Role of anion-cation interactions on the pre-steady-state currents of the rat Na(+)-Cl(-)-dependent GABA cotransporter rGAT1.
The effects of sodium and chloride on the properties of the sodium-dependent component of the 'pre-steady-state' currents of rGAT1, a GABA cotransporter of the Na(+)-Cl(-)-dependent family, were studied using heterologous oocyte expression and voltage clamp. Reductions in either extracellular sodium or chloride shifted the charge-voltage (Q-V) and time constant-voltage (tau-V) characteristics of the process towards more negative potentials. The shift induced by sodium (TMA(+), tetramethylammonium substitution) was stronger than that induced by chloride (acetate substitution), and the shift of tau was accompanied by a decrease in its maximum value. Increasing extracellular Ca(2+) did not produce significant shifts in Q-V and tau-V curves. The negative shift of the Q-V curve upon chloride reduction and the decrease in the value of the relaxation time constant, tau, when either sodium or chloride were lowered, contrasted with the prediction of the Hill-Boltzmann interpretation of the process. Analysis of the unidirectional rate constants under different conditions revealed that both sodium and chloride shifted the outward rate more than the inward rate; furthermore, the shifts induced by sodium were larger than those induced by chloride. These observations are qualitatively compatible with the existence of a selective vestibule at the mouth of the transporters, acting similarly to a Donnan system.
Bossi
Elena
E
Laboratory of Cellular and Molecular Physiology, Department of Structural and Functional Biology, University of Insubria, Via Dunant 3, 21100 Varese, Italy.
Giovannardi
Stefano
S
Binda
Francesca
F
Forlani
Greta
G
Peres
Antonio
A
D000465:Algorithms; D000818:Animals; D002118:Calcium / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D004594:Electrophysiology; D050485:GABA Plasma Membrane Transport Proteins; D008564:Membrane Potentials / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D009865:Oocytes / Q000378:metabolism; D027361:Organic Anion Transporters; D018408:Patch-Clamp Techniques; D012313:RNA / Q000096:biosynthesis; D051381:Rats; D012964:Sodium / Q000378:metabolism*; D015222:Sodium Channels / Q000378:metabolism; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D002352:Carrier Proteins; D002712:Chlorides; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; C493415:Slc6a1 protein, rat; D015222:Sodium Channels; D005680:gamma-Aminobutyric Acid; D012313:RNA; D012964:Sodium; D002118:Calcium