Epilepsy affects nearly 3 million Americans, with 10 percent of all new patients failing to gain control of their seizures with correct medicinal management. Current anti-epileptic therapies restore normal neurotransmission by targeting ion channels in the brain. Many individuals develop tolerance to their prescriptions, experience side effects and require continuous modification of their treatment regimen over time. Today, there is an inherent need for anti-epileptic drug therapies that can reduce these side effects and maintain effectiveness over longer periods of time. G-protein inwardly rectifying potassium (GIRK) channels regulate neurotransmission in the brain. Due to their regional distribution throughout the brain, they have been implicated in many neurological disorders including addiction, anxiety and epilepsy and Parkinson's disease. Recent studies have demonstrated that GIRK channels can be directly activated by alcohol through an isolated binding pocket formed by the N- terminus, ? D- ? E and ? L- ? M. This binding site, which lies at the interface between adjacent subunits, could be a target for other ligands. ML297, an anti-epileptic compound, was recently identified and released as a tool to study GIRK channel function. Both the mechanism by which ML297 activates GIRK channels and the binding site on the channel are not known. Isolating this mechanism could be used to develop better anti-epileptic therapies in the future. We hypothesize that ML297 induces channel activation through direct binding to the alcohol binding site. Preliminary studies have shown that ML297 is inactive at GIRK channels lacking the GIRK1 subunit. In this proposal, I have designed a series of experiments to isolate the binding site for ML297 at different GIRK channels through patch clamp electrophysiology and site-directed mutagenesis under three aims.
For aims 1 and 2, I will use whole-cell patch clamp electrophysiology to examine macroscopic changes in gating induced by ML297.
In aim 1, I will establish a mechanism for ML297 binding to the GIRK1 subunit by testing the effect of mutations In aim 2, I will demonstrate that ML297 binds at the interface between subunits in heteromeric channels and focus on the role of the GIRK2 subunit in ML297 activation of GIRK1/2 channels. Finally, in aim 3, I will examine the efficacy of ML297 at wild-type and mutant channels addressed in the first two aims using single channel recordings. Together, these experiments will isolate key regions of the GIRK channel important for activation by ML297 and establish a discrete mechanism for activating GIRK channels in the absence of GPCRs. Understanding the mechanism of allosteric activation of GIRK channels by anti-epileptic therapies could provide a novel pathway for the development of future anti-epileptic drugs, as well as potential therapies for other disorders such as addiction and Parkinson's disease, where GIRK channels have also been implicated as important drug targets.
The long term goal of this project is to investigate a mechanism of direct activation of GIRK channels by combining assays in electrophysiology and molecular biology. GIRK channels play an inherent role in regulating neuronal transmission in the brain and also electrical activity in the heart. Establishing a method of directly activating functionally distinct GIRK channels will provide a novel pathway for drug treatment of many neurological disorders including epilepsy.