Hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) ion channels play a fundamental role in electric signaling in nerves, muscles, and synapses; however, their ligand gating mechanism is not well understood. There is little structural information on HCN2, as only its cytoplasmic domain in the holo form has been solved by X-ray crystallography. At the N-terminus, this structure contains the C-linker (helices A’-F’) and the cyclic nucleotide binding domain (CNBD), comprising of eight β-strands and helices A-C. cAMP modulates the HCN2 channel opening and it also promotes oligomerization. Through collaboration between experimental and computational methods, this study brings insight into the mechanism of cyclic adenosine monophosphate (cAMP) modulation of the HCN2 channel by exploring the monomer and tetramer dynamics in the apo and holo states. We performed all-atom, as well as coarse-grained (CG) molecular dynamics simulations. In the CNBD the distance between the C-helix and the β-roll in the binding pocket increases in the absence of cAMP. Our results corroborate recent transition metal ion Förster resonance energy transfer and double electron-electron resonance studies showing the outward movement of the C-helix in the absence of cAMP. The most striking dynamics observed in simulations of the apo monomer is the unfolding of the C’-helix together with loss of contact between A’ and B’ helices in the C-linker, and the CNBD. In contrast, in the holo monomer, these contacts are retained, although the C’-helix is unstable. In both the apo and the holo tetramer, the C’-helix remains folded. We inferred that this is due to a reduced solvent accessible surface area, as the C’-helix of a subunit is sheltered by the adjacent subunit. The folding/unfolding interplay of the C’-helix translates into the vertical movement of the CNBD. As recently observed in MloK1, an analogous prokaryotic ion channel, this vertical displacement induces CNBD disinhibition, which promotes channel opening.

Cyclic, nucleotide modulation of structure and dynamics of the cytoplasmic domanin of the HCN2 ion channel

251st National Meeting (2016)

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