PT - JOURNAL ARTICLE AU - Mahesh Kondapuram AU - Benedikt Frieg AU - Sezin YĆ¼ksel AU - Tina Schwabe AU - Christian Sattler AU - Marco Lelle AU - Andrea Schweinitz AU - Ralf Schmauder AU - Klaus Benndorf AU - Holger Gohlke AU - Jana Kusch TI - Functional and structural characterization of interactions between opposite subunits in HCN pacemaker channels AID - 10.1101/2020.09.21.305797 DP - 2020 Jan 01 TA - bioRxiv PG - 2020.09.21.305797 4099 - http://biorxiv.org/content/early/2020/09/21/2020.09.21.305797.short 4100 - http://biorxiv.org/content/early/2020/09/21/2020.09.21.305797.full AB - 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.Competing Interest StatementThe authors have declared no competing interest.