Residues in the extracellular loop 4 are critical for maintaining the conformational equilibrium of the gamma-aminobutyric acid transporter-1

The Journal of Biological Chemistry
N MacAulayUlrik Gether

Abstract

We mutated residues Met345 and Thr349 in the rat gamma-aminobutyric acid transporter-1 (GAT-1) to histidines (M345H and T349H). These two residues are located four amino acids apart at the extracellular end of transmembrane segment 7 in a region of GAT-1 that we have previously suggested undergoes conformational changes critical for the transport process. The two single mutants and the double mutant (M345H/T349H) were expressed in Xenopus laevis oocytes, and their steady-state and presteady-state kinetics were examined and compared with wild type GAT-1 by using the two-electrode voltage clamp method. Oocytes expressing M345H showed a decrease in apparent GABA affinity, an increase in apparent affinity for Na+, a shift in the charge/voltage (Q/Vm) relationship to more positive membrane potentials, and an increased Li+-induced leak current. Oocytes expressing T349H showed an increase in apparent GABA affinity, a decrease in apparent Na+ affinity, a profound shift in the Q/Vm relationship to more negative potentials, and a decreased Li+-induced leak current. The data are consistent with a shift in the conformational equilibrium of the mutant transporters, with M345H stabilized in an outward-facing conformation and T349H in an inwa...Continue Reading

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Citations

Nov 13, 2003·European Journal of Pharmacology·Naomi R GoldbergJonathan A Javitch
Nov 13, 2003·European Journal of Pharmacology·Claus Juul LolandUlrik Gether
Apr 14, 2009·The Journal of Biological Chemistry·Anne-Kristine MeinildNanna MacAulay
Jun 7, 2006·Nihon yakurigaku zasshi. Folia pharmacologica Japonica·Yasushi Shigeri, Keiko Shimamoto
Apr 7, 2004·The European Journal of Neuroscience·Anders S KristensenOve Wiborg
May 14, 2004·The Journal of Biological Chemistry·Susan M MitchellMegan M Stephan

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