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 (MD) simulations to show that residue K464 of the C-linker is relevant for stabilizing the closed state of the mHCN2 channel by forming interactions with the opposite subunit. MD simulations revealed that in the K464E channel, a rotation of the intracellular domain relative to the channel pore is induced, which is similar to the cAMP-induced rotation, weakening the autoinhibitory effect of the unoccupied CL-CNBD region. We suggest that this CL-CNBD rotation is considerably involved in activation-induced affinity increase but only indirectly involved in gate modulation. The adopted poses shown herein are in excellent agreement with previous structural results.

Opening of hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels is controlled by membrane hyperpolarization and binding of cyclic nucleotides to the tetrameric cyclic nucleotide-binding domain (CNBD), attached to the C-linker disk (CL). Confocal patch-clamp fluorometry revealed a pronounced cooperativity of ligand binding among protomers. However, by which pathways allosteric signal transmission occurs remained elusive. Here, we investigate how changes in the structural dynamics of the CL-CNBD of mouse HCN2 upon cAMP binding relate to inter-and intrasubunit signal transmission. Applying a rigidity theory-based approach, we identify two intersubunit and one intrasubunit pathways that differ in allosteric coupling strength between cAMP binding sites or towards the CL. These predictions agree with results from electrophysiological and patch-clamp fluorometry experiments. Our results map out distinct routes within the CL-CNBD that modulate different cAMP binding responses in HCN2 channels. They signify that functionally relevant submodules may exist within and across structurally discernable subunits in HCN channels.

Cyclic nucleotide-gated (CNG) ion channels generate the primary electrical responses in the visual and olfactory signal transduction pathways of vertebrates. CNG channels are tetramers of the ''Kv'' superfamily, but are distinct in that they are essentially voltage-independent and are activated by direct binding of cyclic nucleotides (cAMP/cGMP) to a cytoplasmic cyclic nucleotidebinding domain (CNBD). Cyclic nucleotide-dependent conformational changes in the CNBD are thought to be allosterically coupled to the pore by the so-called C-linker domain, but the molecular mechanism by which this is achieved is not understood. Here we employ double electron-electron resonance (DEER) spectroscopy to measure select intersubunit distance distributions in a CNG channel from Spirochaeta thermophila, termed SthK. DEER distributions recorded both in detergent micelles and in lipid nanodiscs reveal a rearrangement of the C-linker in the presence of activating cAMP, resulting in an outward movement the C-linkers relative to the channel pore. The amplitude of this radial movement is particularly large at the C-terminal segment of the B'-helix. In the presence of saturating amounts of cGMP, very small relative populations of the dilated -or ''active'' -conformation of the C-linker can be observed in some distributions, consistent with its classification as a weak partial agonist. Our structural results are linked with functional states of the channel through electrophysiological recordings from giant E. coli spheroplasts expressing our DEER constructs. Finally, we employ constrained molecular modeling to predict the structure of the activated C-linker domain and discuss these results in terms of current hypotheses for cyclic nucleotide-dependent gating. Together our results reveal an agonist-dependent structural rearrangement of the C-linker domain of a CNG channel and may provide new insight into the complex gating mechanisms in this important class of channels.

overall goal of developing this high throughput assay is to identify ACHM2associated mutants that are misfolded. Candidates will be expressed with the CNGB3 subunit and screened for compounds that assist folding to recover channel function.

30Opening of hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels 31 is controlled by membrane hyperpolarization and binding of cyclic nucleotides to the 32 tetrameric cyclic nucleotide-binding domain (CNBD), attached to the C-linker disk (CL). 33Confocal patch-clamp fluorometry revealed a pronounced cooperativity of ligand binding 34 among protomers. However, by which pathways allosteric signal transmission occurs 35 remained elusive. Here, we investigate how changes in the structural dynamics of the CL-36CNBD of mouse HCN2 upon cAMP binding relate to inter-and intrasubunit signal 37transmission. Applying a rigidity theory-based approach, we identify two intersubunit 38and one intrasubunit pathways that differ in allosteric coupling strength between cAMP 39 binding sites or towards the CL. These predictions agree with results from 40 electrophysiological and patch-clamp fluorometry experiments. Our results map out 41 distinct routes within the CL-CNBD that modulate different cAMP binding responses in 42HCN2 channels. They signify that functionally relevant submodules may exist within and 43 across structurally discernable subunits in HCN channels.