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Gating of the TrkH ion channel by its associated RCK protein TrkA

Abstract

TrkH belongs to a superfamily of K+ transport proteins required for growth of bacteria in low external K+ concentrations. The crystal structure of TrkH from Vibrio parahaemolyticus showed that TrkH resembles a K+ channel and may have a gating mechanism substantially different from K+ channels. TrkH assembles with TrkA, a cytosolic protein comprising two RCK (regulate the conductance of K+) domains, which are found in certain K+ channels and control their gating. However, fundamental questions on whether TrkH is an ion channel and how it is regulated by TrkA remain unresolved. Here we show single-channel activity of TrkH that is upregulated by ATP via TrkA. We report two structures of the tetrameric TrkA ring, one in complex with TrkH and one in isolation, in which the ring assumes two markedly different conformations. These results suggest a mechanism for how ATP increases TrkH activity by inducing conformational changes in TrkA.

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Figure 1: Single-channel activities of the TrkH–TrkA complex.
Figure 2: Structure of the TrkH–TrkA complex.
Figure 3: Comparison of TrkA in the TrkA-only and TrkH–TrkA complex structures.
Figure 4: Proposed mechanism of regulation of TrkH gating by TrkA.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors have been deposited with the Protein Data Bank under accession numbers 4J9U and 4J9V.

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Acknowledgements

Diffraction data for this study were measured at beamlines X4A, X4C, X25 and X29 of the National Synchrotron Light Source, ID-17 and ID-24 at the Advanced Photon Source, 5.0.2 and 8.2.2 at the Advanced Light Source, and A1 and F1 at the Cornell High Energy Synchrotron Source. M.Z. thanks E. Bakker, I. Booth, R. Krämer and C. Slayman for discussions and insights, and K. R. Rajashankar for help with X-ray crystallography. This work was supported by the US National Institutes of Health (DK088057, GM098878 and HL086392 to M.Z., and a subcontract (sub 5808 to M.Z.) from the PSI: Biology grant U54GM095315 to W. Hendrickson), the American Heart Association (12EIA8850017 to M.Z. and 0826067D to Y.P.), and the Irma T. Hirschl Trust Award to M.Z. M.Z. was a Pew Scholar in Biomedical Sciences.

Author information

Authors and Affiliations

Authors

Contributions

Y.C., Y.P., H.H., E.J.L., X.J. and M.Z. designed the experiments. Y.C. and H.H. expressed, purified and crystallized the proteins, and collected and processed the diffraction data. H.H., X.J. and E.J.L. solved and refined the structures. Y.P. prepared spheroplasts and performed patch-clamp recordings. B.K. provided reagents and the VpTrkH clone. All authors participated in data analysis. Y.P., E.J.L. and M.Z. wrote the manuscript with inputs from all authors.

Corresponding author

Correspondence to Ming Zhou.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1-2 and Supplementary Figures 1-13. (PDF 9853 kb)

Movement of the N2-N2 dimer interfaces

Animations showing linear interpolations between the conformations of two TrkA protomers forming the two N2-N2 interfaces in the TrkA-TrkH complex structure and the TrkA structure. The beginning and end states are aligned on both N2 subdomains. The coordinates of the intermediate states for all animations were calculated by linear interpolation with the program LSQMAN. The intermediate states in linear interpolations are not physically realistic and should not be used for drawing mechanistic conclusions. (MOV 3384 kb)

Movement of the N2-N2 dimer interfaces

Animations showing linear interpolations between the conformations of two TrkA protomers forming the two N2-N2 interfaces in the TrkA-TrkH complex structure and the TrkA structure. The beginning and end states are aligned on both N2 subdomains. The coordinates of the intermediate states for all animations were calculated by linear interpolation with the program LSQMAN. The intermediate states in linear interpolations are not physically realistic and should not be used for drawing mechanistic conclusions. (MOV 3513 kb)

Movement of the N1-N1 dimer interface

Animation showing a linear interpolation between the conformations of two TrkA protomers forming an N1-N1 interface in the TrkA-TrkH complex structure and the TrkA structure. The beginning and end states are aligned on both N1 subdomains. (MOV 2981 kb)

Conformational change of the TrkA protomer

Animation showing a linear interpolation between the conformations of one TrkA protomer in the TrkA-TrkH complex structure and the TrkA structure. The beginning and end states are aligned on their N1 subdomains. (MOV 3098 kb)

Conformational change of the TrkA tetramer

Animation showing a linear interpolation between the conformations of the TrkA tetramer in the TrkA-TrkH complex structure and the TrkA structure. The beginning and end states are aligned on their N1 and N2 subdomains. The yellow spheres represent residues that form salt bridges with TrkH in the structure of the complex. (MOV 7480 kb)

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Cao, Y., Pan, Y., Huang, H. et al. Gating of the TrkH ion channel by its associated RCK protein TrkA. Nature 496, 317–322 (2013). https://doi.org/10.1038/nature12056

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