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 ZACL9'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.

Discovery and functional characterization of N-(thiazol-2-yl)-benzamide analogs as the first class of selective antagonists of the Zinc-Activated Channel (ZAC).

The Zinc-Activated Channel (ZAC) is an atypical member of the Cys-loop receptor (CLR) superfamily of pentameric ligand-gated ion channels, with its very different endogenous agonists and signalling properties. In this study, a compound library screening at ZAC resulted in the identification of 2-(5-bromo-2-chlorobenzamido)-4-methylthiazole-5-methyl ester (1) as a novel ZAC antagonist. The structural determinants for ZAC activity in 1 were investigated by functional characterization of 61 analogs at ZAC expressed in Xenopus oocytes by two-electrode voltage clamp electrophysiology, and couple of analogs exerting more potent ZAC inhibition than 1 were identified (IC50 values: 1-3 µM). 1 and N-(4-(tert-butyl)thiazol-2-yl)-3-fluorobenzamide (5a, TTFB) were next applied in studies of the functional properties and the mode of action of this novel class of ZAC antagonists. TTFB was a roughly equipotent antagonist of Zn+- and H+-evoked ZAC signaling and of spontaneous ZAC activity, and the slow on-set of its channel block suggested that its ZAC inhibition is state-dependent. TTFB was found to be a selective ZAC antagonist, exhibiting no significant agonist, antagonist or modulatory activity at 5-HT3A, α3ß4 nicotinic acetylcholine, α1ß2γ2S GABAA or α1 glycine receptors at 30 µM. 1 displayed largely non-competitive antagonism of Zn2+-induced ZAC signalling, and TTFB was demonstrated to target the transmembrane and/or intracellular domains of the receptor, which collectively suggests that the N-(thiazol-2-yl)-benzamide analog acts a negative allosteric modulator of ZAC. We propose that this first class of selective ZAC antagonists could constitute useful pharmacological tools in future explorations of the presently poorly elucidated physiological functions governed by this CLR.

Delineation of the functional properties exhibited by the Zinc-Activated Channel (ZAC) and its high-frequency Thr128Ala variant (rs2257020) in Xenopus oocytes.

The signalling characteristics of the Zinc-Activated Channel (ZAC), a member of the Cys-loop receptor (CLR) superfamily, are presently poorly elucidated. The ZACN polymorphism c.454G>A encoding for the Thr128Ala variation in ZAC is found in extremely high allele frequencies across ethnicities. In this, the first study of ZAC in Xenopus oocytes by TEVC electrophysiology, ZACThr128 and ZACAla128 exhibited largely comparable pharmacological and signalling characteristics, but interestingly the Zn2+- and H+-evoked current amplitudes in ZACAla128-oocytes were dramatically smaller than those in ZACThr128-oocytes. While the variation thus appeared to impact cell surface expression and/or channel properties of ZAC, the similar expression properties exhibited by ZACThr128 and ZACAla128 in transfected mammalian cells indicated that their distinct functionalities could arise from the latter. In co-expression experiments, wild-type and variant ZAC subunits assembled efficiently into "heteromeric" complexes in HEK293 cells, while the concomitant presence of ZACAla128 in ZACThr128:ZACAla128-oocytes did not exert a dominant negative effect on agonist-evoked current amplitudes compared to those in ZACThr128-oocytes. Finally, the structural determinants of the functional importance of the 1-hydroxyethyl side-chain of Thr128 appeared to be subtle, as agonist-evoked current amplitudes in ZACSer128-, ZACVal128- and ZACIle128-oocytes also were substantially lower than those in ZACThr128-oocytes. In conclusion, the functional properties exhibited by ZAC in this work substantiate the notion of it being an atypical CLR. While the impact of the Thr128Ala variation on ZAC functionality in oocytes is striking, it remains to be investigated whether and to which extent this translates into an in vivo setting and thus could constitute a source of inter-individual variation in ZAC physiology.

Delineation of the functional properties and the mechanism of action of AA29504, an allosteric agonist and positive allosteric modulator of GABAA receptors.

The retigabine analog 2-amino-4-[(2,4,6-trimethylbenzylamino)-phenyl]-carbamic acid ethyl ester (AA29504) is a positive allosteric modulator (PAM) of γ-aminobutyric acidA receptors (GABAARs), and the modulator has been used in ex vivo/in vivo studies to probe the physiological roles of native δ-containing GABAARs. In this study, the functional properties and mode of action of AA29504 were investigated at human GABAARs expressed in Xenopus oocytes by two-electrode voltage clamp electrophysiology. AA29504 was found to be an allosteric GABAAR agonist displaying low intrinsic activities at 3-30 µM. AA29504 was essentially equipotent as a PAM at the 13 GABAAR subtypes tested (EC50: 0.45-5.2 µM), however GABA EC5-evoked currents through αßδ subtypes were modulated to substantially higher levels than those through αßγ2S subtypes (relative to GABA Imax). While the δ/γ2S-difference clearly was key for this differential GABA efficacy modulation, studies of the AA29504-mediated modulation of different α4,5,6-containing αß, αßγ2S and αßδ GABAARs revealed the α-subunit identity to be another important determinant. Based on its functional properties at numerous mutant GABAARs and on in silico analysis of its low-energy conformations, AA29504 is proposed to act through an allosteric site in the transmembrane ß(+)/α(-) interface in the GABAAR also targeted by etomidate and several other modulators. In contrast to these modulators, however, AA29504 did not display substantial ß2/ß3-over-ß1 GABAAR preference, which challenges the notion of ligands targeting this site always possessing this subtype-selectivity profile. Hence, the detailed pharmacological profiling of AA29504 both highlights the complexity of allosteric GABAAR modulation and provides valuable information about this modulator as a pharmacological tool.

Functional properties and mechanism of action of PPTQ, an allosteric agonist and low nanomolar positive allosteric modulator at GABAA receptors.

The former sedative-hypnotic and recreational drug methaqualone (Quaalude) is a moderately potent, non-selective positive allosteric modulator (PAM) at GABAA receptors (GABAARs) (Hammer et al., 2015). In the present study, we have identified a novel methaqualone analog, 2-phenyl-3-(p-tolyl)quinazolin-4(3H)-one (PPTQ), in a screening of 67 analogs at five αß2γ2S GABAAR subtypes and delineated its functional properties and mechanism of action at wild-type and mutant GABAARs expressed in Xenopus laevis oocytes by two-electrode voltage clamp electrophysiology. PPTQ was found to be an allosteric agonist and a PAM (ago-PAM) at human α1ß2γ2S and α4ß2δ GABAARs, exhibiting intrinsic activity at micromolar concentrations and potentiating the GABA-evoked signaling through the receptors at concentrations down to the low-nanomolar range. Whereas PPTQ exclusively increased the potency of GABA at the α1ß2γ2S receptor, it increased both GABA potency and efficacy at α4ß2δ and displayed modest potency-based preference for α4ß2δ over α1ß2γ2S. In elaborate mutagenesis and competition experiments PPTQ was found to act through the same or an overlapping site as etomidate in the transmembrane ß(+)/α(-) subunit interfaces, whereas it did not seem to target the other three transmembrane interfaces in the GABAAR. Finally, the PPTQ site was shown to be allosterically linked with sites targeted by neurosteroids and barbiturates but not with the high-affinity benzodiazepine site in the α1ß2γ2S receptor. In conclusion, the development of a highly potent, bioavailable GABAAR ago-PAM by subtle modifications to the methaqualone scaffold demonstrates that derivatization of this infamous drug from the past can lead to modulators with distinct functional characteristics at the receptors.