Presteady-state and steady-state kinetics and turnover rate of the mouse gamma-aminobutyric acid transporter (mGAT3).
We expressed mouse gamma-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 microM. The Hill coefficient for GABA activation and Cl(-) enhancement of the inward current was 1, and that for Na+ activation was > or =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 (V(0.5)) was +25 mV, and the effective valence of the moveable charge (zdelta) was 1.6. In contrast to the ON transients, which relaxed with a time constant of < or =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 approximately 30% of the maximal charge (Q(max)). 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. Q(max) and I(max) 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.
Sacher
A
A
Department of Biochemistry, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
Nelson
N
N
Ogi
J T
JT
Wright
E M
EM
Loo
D D F
DD
Eskandari
S
S
D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D005260:Female; D050485:GABA Plasma Membrane Transport Proteins; D006706:Homeostasis / Q000502:physiology*; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D051379:Mice; D009865:Oocytes / Q000378:metabolism / Q000502:physiology*; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014982:Xenopus laevis
D050485:GABA Plasma Membrane Transport Proteins; D026901:Membrane Transport Proteins; C493419:Slc6a13 protein, mouse