Search

Most cotransporters characteristically display two main kinds of electrical activity: in the absence of organic substrate, transient presteady-state currents (I(pre)) are generated by charge relocation during voltage steps; in the presence of substrate, sustained, transport-associated currents (I(tr)) are recorded. Quantitative comparison of these two currents, in Xenopus oocytes expressing the neural GABA cotransporter rGAT1, revealed several unforeseen consistencies between I(pre) and I(tr), in terms of magnitude and kinetic parameters. The decay rate constant (r) of I(pre) and the quantity of charge displaced to an inner position in the transporter (Q(in)(0)) depended on voltage and ionic conditions. Saturating GABA concentrations, applied under the same conditions, suppressed I(pre) (i.e. Q(in)( infinity ) = 0) and produced a transport-associated current with amplitude I(tr) = Q(in)(0)r. At non-saturating levels of GABA, changes of I(tr) were compensated by corresponding variations in Q(in), such that I(pre) and I(tr) complemented each other, according to the relation: I(tr) = (Q(in)(0) - Q(in)) r. Complementarity of magnitude, superimposable kinetic properties and equal dependence on voltage and [Na(+)](o) point to the uniqueness of the charge carrier for both processes, suggesting that transport and charge migration arise from the same molecular mechanism. The observed experimental relations were correctly predicted by a simple three-state kinetic model, in which GABA binding takes place after charge binding and inward migration have occurred. The model also predicts the observed voltage dependence of the apparent affinity of the transporter for GABA, and suggests a voltage-independent GABA binding rate with a value around 0.64 microM(-1) s(-1).

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.

1. The effect of the mutation K448E in the rat GABA transporter rGAT1 was studied using heterologous expression in Xenopus oocytes and voltage clamp. 2. At neutral pH, the transport-associated current vs. voltage (I-V) relationship of the mutated transporter was different from wild-type, and the pre-steady-state currents were shifted towards more positive potentials. The mutated transporter showed an increased apparent affinity for Na+ (e.g. 62 vs. 152 mM at -60 mV), while the opposite was true for GABA (e.g. 20 vs. 13 microM at -60 mV). 3. In both isoforms changes in [Na+]o shifted the voltage dependence of the pre-steady-state and of the transport-associated currents by similar amounts. 4. In the K448E form, the moved charge and the relaxation time constant were shifted by increasing pH towards positive potentials. The transport-associated current of the mutated transporter was strongly reduced by alkalinization, while acidification slightly decreased and distorted the shape of the I-V curve. Accordingly, uptake of [3H]GABA was strongly reduced in K448E at pH 9.0. The GABA apparent affinity of the mutated transporter was reduced by alkalinization, while acidification had the opposite result. 5. These observations suggest that protonation of negatively charged residues may regulate the Na+ concentration in the proximity of the transporter. Calculation of the unidirectional rate constants for charge movement shows that, in the K448E form, the inward rate constant is increased at alkaline pH, while the outward rate constant does not change, in agreement with an effect due to mass action law. 6. A possible explanation for the complex effect of pH on the transport-associated current may be found by combining changes in local [Na+]o with a direct action of pH on GABA concentration or affinity. Our results support the idea that the extracellular loop 5 may participate to form a vestibule to which sodium ions must have access before proceeding to the steps involving charge movement.