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. Continuouswave 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.hyperpolarization-activated ion channels | allosteric regulation

The contribution of modern EPR to structural biology

“…In modelling such conformational transitions, the rigid-domain assumption can be somewhat relaxed and subjective identification of the domains can be avoided by using an elastic network model [40]. An implementation into MMM that accounts for the spin labels [41] has revealed conformational changes in the nucleotide-gated HCN ion channel upon binding of 3 0 ,5 0 -cyclic adenosine monophosphate [42]. The elastic network model approach performs well only for simple hinge motion or combination of a hinge motion with rotation of one domain [41].…”

Dynamic measurements for funny channels

“…Transition metal ion FRET was previously used to show that the C-helix undergoes a large translation toward the β-roll subsequent to ligand binding and also suggested that binding of cAMP stabilized the secondary structure of the C-helix (4,14). Double electron-electron resonance (DEER) and continuous-wave EPR also confirm the cAMP-induced motions of the C-helix and suggest that the distal C-helix is more conformationally heterogeneous when not bound to agonist (15). Finally, a model of the HCN4 C-terminal region in the absence of agonist was recently obtained using homology restraints along with chemical shifts and residual dipolar couplings obtained from NMR (16).…”

“…FRET can even be measured in intact, functioning channels to directly correlate structural and functional channel states. When coupled with the extensive structural information already accumulated for HCN, even a relative few constraints can accurately model gating conformational changes and follow the chain of dominoes all of the way from ligand binding to channel opening (15).…”

The Synergetic Effects of Combining Structural Biology and EPR Spectroscopy on Membrane Proteins

“…Here, the utilization of distance distribution constraints obtained by pulsed EPR measurements and an initial structure render new models possible. For instance cAMP-bound and unbound models of hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels have been successfully generated based on interspin distance constraints and the cAMP-bound HCN crystal structure as the starting structure [45].…”

“…Besides nowadays various structures a plethora of EPR studies has been performed with regard to conformational changes in the selectivity filter [65,66], the cytoplasmic helical bundle [67,68] and the communication between both features [69]. Further excellent examples for the combined use of structural biology and EPR spectroscopy are present for all classes of ion channels, e.g., for the intracellular Mg 2+ -gated Mg 2+ channel CorA [70][71][72], the nucleotide-and Na + -gated K + channel KtrAB [73], the mechanosensitive channel MscL [74,75], the cyclic AMP-gated HCN channel [45,76], the voltage-gated Na + channel NavMs [77] and the pentameric receptor channels GLIC [78]. In all cases EPR measurements have been applied to validate existing protein structures, determine the structure of missing regions or distorted elements in structures, or add valuable information of the dynamics of the studied systems to provide a model of activation and regulation.…”