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Cryo-EM structures of Arabidopsis CNGC1 and CNGC5 reveal molecular mechanisms underlying gating and calcium selectivity

Nature Plants volume 11, pages 632–642 (2025)Cite this article

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Abstract

Plant cyclic nucleotide-gated channels (CNGCs) belong to the cyclic nucleotide-binding ___domain (CNBD) channel family, but are phylogenetically classified in a distinct branch. In contrast to their animal counterparts of K+-selective or non-selective cation channels, plant CNGCs mainly mediate Ca2+ influx and are involved in various physiological processes, such as stomatal movements, pollen-tube growth and immune responses. Here, we present the cryo-EM structure and electrophysiological analysis of plant CNGC representatives, Arabidopsis CNGC1 and CNGC5. We found that CNGC1 and CNGC5 contain a unique extracellular ___domain featuring disulfide bonds that is essential for channel gating via coupling of the voltage-sensing ___domain with the pore ___domain. The pore ___domain selectivity filter possesses a Gln residue at the constriction site that determines the Ca2+ selectivity. Replacement of this Gln with Glu, typically observed in CNBD-type non-selective cation channels, could convert CNGC1 and CNGC5 from Ca2+-selective channels to non-selective cation channels permeable to Ca2+, Na+ or K+. In addition, we found that the CNGC1 and CNGC5 CNBD homology ___domain contains intrinsic-ligand-like interactions, which may devoid the binding of cyclic nucleotides and lead to gating independent of cAMP or cGMP. This research not only provides a mechanistic understanding of plant CNGCs’ function, but also adds to the comprehensive knowledge of the CNBD channels.

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Fig. 1: Electrophysiology analysis of Arabidopsis CNGC5 and CNGC1.
Fig. 2: Overall structure and ___domain organization of CNGC5 and CNGC1.
Fig. 3: A closed pore with depolarized VSD.
Fig. 4: Ion selectivity of CNGC5 and CNGC1.
Fig. 5: ECD is required for channel gating.
Fig. 6: CNBHDs features a structure devoid of cNMP binding.

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Data availability

The 3D cryo-EM density maps of CNGC1Ca, CNGC5 and CNGC5Ca have been deposited in the Electron Microscopy Data Bank under the accession numbers EMD-61105, EMD-61106 and EMD-61107. Coordinates for structure models have been deposited in the Protein Data Bank (PDB) under the accession codes 9J34, 9J35 and 9J36. Source data are provided with this paper.

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Acknowledgements

We thank M. Zhang, L. Zhu and W. Cai from the Center for Excellence in Molecular Plant Sciences core facility for diagnostic cryo-EM analysis and GFP fluorescence analysis; and A. Dong and H. Zhao from Fudan University for technical assistance in cryo-EM data collection. This work was supported by grants from the National Natural Science Foundation of China (grant no. 32025020 to P.Z. and grant no. 32270279 to Y.-F.W.), the Chinese Academy of Sciences (grant no. XDB0630100 to P.Z.) and the Shanghai Science and Technology Commission (grant no. 23310710100).

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Author notes

  1. These authors contributed equally: Jianping Wang, Bo-Ya Du, Xue Zhang.

Authors and Affiliations

  1. National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China

    Jianping Wang, Bo-Ya Du, Xue Zhang, Xiaomin Qu, Yang Yang, Zhao Yang, Yong-Fei Wang & Peng Zhang

  2. Key Laboratory of Plant Carbon Capture, Chinese Academy of Sciences, Shanghai, China

    Jianping Wang, Xue Zhang, Xiaomin Qu, Yang Yang, Zhao Yang & Peng Zhang

  3. University of Chinese Academy of Sciences, Beijing, China

    Jianping Wang, Bo-Ya Du & Xiaomin Qu

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  1. Jianping Wang
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Contributions

J.W., B.D. and X.Z. designed and performed the bulk of the experiments. J.W. carried out protein expression and purification, and grid sample preparation. X.Z. and J.W. carried out cryo-EM data collection and structure determination, supervised by P.Z. B.D. carried out electrophysiological experiments guided by Y.-F.W. Y.Y. contributed to electrophysiological data analysis. X.Q. and Z.Y. contributed to protein purification and grid sample preparation. P.Z., Y.-F.W. and J.W. wrote the manuscript with inputs from other authors. P.Z. conceived the project.

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Correspondence to Yong-Fei Wang or Peng Zhang.

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Nature Plants thanks Changlin Tian and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Cryo-EM analysis of CNGC5.

a, Gel filtration profile and Coomassie-blue-stained SDS-PAGE analysis of CNGC5 sample prepared in nanodiscs. Independent experiments have been repeated at least three times with similar results. b, Representative micrograph of cryo-EM. c, 2D class averages. d, cryo-EM data analysis pipeline. e, Local resolution estimation and gold-standard Fourier shell correlation (FSC) curves. The color represents the local resolution in Å.

Source data

Extended Data Fig. 2 Cryo-EM analysis of CNGC5Ca.

a, Gel filtration profile and Coomassie-blue-stained SDS-PAGE analysis of CNGC5 in buffer containing 0.01% GDN. Independent experiments have been repeated at least three times with similar results. b, Representative micrograph of cryo-EM. c, 2D class averages. d, cryo-EM data analysis pipeline. e, Local resolution estimation and gold-standard Fourier shell correlation (FSC) curves. The color represents the local resolution in Å.

Extended Data Fig. 3 Cryo-EM analysis of CNGC1Ca.

a, Gel filtration profile and Coomassie-blue-stained SDS-PAGE analysis of CNGC1 in buffer containing 0.01% GDN. Independent experiments have been repeated at least three times with similar results. b, Representative micrograph of cryo-EM. c, 2D class averages. d, cryo-EM data analysis pipeline. e, Local resolution estimation and gold-standard Fourier shell correlation (FSC) curves. The color represents the local resolution in Å.

Extended Data Fig. 4 Structure based sequence alignment of 20 CNGCs from Arabidopsis thaliana, showing exclusively on the segment of ECD.

In the illustration, the numbers highlighted in orange denote the cysteine residues that form the three corresponding disulfide bonds. Sequences are ordered by classification based on phylogenetic relationships.

Extended Data Fig. 5 VSDs of CNGC5 and CNGC1 in comparative perspective with other CNBD Channels.

a, Sequence alignment of S4 helix among 20 Arabidopsis CNGCs. Red asterisks indicate the conserved arginine site. b, VSDs of CNGC5, CNGC1, HCN1(PDB:5U6P) and KAT1(PDB:6V1X) are shown in side view, with the S1 helix omitted for clarity. The positive charged (or polar) residues on S4 and the gating charge transfer center residues on S2 and S3 are shown as sticks.

Extended Data Fig. 6 Ion selectivity of CNGC5 and CNGC1.

a, Sequence alignment of ion selectivity filter motifs in 20 Arabidopsis CNGCs. b-d, Density map at the selectivity filter of CNGC1Ca and CNGC5Ca. Both samples were prepared with a supplementation of 2 mM Ca2+. Selectivity filter regions of two diagonally opposed subunits are shown in sticks, the Ca2+ ions along the ion pathway are in green spheres and water molecules are in red spheres. b, The density at the selectivity filter of CNGC1Ca. Density map is obtained after C4 symmetry processing (contoured at 5.5 σ). c, The density at the selectivity filter of CNGC5Ca. Density map is obtained after C4 symmetry processing (contoured at 5.5 σ). d, The density at the selectivity filter of CNGC5Ca. Density map is obtained after C1 symmetry processing (contoured at 4.0 σ). Chain A, C and chain B, D exhibit distinct conformations due to their mobility or asymmetry, and are shown separately.

Extended Data Fig. 7 Comparison of the selectivity filter of CNGC5, CNGC1 and the selected CNBD channels.

a, Sequence alignment of ion selectivity filter motifs in CNGC5, CNGC1 and other CNBD channels. b, Structure comparison of the selectivity filter of CNGC5Ca, CNGC1Ca and the selected CNBD channels. For clarity, only two diagonally opposed subunits are shown. The PDB accession numbers of the comparison channels are KAT1, 6V1X; HCN1, 5U6P; TAX-4, 6WEK.

Extended Data Fig. 8 Electrophysiological studies on selectivity filter mutants.

Patch clamping experiments were performed in HEK293T cells for the analysis of ion permeability to diverse cations. a and c, The average current-voltage curves show the whole-cell currents of CNGC5Q383A and CNGC1Q371A in 1 mM Ca2+-based bath solution similar to that in 10 mM Ca2+-based bath solution, which are different from the mock control-like small currents of wild-type CNGC5 and CNGC1 in 1 mM Ca2+-based bath solution. b and d, The average current-voltage curves show the whole-cell currents of CNGC5Q383A and CNGC1Q371A in the bath solutions with 120 mM Na+ and 1 mM Ca2+, 8 mM Na+ and 1 mM Ca2+, 8 mM Na+ and 10 mM Ca2+, and 8 mM K+, and 1 mM Ca2+. The currents in different bath solutions show different reversal potentials (zoom-in view), which point to the ion selectivity of CNGC5Q383A and CNGC1Q371A. For the convenience of comparison, the data of mock control in panels a and c of this extended data figure are also shown as the mock control data in Fig. 4d and f, respectively; the data of CNGC5Q383A/CNGC1Q371A 1 mM Ca2+-based bath solution in panels a and c of this extended data figure are also shown as the data of CNGC5Q383A/CNGC1Q371A in 120 mM Na+ plus 1 mM Ca2+ condition in panels b and d, respectively. The letter n denotes the numbers of HEK293T cells tested. Data are presented as means ± SEM.

Source data

Extended Data Fig. 9 Representative fluorescent of CNGC5 wild-type, mutants and GFP in HEK 293T cells.

GFP tag was fused to the N terminus of CNGC5. GFP fluorescence was readily detected in cells expressed CNGC5 wild type (WT) and mutants with similar patterns. Independent experiments have been repeated at least three times with similar results. The varying lengths of the gray bars corresponded to 10 µm in all the indicated variant images.

Extended Data Fig. 10 Sequence alignment of cNMP-binding region.

a, Sequence alignment of cNMP binding region in Arabidopsis CNGC1/5 and other selected CNBD channels. b, Sequence alignment of 20 Arabidopsis CNGCs. Filled red triangles indicate the typical Arg residue required for cNMP binding in cNMP-regulated CNBD channels. Filled red circles indicate another distinctive Arg residue in Arabidopsis CNGCs that occupies the pseudo cNMP-binding pocket.

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Wang, J., Du, BY., Zhang, X. et al. Cryo-EM structures of Arabidopsis CNGC1 and CNGC5 reveal molecular mechanisms underlying gating and calcium selectivity. Nat. Plants 11, 632–642 (2025). https://doi.org/10.1038/s41477-025-01923-z

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