Kondapuram, M. ; Frieg, B.FZJ* ; Yüksel, S. ; Schwabe, T. ; Sattler, C. ; Lelle, M. ; Schweinitz, A. ; Schmauder, R. ; Benndorf, K. ; Gohlke, H.FZJ* ; Kusch, J.

2020
Cold Spring Harbor Laboratory, NY Cold Spring Harbor

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.