Tuning ion channel:drug interactions with unnatural amino acid mutagenesis

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Overview: Ion channel proteins are essential regulators of electrical behavior in all cells, and important targets for the development of therapeutics for diseases of membrane excitability and ion transport. Ion channels form ion-selective pores in cell membranes, enabling regulated diffusion of ions between the intracellular and extracellular compartments. Defects in function of specific ion channel types cause a wide range of disorders, ranging from cardiac arrhythmias, to hormone secretory disorders (diabetes, hyperinsulinism), deafness, ataxias, and others. Background: Most drugs that target ion channel proteins act as inhibitors – they reduce the overall activity of the ion channels. However, there is untapped potential in the development of compounds that increase channel activity, or channel openers. In our proposed research we will investigate the detailed mechanism of action of a recently introduced potassium channel opener (retigabine) that is emerging as an effective treatment for epilepsy in many patients. We will also investigate a second experimental system recently described by research in our lab, in which subtle alterations of drug:ion channel interactions can cause classical ion channel blockers to act as channel openers. We anticipate that by studying these two distinct mechanisms of ion channel potentiation, we will gain important insights into the detailed drug:ion channel interactions that enable some compounds to act as channel openers. These advances will inform the improvement of existing drugs like retigabine, and the development of new ion channel openers, by identifying specific chemical interactions that are essential for their desirable effects. In addition to epilepsy, potassium channel openers are expected to be effective treatments for certain cardiac arrhythmias, hypertension, and other disorders. To approach these problems, we have implemented a very unique experimental method that allows us to make very subtle changes to ion channel structure. This method relies on engineering custom made amino acids that are not normally encoded by our genetic material, and devising a way to incorporate them into ion channels in living cells. This is referred to as unnatural amino acid mutagenesis, and we are one of only a few laboratories that has been able to successfully apply this method. The major advantage is that it allows detailed ‘atom-by-atom’ alteration of ion channel structure. This dramatically expands the palette of tools available to investigate ion channel function, and enables highly specific dissection of chemical forces that control ion channel interactions with therapeutic modulators like retigabine. In addition to our specific research objectives we are hopeful that ongoing development of these methods in our lab will significantly contribute to research by other groups interested in structure function relationships of ion channels or other protein classes. Impact: In summary, our Discovery Grant program will apply unique technology for unnatural amino acid mutagenesis to identify detailed chemical interactions between ion channels and modulators. The long term impact of this research program will be the identification of detailed mechanisms that enable certain compounds to act as ion channel openers. We anticipate that these insights will accelerate the development and improvement of ion channel openers like retigabine, and other compounds that may be effective therapeutics for certain cardiovascular and neurological disorders.