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bioRxiv · 10.1101/2020.09.21.305797

Functional and structural characterization of interactions between opposite subunits in HCN pacemaker channels

Kondapuram, M.·Frieg, B.·Yueksel, S.·Schwabe, T.·Sattler, C.·Lelle, M.·Schweinitz, A.·Schmauder, R.·Benndorf, K.·Gohlke, H.·Kusch, J.
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Abstract

Hyperpolarization-activated and cyclic nucleotide (HCN) modulated channels are tetrameric cation channels. In each of the four subunits, the intracellular cyclic nucleotide-binding domain (CNBD) is coupled to the transmembrane domain via a helical structure, the C-linker. High-resolution channel structures suggest that the C-linker enables functionally relevant interactions with the opposite subunit, which might be critical for coupling the conformational changes in the CNBD to the channel pore. We combined mutagenesis, patch-clamp technique, confocal patch-clamp fluorometry, and molecular dynamics simulations to show that residue K464 of the C-linker is essential for stabilizing the closed state of the mHCN2 channel by forming interactions with the opposite subunit. MD simulations revealed that both cAMP and K464E induce a rotation of the intracellular domain relative to the channel pore, weakening the autoinhibitory effect of the unoccupied CL-CNBD region. The adopted poses are in excellent agreement with structural results.

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Kondapuram, M., Frieg, B., Yueksel, S., Schwabe, T., Sattler, C., Lelle, M., Schweinitz, A., Schmauder, R., Benndorf, K., Gohlke, H., Kusch, J.. 2020-09-21. Functional and structural characterization of interactions between opposite subunits in HCN pacemaker channels. https://doi.org/10.1101/2020.09.21.305797

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