SUMMARYVisualizing conformational dynamics in proteins has been difficult, and the atomic-scale motions responsible for the behavior of most allosteric proteins are unknown. Here, we report that FRET between a small fluorescent dye and a nickel ion bound to a di-histidine motif can be used to monitor small structural rearrangements in proteins. This method provides several key advantages over classical FRET including the ability to measure the dynamics of close range interactions, the use of small probes with short linkers, a low orientation dependence, and the ability to add and remove unique tunable acceptors. We used this ‘transition metal ion FRET’ approach along with x-ray crystallography to determine the structural changes of the gating-ring of the mouse hyperpolarization-activated cyclic nucleotide-regulated ion channel HCN2. Binding of cAMP to the isolated carboxyl-terminal region of HCN2 caused a structural rearrangement involving a movement of the C-helix towards the β-roll of the cAMP-binding domain and a movement of the F′ helix of the C-linker, along with a stabilization of the secondary structure of the helices. Our results suggest a general model for the conformational switch in the cyclic nucleotide-binding site of cyclic nucleotide-regulated ion channels.

In cyclic nucleotide-gated channels (CNG), direct binding of cyclic nucleotides in the carboxy-terminal region is allosterically coupled to opening of the pore. A CNG1 channel pore was probed using site-directed cysteine substitution to elucidate conformational changes associated with channel opening. The effects of cysteine modification on permeation suggest a structural homology between CNG and KcsA pores. We found that intersubunit disulfide bonds form spontaneously between S399C residues in the helix bundle when channels are in the closed but not in the open state. While MTSET modification of pore-lining residues was state dependent, Ag(+) modification of V391C, in the inner vestibule, occurred at the same diffusion-limited rate in both open and closed states. Our results suggest that the helix bundle undergoes a conformational change associated with gating but is not the activation gate for CNG channels.

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) ion channels regulate the spontaneous firing activity and electrical excitability of many cardiac and neuronal cells. The modulation of HCN channel opening by the direct binding of cAMP underlies many physiological processes such as the autonomic regulation of the heart rate. Here we use a combination of X-ray crystallography and electrophysiology to study the allosteric mechanism for cAMP modulation of HCN channels. SpIH is an invertebrate HCN channel that is activated fully by cAMP, but only partially by cGMP. We exploited the partial agonist action of cGMP on SpIH to reveal the molecular mechanism for cGMP specificity of many cyclic nucleotide-regulated enzymes. Our results also elaborate a mechanism for the allosteric conformational change in the cyclic nucleotide-binding domain and a mechanism for partial agonist action. These mechanisms will likely extend to other cyclic nucleotide-regulated channels and enzymes as well.