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Int J Mol Sci
2012 Nov 22;1312:15565-74. doi: 10.3390/ijms131215565.
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An inverse relationship links temperature and substrate apparent affinity in the ion-coupled cotransporters rGAT1 and KAAT1.
Peres A
,
Vollero A
,
Margheritis E
,
D'Antoni F
,
Bossi E
.
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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 (I(max)) and the apparent substrate affinity (K(05)) were measured. In addition to the expected increase in transport rate (Q(10) = 3-6), both transporters showed greater K(05) values (i.e., a decrease in apparent affinity) at higher temperatures. The transport efficiency, estimated as I(max)/K(05), 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.
Figure 1. Temperature effects on the kinetic properties of rGAT1. Top row: currents in the absence (black) or presence (red) of 300 μM GABA at 20 °C (A) and 30 °C (B), in response to the staircase voltage protocol shown in the inset. The arrows point to the presteady-state currents that disappear in the presence of GABA. The bottom row shows the results of the Michaelis-Menten analysis performed on dose-response curves obtained using the same experimental protocol: voltage dependence of Imax (C) and of K05 (D) at the indicated temperatures. Data are means ± SE from seven oocytes (three batches). The current data were normalized to the value at −120 mV and 20 °C for each oocyte before averaging. The K05 value at +40 mV is omitted because its estimate is unreliable. Data marked with asterisks were significantly different at the two temperatures (Student’s t-test, p < 0.05).
Figure 2. Temperature effects on the kinetic properties of KAAT1 with threonine as a substrate. Top row: currents in the absence (black) or presence (red) of 1 mM threonine at 20 °C (A) and 30 °C (B) in response to the staircase voltage protocol shown in the inset. The bottom row shows the results of the Michaelis-Menten analysis performed on dose-response curves obtained using the same experimental protocol: voltage dependence of Imax (C) and of K05 (D) at the indicated temperatures. Data are means ± SE from six oocytes (two batches). The current data were normalized to the value at −120 mV and 20 °C for each oocyte before averaging. Some Imax and K05 values at the most positive potentials are omitted because their estimate is unreliable. All data at 30 °C (except the K05 value at −140 mV) were significantly different (p < 0.05) from those at 20 °C (Student’s t-test).
Figure 3. Temperature dependence of the efficiency of transport. The ratios Imax/K05 have been calculated from the data in Figure 1C,D for rGAT1 and from the data in Figure 2C,D for KAAT1. The values are in arbitrary units because of the normalization of the maximal current in Figures 1 and 2. Error bars are standard errors of the mean.
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