Presteady-State and Steady-State Kinetics and Turnover Rate of the Mouse g-Aminobutyric Acid Transporter (mGAT3)

Presteady-State and Steady-State Kinetics and Turnover Rate of the Mouse g-Aminobutyric Acid... We expressed mouse g-aminobutyric acid (GABA) transporter (mGAT3) in Xenopus laevis oocytes and examined its steady-state and presteady-state kinetics and turnover rate by using tracer flux and electrophysiological methods. In oocytes expressing mGAT3, GABA evoked a Na+-dependent and Cl?-facilitated inward current. The dependence on Na+ was absolute, whereas that for Cl? was not. At a membrane potential of ?50 mV, the half-maximal concentrations for Na+, Cl?, and GABA were 14 mM, 5 mM, and 3 mM. The Hill coefficient for GABA activation and Cl? enhancement of the inward current was 1, and that for Na+ activation was ?2. The GABA-evoked inward current was directly proportional to GABA influx (2.2 ± 0.1 charges/GABA) into cells, indicating that under these conditions, there is tight ion/GABA coupling in the transport cycle. In response to step changes in the membrane voltage and in the absence of GABA, mGAT3 exhibited presteady-state current transients (charge movements). The charge-voltage (Q-V) relation was fitted with a single Boltzmann function. The voltage at half-maximal charge (V0.5) was +25 mV, and the effective valence of the moveable charge (zd) was 1.6. In contrast to the ON transients, which relaxed with a time constant of ?30 msec, the OFF transients had a time constant of 1.1 sec. Reduction in external Na+ ([Na+]o) and Cl? ([Cl?]o) concentrations shifted the Q-V relationship to negative membrane potentials. At zero [Na+]o (106 mM Cl?), no mGAT3-mediated transients were observed, and at zero [Cl?]o (100 mM Na+), the charge movements decreased to ?30% of the maximal charge (Qmax). GABA led to the elimination of charge movements. The half-maximal concentrations for Na+ activation, Cl? enhancement, and GABA elimination of the charge movements were 48 mM, 19 mM, and 5 mM, respectively. Qmax and Imax obtained in the same cells yielded the mGAT3 turnover rate, 1.7 sec?1 at ?50 mV. The low turnover rate of mGAT3 may be due to the slow return of the empty transporter from the internal to the external membrane surface. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Membrane Biology Springer Journals

Presteady-State and Steady-State Kinetics and Turnover Rate of the Mouse g-Aminobutyric Acid Transporter (mGAT3)

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Publisher
Springer-Verlag
Copyright
Copyright © 2002 by Springer-Verlag New York Inc.
Subject
Life Sciences; Biochemistry, general; Human Physiology
ISSN
0022-2631
eISSN
1432-1424
D.O.I.
10.1007/s00232-002-1024-6
Publisher site
See Article on Publisher Site

Abstract

We expressed mouse g-aminobutyric acid (GABA) transporter (mGAT3) in Xenopus laevis oocytes and examined its steady-state and presteady-state kinetics and turnover rate by using tracer flux and electrophysiological methods. In oocytes expressing mGAT3, GABA evoked a Na+-dependent and Cl?-facilitated inward current. The dependence on Na+ was absolute, whereas that for Cl? was not. At a membrane potential of ?50 mV, the half-maximal concentrations for Na+, Cl?, and GABA were 14 mM, 5 mM, and 3 mM. The Hill coefficient for GABA activation and Cl? enhancement of the inward current was 1, and that for Na+ activation was ?2. The GABA-evoked inward current was directly proportional to GABA influx (2.2 ± 0.1 charges/GABA) into cells, indicating that under these conditions, there is tight ion/GABA coupling in the transport cycle. In response to step changes in the membrane voltage and in the absence of GABA, mGAT3 exhibited presteady-state current transients (charge movements). The charge-voltage (Q-V) relation was fitted with a single Boltzmann function. The voltage at half-maximal charge (V0.5) was +25 mV, and the effective valence of the moveable charge (zd) was 1.6. In contrast to the ON transients, which relaxed with a time constant of ?30 msec, the OFF transients had a time constant of 1.1 sec. Reduction in external Na+ ([Na+]o) and Cl? ([Cl?]o) concentrations shifted the Q-V relationship to negative membrane potentials. At zero [Na+]o (106 mM Cl?), no mGAT3-mediated transients were observed, and at zero [Cl?]o (100 mM Na+), the charge movements decreased to ?30% of the maximal charge (Qmax). GABA led to the elimination of charge movements. The half-maximal concentrations for Na+ activation, Cl? enhancement, and GABA elimination of the charge movements were 48 mM, 19 mM, and 5 mM, respectively. Qmax and Imax obtained in the same cells yielded the mGAT3 turnover rate, 1.7 sec?1 at ?50 mV. The low turnover rate of mGAT3 may be due to the slow return of the empty transporter from the internal to the external membrane surface.

Journal

The Journal of Membrane BiologySpringer Journals

Published: Nov 1, 2002

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