Gating Movements and Ion Permeation in HCN4 Pacemaker Channels

HCN channels underlie hyperpolarization-activated cation current If/Ih that controls automaticity in cardiac and neuronal pacemaker cells. We present cryo-EM structures of HCN4 in the presence/absence of bound cAMP, displaying the pore domain in closed and open conformations. Analysis of cAMP-bound and -unbound structures sheds light on how ligand-induced transitions in the channel cytosolic portion mediate the effect of cAMP on channel gating, and highlights the regulatory role of a Mg2+ coordination site formed between the C-linker and the S4-S5 linker. Comparison of open/closed pore states shows that the cytosolic gate opens through concerted movements of the S5 and S6 transmembrane helices. In combination with molecular dynamics, the open pore structures provide insights into the mechanisms of K+/Na+ permeation, revealing distinctive ion-binding dependent adaptation in the selectivity filter. Our results contribute new mechanistic understanding on HCN channel gating, cyclic nucleotide-dependent modulation, and ion permeation.