Probing the molecular basis for signal transduction through the Zinc-Activated Channel (ZAC)

The molecular basis for the signal transduction through the classical Cys-loop receptors (CLRs) has been delineated in great detail. The Zinc-Activated Channel (ZAC) constitutes a so far poorly elucidated fifth branch of the CLR superfamily, and in this study we explore the molecular mechanisms underlying ZAC signaling in Xenopus oocytes by two-electrode voltage clamp electrophysiology. In studies of chimeric receptors fusing either the extracellular domain (ECD) or the transmembrane/intracellular domain (TMD-ICD) of ZAC with the complementary domains of 5-HT₃A serotonin or α₁ glycine receptors, serotonin and Zn²⁺/H⁺ evoked robust concentration-dependent currents in 5-HT₃A/ZAC- and ZAC/α₁-Gly-expressing oocytes, respectively, suggesting that Zn²⁺ and protons activate ZAC predominantly through its ECD. The molecular basis for Zn²⁺-mediated ZAC signaling was probed further by introduction of mutations of His, Cys, Glu and Asp residues in this domain, but as none of the mutants tested displayed substantially impaired Zn²⁺ functionality compared to wild-type ZAC, the location of the putative Zn²⁺ binding site(s) in the ECD was not identified. Finally, the functional importance of Leu²⁴⁶ (Leu9') in the transmembrane M2 α-helix of ZAC was investigated by Ala, Val, Ile and Thr substitutions. In concordance with findings for this highly conserved residue in classical CLRs, the ZAC^L9'X mutants exhibited left-shifted agonist concentration-response relationships, markedly higher degrees of spontaneous activity and slower desensitization kinetics compared to wild-type ZAC. In conclusion, while ZAC is an atypical CLR in terms of its (identified) agonists and channel characteristics, its signal transduction seems to undergo similar conformational transitions as those in the classical CLR.