Presteady-state and steady-state kinetics and turnover rate of the mouse γ-aminobutyric acid transporter (mGAT3)

We expressed mouse γ-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 μM. 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 (V_0.5) was + 25 mV, and the effective valence of the moveable charge (zδ) 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 (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 μM, 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.