An intermediate state of the gamma-aminobutyric acid transporter GAT1 revealed by simultaneous voltage clamp and fluorescence.
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 approximately 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 approximately 20 ms at +60 mV to approximately 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 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.
Li
M
M
Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.
Farley
R A
RA
Lester
H A
HA
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004594:Electrophysiology; D005456:Fluorescent Dyes; D050485:GABA Plasma Membrane Transport Proteins; D008564:Membrane Potentials / Q000502:physiology; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins; D008856:Microscopy, Fluorescence; D009154:Mutation / Q000235:genetics / Q000502:physiology; D009865:Oocytes; D027361:Organic Anion Transporters; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D000644:Quaternary Ammonium Compounds; D051381:Rats; D012235:Rhodamines; D012964:Sodium / Q000378:metabolism; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D002352:Carrier Proteins; D005456:Fluorescent Dyes; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; D000644:Quaternary Ammonium Compounds; D012235:Rhodamines; C493415:Slc6a1 protein, rat; C039795:trimethylethylammonium; D005680:gamma-Aminobutyric Acid; C005358:tetramethylrhodamine; D012964:Sodium