Engineered Zn2+ switches in the gamma-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) base d on structural information derived from Zn2+ binding sites engineered prev iously in the homologous dopamine transporter. Introduction of a histidine (T349H) at the extracellular end of transmembrane segment (TM) 7 together w ith a histidine (E370H) or a cysteine (Q374C) at the extracellular end of T M 8 resulted in potent inhibition of [H-3]GABA uptake by Zn2+ (IC50 = 35 an d 44 muM, respectively). Upon expression in Xenopus laevis oocytes it was s imilarly observed that Zn2+ was a potent inhibitor of the GABA-induced curr ent (IC50 = 21 muM for T349H/E370H and 51 muM for T349H/Q374C), albeit maxi mum 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 tr ansient currents elicited by voltage jumps and thought to reflect capacitiv e charge movements associated with Na+ binding. However, in both mutants Zn 2+ caused a reduction of the inward transient currents upon jumping to hype rpolarized potentials as reflected in rightward-shifted Q/V relationships. This suggests that Zn2+ is inhibiting transporter function by stabilizing t he outward-facing Na+-bound state. Translocation of lithium by the transpor ter does not require GAGA binding and analysis of this uncoupled Li+ conduc tance revealed a potent inhibition by Zn2+ in T349H/E370H, whereas surprisi ngly the T349H/Q374C leak was unaffected. This differential effect supports that the leak conductance represents a unique operational mode of the tran sporter involving conformational changes different from those of the substr ate translocation process. Altogether our results support both an evolution ary conserved structural organization of the TM 7/8 domain and a key role o f this domain in GABA-dependent and -independent conformational changes of the transporter.