In rod photoreceptors of the retina, the cyclic nucleotide-gated (CNG) channel is composed of three CNGA and one CNGB subunits, and it closes in response to light activation to generate an electrical signal that is conveyed to the brain. Here we report the cryo-EM structure of the closed state of the native rod CNG channel isolated from bovine retina. The structure reveals differences between CNGA1 and CNGB1 subunits. Three CNGA1 subunits are tethered at their C terminus by a coiled-coil region. The C-helix in the cyclic nucleotide-binding domain of CNGB1 features a different orientation from that in the three CNGA1 subunits. The arginine residue R994 of CNGB1 reaches into the ionic pathway and blocks the pore, thus introducing an additional gate, which is different from the central hydrophobic gate known from homomeric CNGA channels. These results address the long-standing question of how CNGB1 subunits contribute to the function of CNG channels in visual and olfactory neurons.
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Kaupp, U. B. & Seifert, R. Cyclic nucleotide-gated ion channels. Physiol. Rev. 82, 769–824 (2002).
Kaupp, U. B. Olfactory signalling in vertebrates and insects: differences and commonalities. Nat. Rev. Neurosci. 11, 188–200 (2010).
Kaupp, U. B. & Seifert, R. Molecular diversity of pacemaker ion channels. Annu. Rev. Physiol. 63, 235–257 (2001).
Lee, C. H. & MacKinnon, R. Structures of the human HCN1 hyperpolarization-activated channel. Cell 168, 111–120.e11 (2017).
Haitin, Y., Carlson, A. E. & Zagotta, W. N. The structural mechanism of KCNH-channel regulation by the eag domain. Nature 501, 444–448 (2013).
Codding, S. J., Johnson, A. A. & Trudeau, M. C. Gating and regulation of KCNH (ERG, EAG, and ELK) channels by intracellular domains. Channels 14, 294–309 (2020).
Jarratt-Barnham, E., Wang, L., Ning, Y. & Davies, J. M. The complex story of plant cyclic nucleotide-gated channels. Int. J. Mol. Sci. 22, 874 (2021).
Koch, K. W. & Stryer, L. Highly cooperative feedback control of retinal rod guanylate cyclase by calcium ions. Nature 334, 64–66 (1988).
Stephen, R., Filipek, S., Palczewski, K. & Sousa, M. C. Ca2+‐dependent regulation of phototransduction. Photochem. Photobiol. 84, 903–910 (2008).
Pugh, E. N., Nikonov, S. & Lamb, T. D. Molecular mechanisms of vertebrate photoreceptor light adaptation. Curr. Opin. Neurobiol. 9, 410–418 (1999).
Molday, R. S. Calmodulin regulation of cyclic-nucleotide-gated channels. Curr. Opin. Neurobiol. 6, 445–452 (1996).
Kaupp, U. B. et al. Primary structure and functional expression from complementary DNA of the rod photoreceptor cyclic GMP-gated channel. Nature 342, 762–766 (1989).
Körschen, H. G. et al. A 240 kDa protein represents the complete β subunit of the cyclic nucleotide-gated channel from rod photoreceptor. Neuron 15, 627–636 (1995).
Chen, T. Y. et al. A new subunit of the cyclic nucleotide-gated cation channel in retinal rods. Nature 362, 764–767 (1993).
Bönigk, W. et al. Rod and cone photoreceptor cells express distinct genes for cGMP-gated channels. Neuron 10, 865–877 (1993).
Bönigk, W. et al. The native rat olfactory cyclic nucleotide-gated channel is composed of three distinct subunits. J. Neurosci. 19, 5332–5347 (1999).
Weitz, D., Ficek, N., Kremmer, E., Bauer, P. J. & Kaupp, U. B. Subunit stoichiometry of the CNG channel of rod photoreceptors. Neuron 36, 881–889 (2002).
Zhong, H., Molday, L. L., Molday, R. S. & Yau, K. W. The heteromeric cyclic nucleotide-gated channel adopts a 3A:1B stoichiometry. Nature 420, 193–198 (2002).
Zheng, J., Trudeau, M. C. & Zagotta, W. N. Rod cyclic nucleotide-gated channels have a stoichiometry of three CNGA1 subunits and one CNGB1 subunit. Neuron 36, 891–896 (2002).
Shuart, N. G., Haitin, Y., Camp, S. S., Black, K. D. & Zagotta, W. N. Molecular mechanism for 3:1 subunit stoichiometry of rod cyclic nucleotide-gated ion channels. Nat. Commun. https://doi.org/10.1038/ncomms1466 (2011).
Batra-Safferling, R. et al. Glutamic acid-rich proteins of rod photoreceptors are natively unfolded. J. Biol. Chem. 281, 1449–1460 (2006).
Poetsch, A., Molday, L. L. & Molday, R. S. The cGMP-gated channel and related glutamic acid-rich proteins interact with peripherin-2 at the rim region of rod photoreceptor disc membranes. J. Biol. Chem. 276, 48009–48016 (2001).
Körschen, H. G. et al. Interaction of glutamic-acid-rich proteins with the cGMP signalling pathway in rod photoreceptors. Nature 400, 761–766 (1999).
Haynes, K. W., Kay, L. W. & Yau, A. R. Single cyclic GMP-activated channel activity in excised patches of rod outer segment membrane. Nature 321, 66–70 (1986).
Taylor, W. R. & Baylor, D. A. Conductance and kinetics of single cGMP‐activated channels in salamander rod outer segments. J. Physiol. 483, 567–582 (1995).
Sesti, F., Eismann, E., Kaupp, U. B., Nizzari, M. & Torre, V. The multi‐ion nature of the cGMP‐gated channel from vertebrate rods. J. Physiol. 487, 17–36 (1995).
Stern, J. H., Kaupp, U. B. & MacLeish, P. R. Control of the light-regulated current in rod photoreceptors by cyclic GMP, calcium, and l-cis-diltiazem. Proc. Natl Acad. Sci. USA 83, 1163–1167 (1986).
Hsu, Y.-T. & Molday, R. S. Modulation of the cGMP-gated channel of rod photoreceptor cells by calmodulin. Nature 361, 76–79 (1993).
Chen, T. Y. et al. Subunit 2 (or β) of retinal rod cGMP-gated cation channel is a component of the 240-kDa channel-associated protein and mediates Ca2+-calmodulin modulation. Proc. Natl Acad. Sci. USA 91, 11757–11761 (1994).
Weitz, D. et al. Calmodulin controls the rod photoreceptor CNG channel through an unconventional binding site in the N-terminus of the β-subunit. EMBO J. 17, 2273–2284 (1998).
Grunwald, M. E., Yu, W., Yu, H. & Yau, K. Identification of a domain on the β-subunit of the rod cGMP-gated cation channel that mediates inhibition by calcium-calmodulin. J. Biol. Chem. 273, 9148–9157 (1998).
Gordon, S. E., Downing-Park, J. & Zimmerman, A. L. Modulation of the cGMP‐gated ion channel in frog rods by calmodulin and an endogenous inhibitory factor. J. Physiol. 486, 533–546 (1995).
Sagoo, M. S. & Lagnado, L. The action of cytoplasmic calcium on the cGMP-activated channel in salamander rod photoreceptors. J. Physiol. 497, 309–319 (1996).
Xue, J., Han, Y., Zeng, W., Wang, Y. & Jiang, Y. Structural mechanisms of gating and selectivity of human rod CNGA1 channel. Neuron https://doi.org/10.1016/j.neuron.2021.02.007 (2021).
Cook, N. J., Hanke, W. & Kaupp, U. B. Identification, purification, and functional reconstitution of the cyclic GMP-dependent channel from rod photoreceptors. Proc. Natl Acad. Sci. USA 84, 585–589 (1987).
Molday, R. S. et al. The cGMP-gated channel of the rod photoreceptor cell—characterization and orientation of the amino terminus. J. Biol. Chem. 266, 21917–21922 (1991).
Kang, K. et al. Assembly of retinal rod or Cone Na+/Ca2+-K+ exchanger oligomers with cGMP-gated channel subunits as probed with heterologously expressed cDNAs. Biochemistry 42, 4593–4600 (2003).
Higgins, M. K., Weitz, D., Warne, T., Schertler, G. F. X. & Kaupp, U. B. Molecular architecture of a retinal cGMP-gated channel: the arrangement of the cytoplasmic domains. EMBO J. 21, 2087–2094 (2002).
Zagotta, W. N. et al. Structural basis for modulation and agonist specificity of HCN pacemaker channels. Nature 425, 200–205 (2003).
Zheng, X. et al. Mechanism of ligand activation of a eukaryotic cyclic nucleotide-gated channel. Nat. Struct. Mol. Biol. 27, 625–634 (2020).
Evans, E. G. B., Morgan, J. L. W., DiMaio, F., Zagotta, W. N. & Stoll, S. Allosteric conformational change of a cyclic nucleotide-gated ion channel revealed by DEER spectroscopy. Proc. Natl Acad. Sci. USA 117, 10839–10847 (2020).
Puljung, M. C., DeBerg, H. A., Zagotta, W. N. & Stoll, S. Double electron–electron resonance reveals cAMP-induced conformational change in HCN channels. Proc. Natl Acad. Sci. USA 111, 9816–9821 (2014).
Clayton, G. M., Silverman, W. R., Heginbotham, L. & Morais-Cabral, J. H. Structural basis of ligand activation in a cyclic nucleotide regulated potassium channel. Cell 119, 615–627 (2004).
Ludwiczak, J., Winski, A., Szczepaniak, K., Alva, V. & Dunin-Horkawicz, S. DeepCoil—a fast and accurate prediction of coiled-coil domains in protein sequences. Bioinformatics 35, 2790–2795 (2019).
Root, M. J. & MacKinnon, R. Identification of an external divalent cation-binding site in the pore of a cGMP-activated channel. Neuron 11, 459–466 (1993).
Seifert, R., Eismann, E., Ludwig, J., Baumann, A. & Kaupp, U. B. Molecular determinants of a Ca2+-binding site in the pore of cyclic nucleotide-gated channels: S5/S6 segments control affinity of intrapore glutamates. EMBO J. 18, 119–130 (1999).
Eismann, E., Muller, F., Heinemann, S. H. & Kaupp, U. B. A single negative charge within the pore region of a cGMP-gated channel controls rectification, Ca2+ blockage, and ionic selectivity. Proc. Natl Acad. Sci. USA 91, 1109–1113 (1994).
Nakatani, K. & Yau, K. W. Calcium and magnesium fluxes across the plasma membrane of the toad rod outer segment. J. Physiol. 395, 695–729 (1988).
Frings, S., Seifert, R., Godde, M. & Kaupp, U. B. Profoundly different calcium permeation and blockage determine the specific function of distinct cyclic nucleotide-gated channels. Neuron 15, 169–179 (1995).
Rheinberger, J., Gao, X., Schmidpeter, P. A. M. & Nimigean, C. M. Ligand discrimination and gating in cyclic nucleotide-gated ion channels from apo and partial agonist-bound cryo-EM structures. eLife 7, e39775 (2018).
James, Z. M. et al. CryoEM structure of a prokaryotic cyclic nucleotide-gated ion channel. Proc. Natl Acad. Sci. USA 114, 4430–4435 (2017).
Whicher, J. R. & MacKinnon, R. Structure of the voltage-gated K+ channel Eag1 reveals an alternative voltage sensing mechanism. Science 353, 664–669 (2016).
D’Imprima, E. & Kühlbrandt, W. Current limitations to high-resolution structure determination by single-particle cryoEM. Q. Rev. Biophys. https://doi.org/10.1017/S0033583521000020 (2021).
Tennyson, A. J. D., Palma, R. L., Robertson, H. A., Worthy, T. H. & Gill, B. J. A new species of kiwi (Aves, Apterygiformes) from Okarito, New Zealand. Rec. Auckl. Mus. 40, 55–64 (2003).
Wang, W., Geiger, J. H. & Borhan, B. The photochemical determinants of color vision: revealing how opsins tune their chromophore’s absorption wavelength. Bioessays 36, 65–74 (2014).
Lamb, T. D. Evolution of phototransduction, vertebrate photoreceptors and retina. Prog. Retin. Eye Res. 36, 52–119 (2013).
Tan, B. Z., Huang, H., Lam, R. & Soong, T. W. Dynamic regulation of RNA editing of ion channels and receptors in the mammalian nervous system. Mol. Brain 2, 13 (2009).
Seeburg, P. H., Single, F., Kuner, T., Higuchi, M. & Sprengel, R. Genetic manipulation of key determinants of ion flow in glutamate receptor channels in the mouse. Brain Res. 907, 233–243 (2001).
Sommer, B., Köhler, M., Sprengel, R. & Seeburg, P. H. RNA editing in brain controls a determinant of ion flow in glutamate-gated channels. Cell 67, 11–19 (1991).
Smart, O. S., Neduvelil, J. G., Wang, X., Wallace, B. A. & Sansom, M. S. P. HOLE: a program for the analysis of the pore dimensions of ion channel structural models. J. Mol. Graph. 14, 354–360 (1996).
Crooks, G. E., Hon, G., Chandonia, J.-M. & Brenner, S. E. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004).
Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).
Cardone, G., Heymann, J. B. & Steven, A. C. One number does not fit all: mapping local variations in resolution in cryo-EM reconstructions. J. Struct. Biol. 184, 226–236 (2013).
The PyMOL Molecular Graphics System v.1.2r3pre (Schrödinger, LLC).
Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).
Asarnow, Y., Palovcak, D. & Cheng, E. UCSF pyem v.0.5. Zenodo https://doi.org/10.5281/zenodo.3576630 (2019).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).
Williams, C. J. et al. MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018).
Robert, X. & Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42, W320–W324 (2014).
J.M. received funding from Holcim Stiftung (Holderbank, Switzerland), Promedica Stiftung (no. 1461/M), Swiss National Science Foundation (SNSF) (no. 19082) and from Novartis Stiftung for Biomedical Research (no. 20C198). G.F.X.S. acknowledges SNSF grants no. 173335 and no. 192760. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. We are indebted to M. Peterek at the Scientific Center for Optical and Electron Microscopy (ScopeM) of ETH Zurich, who has provided training and support during acquisition of the datasets on the Titan Krios. We are grateful to our colleagues at the Paul Scherrer Institut who maintain the Merlin HPC cluster.
G.F.X.S. is a co-founder and scientific advisor of the company leadXpro AG and InterAx Biotech AG. The remaining authors declare no competing interests.
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a, Representative micrograph from one of the three datasets. b, Summary of the single-particle pipeline used to obtain the final density map used for model building.
a, On the left, gold-standard FSC curves of the final 3D reconstruction, and on the right, FSC curve for validation of map and model. b, Euler angle distribution of the set of particles that contribute to the final density map. c, Local resolution of the final density map shown at different threshold values (as displayed in Chimera) and different orientations.
The density map relative to one of the CNGA1 subunits (chain C) is shown. The map was contoured at threshold values ranging between 0.03 and 0.065 in Chimera.
The density map relative to the CNGB1 subunits (chain D) is shown. The map was contoured at threshold values ranging between 0.03 and 0.065 in Chimera.
Extended Data Fig. 5 Details of the ab-initio model shown with two different contour levels and two orientations.
In a, the cytoplasmic region of the CNGB1 subunit, and transmembrane region of neighboring CNGA1 is indicated. In b, the transmembrane region of the CNGB1 subunit is indicated. Here, the S2-S3 loop of CNGB1 is visible only at lower threshold. The map thresholds indicated in a are also applied to the density maps shown in b.
Model chain A, B, and C belong to the CNGA1 subunit, and chain D belongs to the CNGB1 subunit.
On top, overview of the CNGA1/CNGB1 structure, with the areas of the interfaces making contacts indicated within boxes. On the bottom, close-up of the interactions taking place between neighboring subunits, with distances between chosen residues indicated (in Å). The structures of TAX-4 in closed (PDB: 6WEJ) and open state (PDB: 6WEK) are shown for comparison.
a, Superposition of single subunits of bovine CNGA1 (chain A, this structure) with TAX-4 in closed-state (PDB: 6WEJ) and human CNGA1 (hCNGA1) in closed-state (PDB: 7LFT). Superposition of the cytoplasmic domains of CNGA1 and CNGB1 (this structure), with TAX-4 in closed (b, d) (PDB: 6WEJ), and TAX-4 in open state (PDB: 6WEK) (c, e).
a, Overview of the CNGA1/CNGB1 structure, with region indicated for the S4-S5 loop and the C-linker of two neighboring CNGA1, and CNGA1/CNGB1 subunits. On the left, panels contain details of model and map and distance (in Å) of selected residues on the S4-S5 loop and on the A’ helix of the C-linker. b, The distance between the S4-S5 loop and the A’ helix of the C-linker of hCNGA1 in closed state (PDB: 7LFT), in open state (PDB: 7LFX), and for TAX-4 in closed state (PDB: 6WEJ).
a, Structural alignment of CNGA1, CNGB1, TAX-4 in closed state (PDB: 6WEJ), HCN1 in closed state (PDB: 5U6O), and SthK (PDB: 6CJQ). The region of the CNBD is shown, with emphasis on the loop that follows the P-helix. b, Details of the density map at the loop connecting the C-helix and the coiled-coiled region in one of the CNGA1 subunits. K554 and D608 are indicated as possible electrostatic interaction c, Sequence alignment of the loop region among CNG subunits (Bos taurus) and other CNG channels. d, The last C-terminal125 residues of the indicated sequences were used as input for DeepCoil for calculating the propensity of the C-terminus to form a coiled-coil.
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Barret, D.C.A., Schertler, G.F.X., Kaupp, U.B. et al. The structure of the native CNGA1/CNGB1 CNG channel from bovine retinal rods. Nat Struct Mol Biol 29, 32–39 (2022). https://doi.org/10.1038/s41594-021-00700-8
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