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 oocyt es was labeled at Cys74, and at one or more other sites, by tetramethylrhod amine-5-maleimide, without significantly altering GAT1 function. Voltage-ju mp relaxation analysis showed that fluorescence increased slightly and mono tonically with hyperpolarization; the fluorescence at -140 mV was similar t o 0.8% greater than at +60 mV. The time course of the fluorescence relaxati ons 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 the most negative potentials tested, and the midpoint of the fluorescence-volta ge relation was at least 50 mV more negative than the midpoint of the charg e-voltage relation previously identified with Na+ binding to GAT1. The pres ence of gamma-aminobutyric acid did not noticeably affect the fluorescence waveforms. The fluorescence signal depended on Na+ concentration with a Hil l coefficient approaching 2. Increasing Cl- concentration modestly increase d and accelerated the fluorescence relaxations for hyperpolarizing jumps. T he fluorescence change was blocked by the GAT1 inhibitor, NO-711. For the W 68L mutant of GAT1, the fluorescence relaxations occurred only during jumps to high positive potentials, in agreement with previous suggestions that t his mutant is trapped in one conformational state except at these potential s. These observations suggest that the fluorescence signals monitor a novel state of GAT1, inter-mediate between the E*(out) and E-out states of Hilge mann, D.W., and C.-C. Lu (1999. J. Gen. Physiol: 114:459-476). Therefore, t he study provides verification that conformational changes occur during GAT 1 function.