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An Intermediate State of the {gamma}-Aminobutyric Acid Transporter GAT1 Revealed by Simultaneous Voltage Clamp and Fluorescence

Li, Ming and Farley, Robert A. and Lester, Henry A. (2000) An Intermediate State of the {gamma}-Aminobutyric Acid Transporter GAT1 Revealed by Simultaneous Voltage Clamp and Fluorescence. Journal of General Physiology, 115 (4). pp. 491-508. ISSN 0022-1295. PMCID PMC2233755. doi:10.1085/jgp.115.4.491.

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The rat {gamma}-aminobutyric acid transporter GAT1 expressed in Xenopus oocytes was labeled at Cys74, and at one or more other sites, by tetramethylrhodamine-5-maleimide, without significantly altering GAT1 function. Voltage-jump relaxation analysis showed that fluorescence increased slightly and monotonically with hyperpolarization; the fluorescence at -140 mV was ~0.8% greater than at +60 mV. The time course of the fluorescence relaxations was mostly described by a single exponential with voltage-dependent but history-independent time constants ranging from ~20 ms at +60 mV to ~150 ms at -140 mV. The fluorescence did not saturate at the most negative potentials tested, and the midpoint of the fluorescence–voltage relation was at least 50 mV more negative than the midpoint of the charge–voltage relation previously identified with Na+ binding to GAT1. The presence of {gamma}-aminobutyric acid did not noticeably affect the fluorescence waveforms. The fluorescence signal depended on Na+ concentration with a Hill coefficient approaching 2. Increasing Cl- concentration modestly increased and accelerated the fluorescence relaxations for hyperpolarizing jumps. The fluorescence change was blocked by the GAT1 inhibitor, NO-711. For the W68L mutant of GAT1, the fluorescence relaxations occurred only during jumps to high positive potentials, in agreement with previous suggestions that this mutant is trapped in one conformational state except at these potentials. These observations suggest that the fluorescence signals monitor a novel state of GAT1, intermediate between the E*out and Eout states of Hilgemann, D.W., and C.-C. Lu (1999. J. Gen. Physiol. 114:459–476). Therefore, the study provides verification that conformational changes occur during GAT1 function.

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Lester, Henry A.0000-0002-5470-5255
Additional Information:© 2000 The Rockefeller University Press Submitted: 7 December 1999; Revised: 11 February 2000; Accepted: 22 February 2000; Published 27 March 2000. doi:10.1085/jgp.115.4.491 We thank Mike Walsh for excellent technical assistance and Ehud Isacoff, Micah Siegel, and Yong-Xin Li for advice and reagents. This work is supported by grants from the National Institutes of Health (NS-11756, DA-09121).
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Subject Keywords:voltage clamp; Xenopus oocyte; tetramethylrhodamine; conformational change
Issue or Number:4
PubMed Central ID:PMC2233755
Record Number:CaltechAUTHORS:LIMjgp00
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:6577
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Deposited On:13 Dec 2006
Last Modified:08 Nov 2021 20:35

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