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

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 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.

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

Fig. 1.. Chimeric receptors fusing the ECDs and TMD-ICDs of ZAC, m5-HT₃AR and hα₁ GlyR.

A. Topologies of WT m5-HT3A, WT ZAC and WT hα₁ GlyR subunits and chimeric ZAC/m5-HT₃A, m5-HT₃A/ZAC, ZAC/hα₁-Gly and hα₁-Gly/ZAC subunits and illustration of the pentameric complexes assembled from them. The amino acid sequences of the ECD-into-TMD sequences in the three WT subunit proteins are given. The borders between β10 (ECD) and M1 (TMD) reported for m5-HT₃AR [11] and hα₁ GlyR [15] and the fusion points in the four chimeras (“fusion”) are indicated. B. Functionalities of the constructed chimeric receptors. Representative traces from the testing of the putative agonists at ZAC/m5-HT₃A-, m5-HT₃A/ZAC-, ZAC/hα₁-Gly- and hα₁-Gly/ZAC-expressing oocytes by TEVC electrophysiology. C. Averaged agonist-evoked current amplitudes in oocytes expressing the functional m5-HT₃A/ZAC (top) and ZAC/hα₁-Gly (bottom) chimeras and their respective parent receptors. Saturating agonist concentrations for the different receptors were used: 300 μM 5-HT (WT m5-HT₃AR), 3 μM 5-HT (m5-HT₃A/ZAC), 10 mM Zn²⁺ (WT ZAC), 30 μM Zn²⁺ (ZAC/hα₁-Gly), and 1 mM Gly (WT hα₁ GlyR). The averaged data are given as means ± S.E.M. and are based on data from recordings performed on the receptors expressed in at least two diffent oocyte batches (n = 5–19).

Fig. 2.. Functional properties exhibited by the chimeric m5-HT₃A/ZAC receptor.

A. Agonist properties displayed by 5-HT at m5-HT₃A/ZAC. Representative traces for 5-HT-evoked currents in WT m5-HT₃AR- and m5-HT₃A/ZAC-expressing oocytes (left) and averaged concentration-response relationships displayed by 5-HT at WT m5-HT₃AR and m5-HT₃A/ZAC (means ± S.E.M., n = 7–9) (right). B. Modulatory properties displayed by Zn²⁺ at m5-HT₃A/ZAC. Representative traces for the Zn²⁺-mediated modulation of 5-HT (EC₈₀)-evoked currents in WT m5-HT₃AR- and m5-HT₃A/ZAC-expressing oocytes (left) and averaged concentration-effect relationships displayed by Zn²⁺ at the 5-HT (EC₈₀)-mediated currents through WT m5-HT₃AR and m5-HT₃A/ZAC (means ± S.E.M., n = 7–8) (right). 5-HT (30 μM) and 5-HT (1 μM) were used for WT m5-HT₃AR and m5-HT₃A/ZAC, respectively. C. Representative traces of the currents evoked by 4 min-applications of saturating agonist concentrations at WT m5-HT₃AR-, WT ZAC- and m5-HT₃A/ZAC-expressing oocytes. 5-HT (100 μM), Zn²⁺ (10 mM) and 5-HT (3 μM) were used at WT m5-HT₃AR, WT ZAC and m5-HT₃A/ZAC, respectively.

Fig. 3.. Functional properties exhibited by the chimeric ZAC/hα₁-Gly receptor.

A. Agonist properties displayed by Zn⁺ and H⁺ at ZAC/α₁-Gly. Representative traces for Zn²⁺- and H⁺-evoked currents in WT ZAC- and ZAC/α₁-Gly-expressing oocytes (left), and averaged concentration-response relationships displayed by Zn²⁺ and H⁺ at WT ZAC and ZAC/α₁-Gly [means ± S.E.M., n = 8–10 (Zn²⁺) and n = 8–11 (H⁺)] (right). B. Antagonist properties displayed by picrotoxin (PTX) at ZAC/hα₁-Gly. Representative traces for picrotoxin-mediated inhibition of Gly (EC₉₀)-evoked currents in WT hα₁ GlyR and Zn²⁺ (EC₉₀)-evoked currents in ZAC/α₁-Gly-expressing oocytes (left), and averaged concentration-inhibition relationships displayed by picrotoxin at WT hα₁ GlyR, WT ZAC and ZAC/hα₁-Gly (means ± S.E.M., n = 9–12) (right). Zn²⁺ (1 mM), Zn²⁺ (10 μM) and Gly (100 μM) were used for WT ZAC, ZAC/α₁-Gly and WT hα₁ GlyR, respectively. C. Representative traces of the currents evoked by 4 min-application of saturating agonist concentrations at WT hα₁ GlyR, WT ZAC- and ZAC/α₁-Gly-expressing oocytes. Zn²⁺ (10 mM), Zn²⁺ (30 μM) and Gly (100 μM) were used as agonist contrations for WT ZAC, ZAC/α₁-Gly and WT hα₁ GlyR, respectively. The trace for WT ZAC is the same as that shown in Fig. 2C.

Fig. 4.. The alternative ZAC/m5-HT₃A-II and hα₁-Gly/ZAC-II chimeras and the ECD/TMD-ICD chimeras with Cys-loop and/or C-terminus modifications.

A. Topologies of the chimeric ZAC/m5-HT₃A-II and hα₁-Gly/ZAC-II subunits and illustration of the pentameric complexes assembled from them. The amino acid sequences of the ECD-into-TMD sequences in the three WT subunit proteins are given, and the alternative fusion points in ZAC/m5-HT₃A-II and hα₁-Gly/ZAC-II (“fusion-II”) compared to those in the original ZAC/m5-HT₃A and hα₁-Gly/ZAC chimeras (“fusion”) are indicated. B. Schematic outline of the modifications made to the PFX-motif in the Cys-loop and in the C-terminal in the ECD-parts and the TMD/ICD-parts of the ZAC/m5-HT₃A, m5-HT₃A/ZAC, ZAC/hα₁-Gly and hα₁-Gly/ZAC chimeras.

Fig. 5.. Candidate Zn²⁺-binding residues in the extracellular domain of ZAC.

A. Amino acid sequence of the ZAC ECD. The indicated signal peptide, the β-sheet β1-β10 and the M1 α-helix segments in ZAC are predicted based on amino acid sequence aligment of the ZAC and m5-HT3A subunits and these segments in the m5-HT₃AR cryo-EM structure (PDB ID: 6HIN) [11]. The 25 candidate Zn²⁺-binding residues in the ZAC ECD are given in bold with their residue numbers above. The candidate Zn²⁺-binding residues in the four defined clusters are given (Cluster 1: green; Cluster 2: cyan; Cluster 3: red; Cluster 4: dark-blue), with the five candidate residues not included in a cluster given in grey (“X”), and the two cysteines forming the Cys-loop are indicated with asterisks. B. Homology model of ZAC based on the cryo-EM structure of m5-HT₃AR (PDB ID: 6HIN). The pentameric ZAC complex (left) and the ECD for two neighbouring subunits in the ZAC complex viewed from the outside (middle) and from the vestibule (right). The candidate Zn²⁺-binding residues in this domain defined as Cluster 1 (green), Cluster 2 (cyan), Cluster 3 (red) and Cluster 4 (dark-blue) are indicated in the ECD dimer, with the five candidate residues not included in a cluster shown in grey.

Fig. 6.. Probing the importance of candidate Zn²⁺-binding residues in Clusters 1 and 2 of the ZAC ECD for Zn²⁺-mediated ZAC activation.

A. Cluster 1. Left: Cluster 1 residues (in green, detail of ZAC homology model). Middle: Averaged I_{pH 4.0} and I_{10 mM Zn2+} values recorded from oocytes expressing WT ZAC and various ZAC mutants [means ± S.E.M., H⁺: n = 5–8 (mutants), n = 14 (WT); Zn²⁺: n = 6–8 (mutants), n = 16 (WT)]. Right: Averaged concentration-response relationships displayed by Zn²⁺ at oocytes expressing WT ZAC and various ZAC mutants [Top graphs: means ± S.E.M., n = 6–8 (mutants), n = 14 (WT). Bottom graph: means ± S.E.M., n = 6–8]. B. Cluster 2. Left: Cluster 2 residues (in cyan, detail of ZAC homology model). Middle: Averaged I_{pH 4.0} and I_{10 mM Zn2+} values recorded from oocytes expressing WT ZAC and various ZAC mutants [means ± S.E.M., H⁺: n = 6–8; Zn²⁺: n = 6–7]. Right: Averaged concentration-response relationships displayed by Zn²⁺ at oocytes expressing WT ZAC, ZAC^H139A and ZAC^H144A [means ± S.E.M., n = 7–8 (mutants), n = 14 (WT)].

Fig. 7.. Probing the importance of candidate Zn²⁺-binding residues in Clusters 3 and 4 of the ZAC ECD for Zn²⁺-mediated ZAC activation.

A. Cluster 3. Left: Cluster 3 residues (in red, detail of ZAC homology model). Right, top: Averaged I_{pH 4.0} and I_{10 mM Zn2+} values recorded from oocytes expressing WT ZAC and various ZAC mutants [means ± S.E.M., n = 5–8]. Right, bottom: Averaged concentration-response relationships displayed by Zn²⁺ at oocytes expressing WT ZAC and various ZAC mutants [means ± S.E.M., n = 5–8 (mutants), n = 12 (WT)]. B. Cluster 4. Left: Cluster 4 residues (in dark-blue, detail of ZAC homology model). Middle: Averaged I_{pH 4.0} and I_{10 mM Zn2+} values recorded from oocytes expressing WT ZAC and various ZAC mutants [means ± S.E.M., n = 5–6]. Right: Averaged concentration-response relationships displayed by Zn²⁺ at oocytes expressing WT ZAC and ZAC^{E160A/E162A/C195} [means ± S.E.M., n = 5–6]. WT ZAC- and ZAC^{E160A/E162A/C195}-oocytes were injected with 1.84 ng and 3.68 ng cRNA, respectively.

Fig. 8.. Functional importance of the Leu9′ residue in ZAC.

A. Representative traces for Zn²⁺-evoked currents in ZAC^L9′I-expressing oocytes (top), and averaged concentration-response relationships exhibited by Zn²⁺ at WT ZAC, ZAC^L9′A, ZAC^L9′V, ZAC^L9′I and ZAC^L9′T (means ± S.E.M., n = 6–8) (bottom). B. Resting membrane potentials (left) and leak currents (right) recorded from oocytes expressing WT ZAC, ZAC^L9′A, ZAC^L9′T, ZAC^L9′V and ZAC^L9′I. Data are given as mean ± S.E.M. values (n = 40–60). C. Representative traces for Zn²⁺- and TC (100 μM)-evoked currents in WT ZAC- and ZAC^L9′I-expressing oocytes (top), and averaged current amplitudes evoked by a saturating concentration of Zn²⁺ and by TC (100 μM) in WT ZAC-, ZAC^L9′A-, ZAC^L9′V-, ZAC^L9′I-, and ZAC^L9′T-oocytes (means ± S.E.M., n = 6–8) (bottom). 10 mM Zn²⁺ were used for WT ZAC and 1 mM Zn²⁺ were used for ZAC^L9′A, ZAC^L9′V, ZAC^L9′I and ZAC^L9′T. D. Degrees of spontaneous activity [defined as: I_{100 uM TC}/ (I_{Zn2+ max} + I_{100 uM TC})] (left) and total current amplitude windows (defined as: I_{Zn2+ max} + I_{100 uM TC}) (right) exhibited by WT ZAC, ZAC^L9′A, ZAC^L9′V, ZAC^L9′I and ZAC^L9′T expressed in oocytes.

Fig. 9.. Signalling characteristics exhibited by the ZAC^L9′X mutants.

A. Representative traces of currents evoked by saturating Zn²⁺ concentrations in WT ZAC-, ZAC^L9′A-, ZAC^L9′V-, ZAC^L9′I- and ZAC^L9′T-expressing oocytes. 10 mM Zn²⁺ was used for WT ZAC and 1 mM Zn²⁺ was used for the ZAC^L9′X mutants, respectively. B. Representative traces of currents evoked by sustained application of saturating Zn²⁺ concentrations in WT ZAC- and ZAC^L9′I-expressing oocytes. 10 mM Zn²⁺ and 1 mM Zn²⁺ were used for WT ZAC and for ZAC^L9′I, respectively.

Fig. 10.. Key residues involved in signal transduction through the CLR.

A. Residues involved in ECD/TMD cross-talk in the classical CLR. Alignment of the amino acid sequences of the β1-β2, β6-β7 (Cys-loop) and β8-β9 loops, the pre-M1/M1 segments and the M2-M3 linkers in ZAC, selected classical CLRs and the prokaryotic CLRs GLIC and ELIC. The conservation of key residues for the ECD/TMD cross-talk are indicated (negatively charged or charge-neutral, polar residues in red and positively charged residues blue, structural residues in green). B. Residues involved in orthosteric agonist binding to the classical CLR. Alignment of the amino acid sequences of loops A-F in ZAC and selected classical CLRs. The residues in the loops directly involved in orthosteric agonist binding to m5-HT₃AR [12], hα₄β₂ nAChR [14], hα₁β₂γ2 GABA_AR [13] and hα₁ GlyR [10] are indicated in bold and highlighted in yellow.