TY - JOUR

T1 - Conformational basis for the Li(+)-induced leak current in the rat gamma-aminobutyric acid (GABA) transporter-1

AU - MacAulay, Nanna

AU - Zeuthen, Thomas

AU - Gether, Ulrik

PY - 2002/10/15

Y1 - 2002/10/15

N2 - 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.

AB - 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.

KW - Animals

KW - Biological Transport

KW - Carrier Proteins

KW - Electric Conductivity

KW - Female

KW - GABA Plasma Membrane Transport Proteins

KW - Hydrogen-Ion Concentration

KW - Lithium

KW - Membrane Proteins

KW - Membrane Transport Proteins

KW - Molecular Conformation

KW - Oocytes

KW - Organic Anion Transporters

KW - Patch-Clamp Techniques

KW - Permeability

KW - Rats

KW - Sodium

KW - Water

KW - Xenopus laevis

M3 - Journal article

C2 - 12381817

VL - 544

SP - 447

EP - 458

JO - The Journal of Physiology

JF - The Journal of Physiology

SN - 0022-3751

IS - Pt 2

ER -