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.
Binda
Francesca
F
Laboratory of Cellular and Molecular Physiology, Department of Structural and Functional Biology, University of Insubria, Via Dunant 3, 21100, Varese, Italy.
Bossi
Elena
E
Giovannardi
Stefano
S
Forlani
Greta
G
Peres
Antonio
A
D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D004553:Electric Conductivity; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008956:Models, Chemical; D027361:Organic Anion Transporters; D051381:Rats; D013696:Temperature; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; C493415:Slc6a1 protein, rat; D005680:gamma-Aminobutyric Acid