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HCN domain is required for HCN channel expression and couples voltage- and cAMP-dependent gating mechanisms

Wang, Ze-Jun and Blanco, Ismary and Hayoz, Sebastien and Brelidze, Tinatin I. (2020) HCN domain is required for HCN channel expression and couples voltage- and cAMP-dependent gating mechanisms. . (Unpublished)

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Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are major regulators of synaptic plasticity, and rhythmic activity in the heart and brain. Opening of HCN channels requires membrane hyperpolarization and is further facilitated by intracellular cyclic nucleotides (cNMPs). In HCN channels, membrane hyperpolarization is sensed by the membrane-spanning voltage sensor domain (VSD) and the cNMP-dependent gating is mediated by the intracellular cyclic nucleotide-binding domain (CNBD) connected to the pore-forming S6 transmembrane domain via the C-linker. Previous functional analysis of HCN channels suggested a direct or allosteric coupling between the voltage- and cNMP-dependent activation mechanisms. However, the specifics of the coupling were unclear. The first cryo-EM structure of an HCN1 channel revealed that a novel structural element, dubbed HCN domain (HCND), forms a direct structural link between the VSD and C-linker/CNBD. In this study, we investigated the functional significance of the HCND. Deletion of the HCND prevented surface expression of HCN2 channels. Based on the HCN1 structure analysis, we identified R237 and G239 residues on the S2 of the VSD that form direct interactions with I135 on the HCND. Disrupting these interactions abolished HCN2 currents. We then identified three residues on the C-linker/CNBD (E478, Q382 and H559) that form direct interactions with residues R154 and S158 on the HCND. Disrupting these interactions affected both voltage- and cAMP-dependent gating of HCN2 channels. These findings indicate that the HCND is necessary for the surface expression of HCN channels, and provides a functional link between the voltage- and cAMP-dependent mechanisms of HCN channel gating.

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Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license. Posted March 02, 2020. We are grateful to William N Zagotta for helpful discussions. We would like to thank Gerard Ahern for generously allowing us to use his lab equipment to carry out the initial experiments for the project, and to Robert Yasuda for providing the HEK293 cell cultures for our experiments. This work was supported by the National Institute of General Medicine grant R01GM124020 (T.I.B.). The authors declare that they have no conflicts of interest with the contents of this article. Authorship contributions: T.I.B. conceived the study. Z.J.W, I.B. and S.H. performed the experiments. Z.J.W, I.B. S.H. and T.I.B. performed data analysis. T.I.B. wrote the manuscript with the input from all coauthors.
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Subject Keywords:HCN1, HCN2, cyclic nucleotide binding domain, cyclic nucleotide, gating, cyclic AMP (cAMP), ion channel, ligand-binding protein
Record Number:CaltechAUTHORS:20200303-155957589
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Official Citation:HCN domain is required for HCN channel expression and couples voltage- and cAMP-dependent gating mechanisms. Ze-Jun Wang, Ismary Blanco, Sebastien Hayoz, Tinatin I Brelidze. bioRxiv 2020.03.02.973560; doi:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:101687
Deposited By: Tony Diaz
Deposited On:04 Mar 2020 03:55
Last Modified:16 Nov 2021 18:05

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