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Structural dynamics in the gating ring of cyclic nucleotide–gated ion channels

Nature Structural & Molecular Biology volume 14, pages 854–860 (2007)Cite this article

Abstract

For ligand-gated ion channels, the binding of a ligand to an intracellular or extracellular domain generates changes in transmembrane pore-forming helices, which alters ion flow. The molecular mechanism for this allostery, however, remains unknown. Here we explore the structure and conformational rearrangements of the C-terminal gating ring of the cyclic nucleotide–gated channel CNGA1 during activation by cyclic nucleotides with patch-clamp fluorometry. By monitoring fluorescent resonance energy transfer (FRET) between membrane-resident quenchers and fluorophores attached to the channel, we detected no movement orthogonal to the membrane during channel activation. By monitoring FRET between fluorophores within the C-terminal region, we determined that the C-terminal end of the C-linker and the end of the C-helix move apart when channels open. We conclude that during channel activation, a portion of the gating ring moves parallel to the plasma membrane, hinging toward the central axis of the channel.

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Figure 1: X-ray crystal structure of HCN2 cytoplasmic domain.
Figure 2: FRET between fluorescently labeled ion channels and the membrane probe, DPA, measured with patch-clamp fluorometry.
Figure 3: FRET between fluorescent channels and the membrane probe, DPA, is sensitive to movement.
Figure 4: FRET between the channel and DPA does not change during ligand activation.
Figure 5: Two-color labeling of channels with GFP and Alexa 568.
Figure 6: The C-helix and residue Cys481 separate during ligand activation.
Figure 7: FRET measurements between GFP and residues throughout the C-terminal region of CNGA1.

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Acknowledgements

We thank H. Utsugi, S. Cunnington and G. Sheridan for technical assistance, E.R. Liman (University of Southern California) for the pGEMHE oocyte expression vector, R.T. Moon (University of Washington) for the plasmid encoding EGFP-F, W. Almers, K. Craven, G. Flynn, A. Merz, M. Puljung and N. Shuart for comments on the manuscript, and L. Islas for stimulating discussions. This work was supported by the Howard Hughes Medical Institute, a grant from the National Eye Institute of the US National Institutes of Health (EY10329) to W.N.Z. and a postdoctoral fellowship from the Jane Coffin Childs Foundation to J.W.T.

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  1. Department of Physiology and Biophysics, Howard Hughes Medical Institute, University of Washington, Seattle, 98195, Washington, USA

    Justin W Taraska & William N Zagotta

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  1. Justin W Taraska
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Correspondence to William N Zagotta.

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Taraska, J., Zagotta, W. Structural dynamics in the gating ring of cyclic nucleotide–gated ion channels. Nat Struct Mol Biol 14, 854–860 (2007). https://doi.org/10.1038/nsmb1281

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