Engineered Zn²⁺ switches in the γ-aminobutyric acid (GABA) transporter-1. Differential effects on GABA uptake and currents

Two high affinity Zn²⁺ binding sites were engineered in the otherwise Zn²⁺-insensitive rat γ-aminobutyric acid (GABA) transporter-1 (rGAT-1) based on structural information derived from Zn ²⁺ binding sites engineered previously in the homologous dopamine transporter. Introduction of a histidine (T349H) at the extracellular end of transmembrane segment (TM) 7 together with a histidine (E370H) or a cysteine (Q374C) at the extracellular end of TM 8 resulted in potent inhibition of [ ³H]GABA uptake by Zn²⁺ (IC₅₀ = 35 and 44 μM, respectively). Upon expression in Xenopus laevis oocytes it was similarly observed that Zn²⁺ was a potent inhibitor of the GABA-induced current (IC₅₀ = 21 μM for T349H/E370H and 51 μM for T349H/Q374C), albeit maximum inhibition was only ∼40% in T349H/E370H versus ∼90% in T349H/Q374C. In the wild type, Zn²⁺ did not affect the Na⁺-dependent transient currents elicited by voltage jumps and thought to reflect capacitive charge movements associated with Na⁺ binding. However, in both mutants Zn²⁺ caused a reduction of the inward transient currents upon jumping to hyperpolarized potentials as reflected in rightward-shifted Q/V relationships. This suggests that Zn ²⁺ is inhibiting transporter function by stabilizing the outward-facing Na⁺-bound state. Translocation of lithium by the transporter does not require GABA binding and analysis of this uncoupled Li ⁺ conductance revealed a potent inhibition by Zn²⁺ in T349H/E370H, whereas surprisingly the T349H/Q374C leak was unaffected. This differential effect supports that the leak conductance represents a unique operational mode of the transporter involving conformational changes different from those of the substrate translocation process. Altogether our results support both an evolutionary conserved structural organization of the TM 7/8 domain and a key role of this domain in GABA-dependent and -independent conformational changes of the transporter.