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Structural basis for the coupling between activation and inactivation gates in K+ channels

Abstract

The coupled interplay between activation and inactivation gating is a functional hallmark of K+ channels1,2. This coupling has been experimentally demonstrated through ion interaction effects3,4 and cysteine accessibility1, and is associated with a well defined boundary of energetically coupled residues2. The structure of the K+ channel KcsA in its fully open conformation, in addition to four other partial channel openings, richly illustrates the structural basis of activation–inactivation gating5. Here, we identify the mechanistic principles by which movements on the inner bundle gate trigger conformational changes at the selectivity filter, leading to the non-conductive C-type inactivated state. Analysis of a series of KcsA open structures suggests that, as a consequence of the hinge-bending and rotation of the TM2 helix, the aromatic ring of Phe 103 tilts towards residues Thr 74 and Thr 75 in the pore-helix and towards Ile 100 in the neighbouring subunit. This allows the network of hydrogen bonds among residues Trp 67, Glu 71 and Asp 80 to destabilize the selectivity filter6,7, allowing entry to its non-conductive conformation. Mutations at position 103 have a size-dependent effect on gating kinetics: small side-chain substitutions F103A and F103C severely impair inactivation kinetics, whereas larger side chains such as F103W have more subtle effects. This suggests that the allosteric coupling between the inner helical bundle and the selectivity filter might rely on straightforward mechanical deformation propagated through a network of steric contacts. Average interactions calculated from molecular dynamics simulations show favourable open-state interaction-energies between Phe 103 and the surrounding residues. We probed similar interactions in the Shaker K+ channel where inactivation was impaired in the mutant I470A. We propose that side-chain rearrangements at position 103 mechanically couple activation and inactivation in KcsA and a variety of other K+ channels.

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Figure 1: Conformational coupling between activation and inactivation gates in K + channels.
Figure 2: Structural basis for allosteric coupling in KcsA.
Figure 3: Role of Phe 103 in allosteric coupling with the selectivity filter.
Figure 4: A common gate-coupling mechanism in K + channels.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates of the mutant E71H–F103A in the KcsA–OM background and of KcsA–OM in Rb+ ions have been deposited in the Protein Data Bank under accession codes 3HPL and 3FB7, respectively.

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Acknowledgements

We thank F. Bezanilla, H Mchaourab and R. Nakamoto for discussions and comments on the manuscript. We also thank K. Locher for comments on the manuscript. R. Mackinnon provided the Fab-expressing hybridoma cells. We thank the members of the Perozo laboratory for comments on the manuscript. We thank K. R. Rajashankar, R. Sanishvili and the staff at the NE-CAT 24ID and GM-CA 23ID beamlines at the Advanced Photon Source, Argonne National Laboratory for assistance during data collection. This work was supported in part by NIH grant R01-GM57846 and a gift from the Palmer family to E.P., by grant R01-GM62342 to B.R. and by an NRSA postdoctoral fellowship to A.C.P.

Author information

Authors and Affiliations

Authors

Contributions

E.P. and L.G.C. conceived the project. L.G.C. and D.M.C. generated constructs, performed biochemical analysis, and expressed, purified and crystallized the proteins. L.G.C. performed EPR experiments. L.G.C., V.J. and E.P. collected X-ray diffraction data. L.G.C. and V.J. determined and analysed the structures. A.C.P. and B.R. performed computation analysis. O.D. and L.G.C. conducted FRET measurements. S.C. measured inactivation in truncated KcsA and in Rb+ ions. J.F.C.-M. measured E71H single-channel activity and made the F103A mutation. L.G.C. measured F103X mutant series electrophysiology. D.G.G. made electrophysiology measurements in Shaker channels. E.P., L.G.C. and V.J. analysed the data and wrote the paper.

Corresponding author

Correspondence to Eduardo Perozo.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Table 1 and Supplementary Figures S1-S10 with legends. (PDF 1962 kb)

Supplementary Movie 1

This movie shows coupling between F103-I100-T74/T75 during C-type inactivation. (MPG 3599 kb)

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Cuello, L., Jogini, V., Cortes, D. et al. Structural basis for the coupling between activation and inactivation gates in K+ channels. Nature 466, 272–275 (2010). https://doi.org/10.1038/nature09136

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