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

Abstract

The effects of temperature on the Q Q-aminobutyric acid (GABA) uptake and on the presteady-state and transportassociated currents of the GABA cotransporter, rat Q Q-aminobutyric acid transporter 1 (rGAT1), have been studied using heterologous oocyte expression and voltage-clamp. Increasing temperature from 15 to 30³C 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 d d versus V curve was also negatively shifted by increasing temperatures. Analysis of the outward (K K) and inward (L L) rate constants as functions of temperature showed that they are affected differently, with a Q 10 = 3.4 for K K and Q 10 = 1.5 for L L. 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. ß

GABAergic Spill-Over Transmission onto Hippocampal Mossy Fiber Boutons

“…This difference could be explained by a species-specific difference in hippocampal GABA signaling (Walker et al, 2001;Gutiérrez, 2003), a lower recording temperature (Ruiz et al, 2003) that markedly reduces transmitter uptake by transporters (Mitchell and Silver, 2000;Binda et al, 2002), and a possible contribution of GABA receptors in the surrounding tissue to the observed effects, as discussed in Ruiz et al (2003). However, in accordance with that study, we found that presynaptic GABA A receptors in rat mossy fibers contain the ␣ 2 -subunit.…”

Transport model of the human Na+-coupled l-ascorbic acid (vitamin C) transporter SVCT1

“…Maximal charge (Q max ) was conserved upon progressive removal of Na ϩ , whereas the voltage midpoint (V 0.5 ) shifted to more hyperpolarized potentials. Pre-steady-state currents have been observed for several other Na ϩ -coupled (1,9,14,17,19,27) or H ϩ -coupled (8,12,16,21,22,25) solute transporters. The results of model simulation have led investigators to attribute pre-steady-state currents to two steps in the transport cycle, namely reorientation of the empty charged carrier and binding/dissociation of the driving ion partway within the plane of the membrane electric field (and in some cases also to the binding/dissociation of the ion at the internal face).…”

“…(The order of intracellular substrate dissociation is assumed.) Each carrier state is identified by a number (1)(2)(3)(4)(5)(6)(7)(8) and by a prime denoting that the carrier is at the external face of the membrane or double prime indicating the internal face. A Na ϩ uniport pathway or "internal leak," also electroneutral (n H Na ϭ 1), is shown as step 2 3 7.…”

Residues in the Extracellular Loop 4 Are Critical for Maintaining the Conformational Equilibrium of the γ-Aminobutyric Acid Transporter-1

“…The GABA re-uptake is coupled to the cotransport of 2 Na ϩ and 1 Cl Ϫ , which renders the transport process electrogenic (3,4). Several electrophysiological studies of GAT-1 heterologously expressed in either mammalian cell lines or in Xenopus laevis oocytes have been carried out, and four current-generating modes of the transporter have been described; they are the Na ϩ -coupled substrate-induced current, capacitive Na ϩ -dependent presteady-state currents, a Li ϩ -induced leak conductance, and a not fully documented un-coupled substrate-induced channel activity (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18).…”