Single-channel currents produced by the serotonin transporter and analysis of a mutation affecting ion permeation.
Single-channel activities were observed in outside-out patches excised from oocytes expressing a mammalian 5-hydroxytryptamine (5-HT) transporter. Channel conductance was larger for a mutant in which asparagine177 of the third putative transmembrane domain was replaced by glycine, suggesting that this residue lies within or near the permeation pathway. The N177G mutant enables quantitative single-channel measurements; it displays two conducting states. One state, with conductance of approximately 6 pS, is induced by 5-HT and is permeable to Na+. The other state (conductance of approximately 13 pS) is associated with substrate-independent leakage current and is permeable to both Na+ and Li+. Cl- is not a major current carrier. Channel lifetimes under all conditions measured are approximately 2.5 ms. The single-channel phenomena account for previously observed macroscopic electrophysiological phenomena, including 5-HT-induced transport-associated currents and substrate-independent leakage currents. The channel openings occur several orders of magnitude less frequently than would be expected if one such opening occurred for each transport cycle and therefore do not represent an obligatory step in transport. Nevertheless, single-channel events produced by neurotransmitter transporters indicate the functional and structural similarities between transporters and ion channels and provide a new tool, at single-molecule resolution, for detailed structure-function studies of transporters.
Lin
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Division of Biology, California Institute of Technology, Pasadena 91125, USA.
Lester
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HA
Mager
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D000818:Animals; D001216:Asparagine; D002352:Carrier Proteins / Q000737:chemistry / Q000502:physiology*; D004553:Electric Conductivity; D005260:Female; D005473:Fluoxetine / Q000494:pharmacology; D005998:Glycine; D007473:Ion Channels / Q000737:chemistry / Q000502:physiology*; D008322:Mammals; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects; D026901:Membrane Transport Proteins; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012701:Serotonin / Q000494:pharmacology*; D050486:Serotonin Plasma Membrane Transport Proteins; D012964:Sodium / Q000494:pharmacology; D014982:Xenopus laevis
D002352:Carrier Proteins; D007473:Ion Channels; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D050486:Serotonin Plasma Membrane Transport Proteins; D005473:Fluoxetine; D012701:Serotonin; D001216:Asparagine; D012964:Sodium; D005998:Glycine