Identification of potential drug binding sites within allosteric networks in cyclic nucleotide modulated channels

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PROJECT SUMMARY Cyclic nucleotide modulated ion channels are a class of proteins that have important roles in many physiological processes, including regulation of the heart, neuronal signaling, and pain perception. The discovery of new drugs targeting different ion channels is notoriously difficult for a host of reasons, as the focus on traditional orthosteric agonists and antagonists has been dominant. Overall, this proposal unifies genomic, functional, and structural methods to reveal how specific allosteric interactions govern mechanistic function. The ability to detect and isolate the function of networks of allosteric interactions can provide a more focused approach in the design of allosteric drugs for cyclic nucleotide modulated channels. In the first aim, I will identify and classify allosteric networks using coevolution analysis. Mutagenesis and quantitative electrophysiology measurements will be used to probe how different residue positions contribute to requisite energetic coupling in channel gating. Next, we will use this unique information to obtain novel channel transition states. The overall goal of this aim is to define allosteric networks that functionally regulate cyclic nucleotide modulated ion channels, experimentally validate these networks functionally, and use this information to obtain difficult-to-resolve conformational states. In the second aim, I will seek to uncover unknown binding sites for known allosteric modulators of cyclic nucleotide modulated ion channels. Using cryoEM, we will determine the structure of channels in complex with established allosteric modulators and define their interaction with the allosteric networks in aim 1. We will validate the binding site using mutagenesis and two electrode voltage clamp. This aim will demonstrate feasibility of drug design strategies targeting allosteric networks.