function, including agonist-independent leakage current, voltage-dependent transient current, and H+ current. This region may therefore be part of an external gate required for rSERT function. The data and analyses show that, in the absence of detailed structural information, a gate-lumen-gate scheme is useful for interpreting results from mutations that alter functional properties of neurotransmitter transporters.
Cao
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Y
Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.
Li
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M
Mager
S
S
Lester
H A
HA
D000143:Acids / Q000494:pharmacology; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D004594:Electrophysiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating / Q000187:drug effects / Q000235:genetics*; D008322:Mammals; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D017354:Point Mutation; D011522:Protons; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012701:Serotonin / Q000235:genetics / Q000378:metabolism; D050486:Serotonin Plasma Membrane Transport Proteins; D014981:Xenopus
D000143:Acids; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011522:Protons; D011993:Recombinant Fusion Proteins; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; C493232:Slc6a4 protein, rat; D012701:Serotonin