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Role of anion-cation interactions on the pre-steady-state currents of the rat Na(+)-Cl(-)-dependent GABA cotransporter rGAT1.
Bossi E
,
Giovannardi S
,
Binda F
,
Forlani G
,
Peres A
.
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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.
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