Elsevier

Biochemical Pharmacology

Volume 193, November 2021, 114781
Biochemical Pharmacology

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

https://doi.org/10.1016/j.bcp.2021.114781Get rights and content
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Abstract

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.

Keywords

Cys-loop receptor (CLR)
Pentameric ligand-gated ion channel (pLGIC)
Zinc-Activated Channel (ZAC)
Agonist binding
Chimeric subunits
Leu9′ residue

Abbreviations

5-HT
5-hydroxytryptamine
5-HT3R
5-HT3 receptor
CLR
Cys-loop receptor
ECD
extracellular domain
GABA
γ-aminobutyric acid
GABAAR
GABAA receptor
GlyR
glycine receptor
nAChR
nicotinic acetylcholine receptor
TEVC
two-electrode voltage clamp
TMD
transmembrane domain
ICD
intracellular domain
ZAC
Zinc-Activated Channel

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1

N.M. and E.M. contributed equally to this work and are co-first-authors.