Novel properties of a mouse γ-aminobutyric acid transporter (GAT4)

We expressed the mouse γ-aminobutyric acid (GABA) transporter GAT4 (homologous to rat/human GAT-3) in Xenopus laevis oocytes and examined its functional and pharmacological properties by using electrophysiological and tracer uptake methods. In the coupled mode of transport (Na⁺/Cl ^-/GABA cotransport), there was tight coupling between charge flux and GABA flux across the plasma membrane (2 charges/GABA). Transport was highly temperature-dependent with a temperature coefficient (Q ₁₀) of 4.3. The GAT4 turnover rate (1.5 s^-1; -50 mV, 21°C) and temperature dependence suggest physiological turnover rates of 15-20 s^-1. No uncoupled current was observed in the presence of Na⁺. In the absence of external Na⁺, GAT4 exhibited two distinct uncoupled currents. (i) A Cl^- leak current (I_leak^Cl ) was observed when Na⁺ was replaced with choline or tetraethylammonium. The reversal potential of (I_leak^Cl) followed the Cl ^- Nernst potential. (ii) A Li⁺ leak current (I _leak^Li) was observed when Na⁺ was replaced with Li⁺. Both leak currents were inhibited by Na⁺, and both were temperature-independent (Q ₁₀ ≈ 1). The two leak modes appeared not to coexist, as Li⁺ inhibited (I_leak ^Cl). The results suggest the existence of cation- and anion-selective channel-like pathways in GAT4. Flufenamic acid inhibited GAT4 Na ⁺/C1^-/GABA cotransport, I_leak^Li), and I_leak^Cl), (K _i ≈ 30 μM), and the voltage-induced presteady-state charge movements (K _i ≈ 440 μM). Flufenamic acid exhibited little or no selectivity for GAT1, GAT2, or GAT3. Sodium and GABA concentration jumps revealed that slow Na⁺ binding to the transporter is followed by rapid GABA-induced translocation of the ligands across the plasma membrane. Thus, Na⁺ binding and associated conformational changes constitute the rate-limiting steps in the transport cycle.