Cyclic nucleotide-regulated ion channels are exquisite molecular machines that underlie important physiological functions. Cyclic nucleotide-gated (CNG) channels generate the primary electrical response to light in photoreceptors and to odorant in olfactory receptors. The related hyperpolarization-activated cyclic nucleotide-gated (HCN) channels underlie the pacemaker activity of the heart and many neurons in the brain. These cation selective channels are opened by the direct binding of cyclic nucleotides (cAMP and cGMP) to an intracellular domain of the channel. Our goal is to reveal the molecular mechanism for this allostery in CNG channels. Our approach will be to study bacterial CNG channels as a model system for the eukaryotic channels because of the huge advantages they provide for our biochemical methods . We will leverage the power of four different methodologies to determine the structure, conformational heterogeneity, and dynamics of these channels: 1) cryoelectron microscopy (cryo-EM), 2) double electron-electron resonance (DEER), 3) microfluidic rapid freeze quench (µRFQ) in combination with DEER, and 4) Rosetta-based molecular modeling. The proposal includes four investigators who are pioneers in each of these methods. The use of all four methods on the same ion channel under the same conditions is synergistic and ultimately will lead to a comprehensive structural and energetic model for the allostery of this channel. Ultimately a molecular understanding of these channels would inform not only the physiology and pathophysiology of the heart and brain, but also the general mechanisms for allosteric control of many enzymes.