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 gamma -aminobutyric acid (GABA) transporter-1 (rGAT-1) based on structural information derived from Zn2+ 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 [H-3]GABA uptake by Zn2+ (IC50 = 35 and 44 muM, respectively). Upon expression in Xenopus laevis oocytes it was similarly observed that Zn2+ was a potent inhibitor of the GABA-induced current (IC50 = 21 muM for T349H/E370H and 51 muM for T349H/Q374C), albeit maximum inhibition was only similar to 40% in T349H/E370H versus similar to 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 Zn2+ is inhibiting transporter function by stabilizing the outward-facing Na+-bound state. Translocation of lithium by the transporter does not require GAGA 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.