Abstract
The effects of temperature on the operation of two ion-coupled cotransporters of the SLC6A family, namely rat GAT1 (SLC6A1) and KAAT1 (SLC6A19) from Manduca sexta, have been studied by electrophysiological means in Xenopus laevis oocytes expressing these proteins. The maximal transport-associated current (Imax) and the apparent substrate affinity (K05) were measured. In addition to the expected increase in transport rate (Q10 = 3–6), both transporters showed greater K05 values (i.e., a decrease in apparent affinity) at higher temperatures. The transport efficiency, estimated as Imax/K05, increased at negative potentials in both transporters, but did not show statistically significant differences with temperature. The observation that the apparent substrate affinity is inversely related to the transport rate suggests a kinetic regulation of this parameter. Furthermore, the present results indicate that the affinities estimated at room temperature for mammalian cotransporters may not be simply extrapolated to their physiological operating conditions.
Highlights
Functional studies of ion-coupled cotransporters have strongly taken advantage of the possibility of heterologous expression in Xenopus oocytes, which may be studied with relatively simple electrophysiological techniques, such as the two-electrode voltage-clamp (TEVC)
To better investigate these points, we have studied the effects of temperature on the apparent substrate affinity in two other transporters, namely the rat neuronal GABA transporter GAT1 and the neutral amino acid transporter KAAT1, cloned from the gut of the invertebrate Manduca sexta [17]
Apparent Affinity Changes Induced by Temperature in rGAT1
Summary
Functional studies of ion-coupled cotransporters have strongly taken advantage of the possibility of heterologous expression in Xenopus oocytes, which may be studied with relatively simple electrophysiological techniques, such as the two-electrode voltage-clamp (TEVC) This approach suffers, of an important drawback when mammalian transporters are investigated, because temperatures above 30 °C are not tolerated by the oocytes, and most studies have been performed at room temperature (20 to 25 °C) [1,2,3,4]. A decrease in apparent affinity was measured in the physiological voltage range when increasing the temperature from 20 to 30 °C This effect was associated with a strong increase in the maximal transport current and with an acceleration of the kinetics of the presteady-state currents. The aim of the investigation was first of all to verify if the apparent substrate affinity was affected by temperature and, secondarily, to examine the overall efficiency of the transport, considering in addition that, while rGAT1 (and PepT1) are from mammalian (homeotherm) animals, KAAT1 originates from a poikilotherm invertebrate [18]
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