title: Double electron-electron resonance reveals cAMP-induced conformational change in HCN channels.
abstract: Binding of 3',5'-cyclic adenosine monophosphate (cAMP) to hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels regulates their gating. cAMP binds to a conserved intracellular cyclic nucleotide-binding domain (CNBD) in the channel, increasing the rate and extent of activation of the channel and shifting activation to less hyperpolarized voltages. The structural mechanism underlying this regulation, however, is unknown. We used double electron-electron resonance (DEER) spectroscopy to directly map the conformational ensembles of the CNBD in the absence and presence of cAMP. Site-directed, double-cysteine mutants in a soluble CNBD fragment were spin-labeled, and interspin label distance distributions were determined using DEER. We found motions of up to 10 Å induced by the binding of cAMP. In addition, the distributions were narrower in the presence of cAMP. Continuous-wave electron paramagnetic resonance studies revealed changes in mobility associated with cAMP binding, indicating less conformational heterogeneity in the cAMP-bound state. From the measured DEER distributions, we constructed a coarse-grained elastic-network structural model of the cAMP-induced conformational transition. We find that binding of cAMP triggers a reorientation of several helices within the CNBD, including the C-helix closest to the cAMP-binding site. These results provide a basis for understanding how the binding of cAMP is coupled to channel opening in HCN and related channels.
authors: Departments of Physiology and Biophysics and stst@uw.edu.
authors: Michael C
authors: MC
authors: Puljung
authors: Departments of Physiology and Biophysics andChemistry, University of Washington, Seattle, WA 98195 stst@uw.edu.
authors: Hannah A
authors: HA
authors: DeBerg
authors: Departments of Physiology and Biophysics and.
authors: William N
authors: WN
authors: Zagotta
authors: Chemistry, University of Washington, Seattle, WA 98195 stst@uw.edu.
authors: Stefan
authors: S
authors: Stoll
mesh_terms: D000818:Animals; D000242:Cyclic AMP / Q000737:chemistry*; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels / Q000737:chemistry*; D051379:Mice; D008958:Models, Molecular; D015221:Potassium Channels / Q000737:chemistry*; D011487:Protein Conformation; D013057:Spectrum Analysis / Q000379:methods*; D013113:Spin Labels
chemical_list: C578711:Hcn2 protein, mouse; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D015221:Potassium Channels; D013113:Spin Labels; D000242:Cyclic AMP
keywords: allosteric regulation; hyperpolarization-activated ion channels