Engineered Zn(2+) switches in the gamma-aminobutyric acid (GABA) transporter-1. Differential effects on GABA uptake and currents.

Two high affinity Zn(2+) binding sites were engineered in the otherwise Zn(2+)-insensitive rat gamma-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 Zn(2+) (IC(50) = 35 and 44 microM, respectively). Upon expression in Xenopus laevis oocytes it was similarly observed that Zn(2+) was a potent inhibitor of the GABA-induced current (IC(50) = 21 microM for T349H/E370H and 51 microM for T349H/Q374C), albeit maximum inhibition was only approximately 40% in T349H/E370H versus approximately 90% in T349H/Q374C. In the wild type, Zn(2+) 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 Zn(2+) 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 Zn(2+) 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.

Delineating structure-function relationships in the dopamine transporter from natural and engineered Zn2+ binding sites.

The dopamine transporter is member of a large family of Na+/Cl- dependent neurotransmitter and amino acid transporters. Little is known about the molecular basis for substrate translocation in this class of transporters as well as their tertiary structure remains elusive. In this report, we provide the first crude insight into the structural organization of the human dopamine transporter (hDAT) based on the identification of an endogenous high affinity Zn2+ binding site followed by engineering of an artificial Zn2+ binding site. By binding to the endogenous site, Zn2+ acts as a potent non-competitive inhibitor of dopamine uptake mediated by the hDAT transiently expressed in COS-7 cells. Systematic mutagenesis of potential Zn2+ coordinating residues lead to the identification of three residues on the predicted extracellular face of the transporter, 193His in the second extracellular loop, 375His at the external end of the putative transmembrane segment (TM) 7, and 396Glu at the external end of TM 8, forming three coordinates in the endogenous Zn2+ binding site. The three residues are separate in the primary structure but their common participation in binding the small Zn(II) ion define their spatial proximity in the tertiary structure of the transporter. Finally, an artificial inhibitory Zn2+ binding site was engineered between TM 7 and TM 8. This binding site both verify the proximity between the two domains as wells as it supports an alpha-helical configuration at the top of TM 8 in the hDAT.

Structural probing of a microdomain in the dopamine transporter by engineering of artificial Zn2+ binding sites.

Previously, we have identified three Zn(2+) binding residues in an endogenous Zn(2+) binding site in the human dopamine transporter (hDAT): (193)His in extracellular loop 2 (ECL 2), (375)His at the external end of transmembrane segment (TM) 7, and (396)Glu at the external end of TM 8. Here we have generated a series of artificial Zn(2+) binding sites in a domain situated around the external ends of TMs 7 and 8 by taking advantage of the well-defined structural constraints for binding of the zinc(II) ion. Initially, we found that the Zn(2+)-coordinating (193)His in ECL 2 could be substituted with a histidine inserted at the i - 4 position relative to (375)His in TM 7. In this mutant (H193K/M371H), Zn(2+) potently inhibited [(3)H]dopamine uptake with an IC(50) value of 7 microM as compared to a value of 300 microM for the control (H193K). These data are consistent with the presence of an alpha-helical configuration of TM 7. This inference was further corroborated by the observation that no increase in the apparent Zn(2+) affinity was observed following introduction of histidines at the i - 2, i - 3, and i - 5 positions. In contrast, introduction of histidines at positions i + 2, i + 3, and i + 4 all resulted in potent inhibition of [(3)H]dopamine uptake by Zn(2+) (IC(50) = 3-32 microM). These observations are inconsistent with continuation of the helix beyond position 375 and indicate an approximate boundary between the end of the helix and the succeeding loop. In summary, the data presented here provide new insight into the structure of a functionally important domain in the hDAT and illustrate how engineering of Zn(2+) binding sites can be a useful approach for probing both secondary and tertiary structure relationships in membrane proteins of unknown structure.

Defining proximity relationships in the tertiary structure of the dopamine transporter. Identification of a conserved glutamic acid as a third coordinate in the endogenous Zn(2+)-binding site.

Recently, we have described a distance constraint in the unknown tertiary structure of the human dopamine transporter (hDAT) by identification of two histidines, His(193) in the second extracellular loop and His(375) at the top of transmembrane (TM) 7, that form two coordinates in an endogenous, high affinity Zn(2+)-binding site. To achieve further insight into the tertiary organization of hDAT, we set out to identify additional residues involved in Zn(2+) binding and subsequently to engineer artificial Zn(2+)-binding sites. Ten aspartic acids and glutamic acids, predicted to be on the extracellular side, were mutated to asparagine and glutamine, respectively. Mutation of Glu(396) (E396Q) at the top of TM 8 increased the IC(50) value for Zn(2+) inhibition of [(3)H]dopamine uptake from 1.1 to 530 microM and eliminated Zn(2+)-induced potentiation of [(3)H]WIN 35,428 binding. These data suggest that Glu(396) is involved in Zn(2+) binding to hDAT. Importantly, Zn(2+) sensitivity was preserved following substitution of Glu(396) with histidine, indicating that the effect of mutating Glu(396) is not an indirect effect because of the removal of a negatively charged residue. The common participation of Glu(396), His(193), and His(375) in binding the small Zn(2+) ion implies their proximity in the unknown tertiary structure of hDAT. The close association between TM 7 and 8 was further established by engineering of a Zn(2+)-binding site between His(375) and a cysteine inserted in position 400 in TM 8. Summarized, our data define an important set of proximity relationships in hDAT that should prove an important template for further exploring the molecular architecture of Na(+)/Cl(-)-dependent neurotransmitter transporters.