Abstract
Potassium channels selectively conduct K+ ions across cell membranes, and use diverse mechanisms to control their gating. We studied ion permeation and gating of an inwardly rectifying K+ channel by individually changing the amide carbonyls of two conserved glycines lining the selectivity filter to ester carbonyls using nonsense suppression. Surprisingly, these backbone mutations do not significantly alter ion selectivity. However, they dramatically change the kinetics of single-channel gating and produce distinct subconductance levels. The mutation at the glycine closer to the inner mouth of the pore also abolishes high-affinity binding of Ba2+ to the channel, indicating the importance of this position in ion stabilization in the selectivity filter. Our results demonstrate that K+ ion selectivity can be retained even with significant reduction of electronegativity in the selectivity filter, and that conformational changes of the filter arising from interactions between permeant ions and the backbone carbonyls contribute directly to channel gating.
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Acknowledgements
We thank V.W. Cornish for her participation in the early stage of this work, S. Siegelbaum and members of his lab for discussions, and S. Siegelbaum and W. Sather for comments on a previous version of the manuscript. We also thank H. Lester and D. Dougherty for the tRNA gene and Z. Lu for a gift of tertiapin. This work was supported by grants from the NIH (J.Y., L.Y. J. and P.G.S.), the NSF (A.Y.T.), the Alfred Sloan Foundation (J.Y.) and the HHMI (L.Y. J. and P.G.S.).
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Lu, T., Ting, A., Mainland, J. et al. Probing ion permeation and gating in a K+ channel with backbone mutations in the selectivity filter. Nat Neurosci 4, 239–246 (2001). https://doi.org/10.1038/85080
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DOI: https://doi.org/10.1038/85080
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