Double Electron-Electron Resonance Studies of Ligand Induced Rearrangements of HCN Channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) ion channels are members of the superfamily of voltage-gated potassium channels. HCN channels play a role in controlling neuronal and cardiac pacemaking activity. They are regulated by the binding of cyclic nucleotides to a conserved, intracellular cyclic nucleotide-binding domain (CNBD). The CNBD is connected to the transmembrane domain through a C-linker. Crystallization studies of intracellular fragments of the C-linker and CNBD show little conformational difference in cAMP bound and unbound states. However, fluorescence and electrophysiology experiments suggest a significant conformational change in the C-linker and CNBD. Here, we use double electron-electron resonance (DEER) to study conformational changes of a soluble fragment of HCN channels in response to binding different cyclic nucleotide species. DEER is an electron paramagnetic resonance technique capable of measuring absolute distances and distance distributions between two spin-labeled protein residues separated by 2 to 8 nanometers. The ability to measure distance distributions allows us to probe whether binding of a partial agonist induces a conformational change smaller compared to the full agonist or results only in a shift of the conformational equilibrium. We find that in a soluble fragment of HCN2 consisting of the C-linker and CNBD, cAMP, the physiological agonist, leads to conformational changes of the CNBD that are much greater than suggested by crystallization. Binding of cCMP, known to be a partial agonist of the full channel, leads to conformational changes similar to cAMP. We also find that cGMP, known to be a full agonist on intact channels, acts as a partial agonist on the fragment, causing only a fraction of protein to undergo conformational change.