Temperature effects on the presteady-state and transport-associated currents of GABA cotransporter rGAT1

The effects of temperature on the gamma-aminobutyric acid (GABA) uptake and on the presteady-state and transport-associated currents of the GABA cotransporter, rat gamma-aminobutyric acid transporter 1 (rGAT1), have been studied using heterologous oocyte expression and voltage-clamp. Increasing temperature from 15 to 30 degrees C increased GABA uptake, diminished the maximal value of the relaxation time constant of the presteady-state currents and increased the amplitude of the current associated with the transport of GABA. The curve of the presteady-state charge versus voltage was shifted toward negative potentials by increasing the temperature, while the maximal amount of charge (Q(max)) remained constant; the tau versus V curve was also negatively shifted by increasing temperatures. Analysis of the outward (alpha) and inward (beta) rate constants as functions of temperature showed that they are affected differently, with a Q(10)=3.4 for alpha and Q(10)=1.5 for beta. The different temperature coefficients of the rate constants account for the observed shifts. These observations are consistent with a charge moving mechanism based on a conformational change of the protein; the weaker temperature sensitivity of the inward rate constant suggests a rate-limiting diffusional component on this process