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

Abstract

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 Thr 128 Ala 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 , ZAC Thr128 and ZAC Ala128 exhibited largely comparable pharmacological and signalling characteristics, but interestingly the Zn 2+ - and H + -evoked current amplitudes in ZAC Ala128 -oocytes were dramatically smaller than those in ZAC Thr128 -oocytes. While the variation thus appeared to impact cell surface expression and/or channel properties of ZAC, the similar expression properties exhibited by ZAC Thr128 and ZAC Ala128 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 ZAC Ala128 in ZAC Thr128 :ZAC Ala128 -oocytes did not exert a dominant negative effect on agonist-evoked current amplitudes compared to those in ZAC Thr128 -oocytes. Finally, the structural determinants of the functional importance of the 1-hydroxyethyl side-chain of Thr 128 appeared to be subtle, as agonist-evoked current amplitudes in ZAC Ser128 -, ZAC Val128 - and ZAC Ile128 -oocytes also were substantially lower than those in ZAC Thr128 -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 Thr 128 Ala 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.