Transmembrane domain I of the gamma-aminobutyric acid transporter GAT-1 plays a crucial role in the transition between cation leak and transport modes.
The sodium- and chloride-dependent gamma-aminobutyric acid (GABA) transporter is essential for synaptic transmission by this neurotransmitter. GAT-1 expressed in Xenopus laevis oocytes exhibits sodium-dependent GABA-induced inward currents reflecting electrogenic sodium-coupled transport. In lithium-containing medium, GAT-1 mediates GABA-independent currents, the relationship of which to the physiological transport process is poorly understood. In this study, mutants are described that appear to be locked in this cation leak mode. When Gly(63), located in the middle of the highly conserved transmembrane domain I, was mutated to serine or cysteine, sodium-dependent GABA currents were abolished. Strikingly, these mutants exhibited robust inward currents in lithium- as well as potassium-containing media. Membrane-impermeant sulfhydryl reagents inhibited these currents of the cysteine but not of the serine mutant, indicating that this position was accessible to the external aqueous medium. The cation leak currents mediated by wild-type GAT-1 were inhibited by low millimolar sodium concentrations in a noncompetitive manner. Mutations at other positions of transmembrane domain I increased or decreased the apparent sodium affinity, as monitored by the sodium-dependent steady-state GABA currents or transient currents. In parallel, the ability of sodium to inhibit the cation leak currents was increased or decreased, respectively. Thus, transmembrane domain I of GAT-1 contains determinants controlling both sodium-coupled GABA flux and the cation leak pathway as well as the interconversion of these distinct modes. Our observations suggest the possibility that the permeation pathway in both modes shares common structural elements.
Kanner
Baruch I
BI
Department of Biochemistry, Hadassah Medical School, Hebrew University, Jerusalem 91120, Israel. kannerb@cc.huji.ac.il
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D002412:Cations; D050485:GABA Plasma Membrane Transport Proteins; D006367:HeLa Cells; D006801:Humans; D017136:Ion Transport; D008094:Lithium / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D027361:Organic Anion Transporters; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D014982:Xenopus laevis
D002352:Carrier Proteins; D002412:Cations; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; C493413:SLC6A1 protein, human; D008094:Lithium; D012964:Sodium; D011188:Potassium