Conformational basis for the Li(+)-induced leak current in the rat gamma-aminobutyric acid (GABA) transporter-1.
The rat gamma-aminobutyric acid transporter-1 (GAT-1) was expressed in Xenopus laevis oocytes and the substrate-independent Li(+)-induced leak current was examined using two-electrode voltage clamp. The leak current was not affected by the addition of GABA and was not due to H(+) permeation. The Li(+)-bound conformation of the protein displayed a lower passive water permeability than that of the Na(+)- and choline (Ch(+))-bound conformations and the leak current did not saturate with increasing amounts of Li(+) in the test solution. The mechanism that gives rise to the leak current did not support active water transport in contrast to the mechanism responsible for GABA translocation (approximately 330 water molecules per charge). Altogether, these data support the distinct nature of the leak conductance in relation to the substrate translocation process. It was observed that the leak current was inhibited by low millimolar concentrations of Na(+) (the apparent affinity constant, K'(0.5) = 3 mM). In addition, it was found that the GABA transport current was sustained at correspondingly low Na(+) concentrations if Li(+) was present instead of choline. This is consistent with a model in which Li(+) can bind and substitute for Na(+) at the putative "first" apparently low-affinity Na(+) binding site. In the absence of Na(+), this allows a Li(+)-permeable channel to open at hyperpolarized potentials. Occupancy of the "second" apparently high-affinity Na(+) binding site by addition of low millimolar concentrations of Na(+) restrains the transporter from moving into a leak conductance mode as well as allowing maintenance of GABA-elicited transport-associated current.
MacAulay
Nanna
N
Department of Medical Physiology 12.5, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark. nmacaulay@mfi.ku.dk
Zeuthen
Thomas
T
Gether
Ulrik
U
D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry* / Q000502:physiology*; D004553:Electric Conductivity; D005260:Female; D050485:GABA Plasma Membrane Transport Proteins; D006863:Hydrogen-Ion Concentration; D008094:Lithium / Q000494:pharmacology*; D008565:Membrane Proteins / Q000737:chemistry* / Q000502:physiology*; D026901:Membrane Transport Proteins; D008968:Molecular Conformation; D009865:Oocytes; D027361:Organic Anion Transporters; D018408:Patch-Clamp Techniques; D010539:Permeability; D051381:Rats; D012964:Sodium / Q000494:pharmacology; D014867:Water / Q000378:metabolism; D014982:Xenopus laevis
D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; C493415:Slc6a1 protein, rat; D014867:Water; D008094:Lithium; D012964:Sodium