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Titolo:
An intermediate state of the gamma-aminobutyric acid transporter GAT1 revealed by simultaneous voltage clamp and fluorescence
Autore:
Li, M; Farley, RA; Lester, HA;
Indirizzi:
CALTECH, Div Biol, Pasadena, CA 91125 USA CALTECH Pasadena CA USA 91125CALTECH, Div Biol, Pasadena, CA 91125 USA
Titolo Testata:
JOURNAL OF GENERAL PHYSIOLOGY
fascicolo: 4, volume: 115, anno: 2000,
pagine: 491 - 508
SICI:
0022-1295(200004)115:4<491:AISOTG>2.0.ZU;2-S
Fonte:
ISI
Lingua:
ENG
Soggetto:
SHAKER K+ CHANNEL; BRAIN GABA TRANSPORTER; RAT-BRAIN; SEROTONIN TRANSPORTER; DOPAMINE TRANSPORTER; NEUROTRANSMITTER TRANSPORTERS; CONFORMATIONAL-CHANGES; XENOPUS OOCYTES; ION-BINDING; EXPRESSION;
Keywords:
voltage clamp; Xenopus oocyte; tetramethylrhodamine; conformational change;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
52
Recensione:
Indirizzi per estratti:
Indirizzo: Lester, HA CALTECH, Div Biol, 1201 E Calif Blvd, Pasadena, CA 91125 USA CALTECH 1201 E Calif Blvd Pasadena CA USA 91125 a, CA 91125 USA
Citazione:
M. Li et al., "An intermediate state of the gamma-aminobutyric acid transporter GAT1 revealed by simultaneous voltage clamp and fluorescence", J GEN PHYSL, 115(4), 2000, pp. 491-508

Abstract

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 similar to 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 similar to 20 ms at +60 mV to similar to 150 ms at -140 mV. The fluorescence did not saturate at themost 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 fluorescencewaveforms. 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, inter-mediate between the E*(out) and E-out 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.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 05/04/20 alle ore 07:14:17