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

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 antagonism displayed by 1 at the ZAC-HEK293 cells in the FMP assay was subsequently verified at ZAC expressed in Xenopus oocytes in TEVC recordings. The signalling properties exhibited by ZAC in this assay has been characterized in detail in a previous study [ 25 ], and insights gained from that study guided the design of these experiments. 1 was found to inhibit the currents evoked by 1 mM Zn 2+ (~EC 50 ) through ZAC-expressing oocytes in a concentration-dependent manner.…”

“…Analogously to the robust spontaneous activity exhibited by ZAC in mammalian cells [ 21 , 22 ], the channel also displays this characteristic when expressed in oocytes, albeit to a smaller degree [ 25 ], As can be seen from the traces in Fig. 2A , the 30 s preincubation of the ZAC-expressing oocyte with compound 1 prior to the co-application of 1 and Zn 2+ onto the oocyte produced small but significant outward currents, reflecting a block of this spontaneous activity ( Fig.…”

“…The antagonist properties exhibited by TC at ZAC-expressing oocytes in parallel recordings are given in Fig. 2A and 2B for comparison (data are from a recent study [ 25 ]). In contrast to 1, TC mediated complete inhibition of the Zn 2+ (1 mM)-induced response through ZAC (a fitted ~94% inhibition range), but with an IC 50 of 3.2 μM (pIC 50 ± S.E.M: 5.49 ± 0.04, n = 8) TC was equipotent with 1 as a ZAC antagonist ( Fig.…”

“…Defolliculated stage V-VI oocytes harvested from female Xenopus laevis frogs were obtained from Lohmann Research Equipment (Castrop-Rauxel, Germany). The construction of cDNAs for wild-type (WT) human ZAC (ZAC Thl128 [ 25 ]) and other CLR subunits (ZAC-pCIneo, ZAC-pUNIV, m5-HT3A-pUNIV, hαl-GlyR-pUNIV, hαl -GABA A R-pCDNA3.1 hβ2-GABA A R-pCDNA3.1, hγ2S-GABA A R-pCDNA3.1, hα3-nAChR-pCDNA3.1 and hβ4-nAChR-pCDNA3.1) and for chimeric subunits (m5-HT 3 A/ZAC-pUNIV and ZAC/hα 1 -Gly-pUNIV) have been described previously (the prefixes “h”, “m” and “r” denote himian, mouse and rat, respectively) [ 25 , 29 – 31 ]. The HEK293, h5-HT 3 A-HEK293 and rα 3 β 4 -nAChR-HEK293 cell lines were generous gifts from Drs.…”

“…Explorations into the in vivo functions governed by ZAC and the therapeutic potential in the receptor would be facilitated by the availability of potent and selective pharmacological tools. The curare alkaloid tubocurarine (TC) has been shown to inhibit ZAC signalling with low-micromolar IC 50 values [ 21 , 25 ], but the fact that TC is a promiscuous antagonist displaying comparable or even higher antagonist potencies at several other CLRs makes it unsuited as a pharmacological tool for ZAC [ 26 , 27 ]. A recent patent application has reported that a wide range of structurally diverse ligands (including ATP, dye compounds, heparin and tricyclic antidepressants) modulate ZAC signalling at mid-micromolar to millimolar concentration ranges [ 24 ].…”