Pharmacological and Fluorometric Assessment of Neuronal KCNQ Channels, and Implications for Understanding Neurological Disease

Abstract: Epilepsy affects 60 million people worldwide, and encompasses the most common forms of neurological disorders. Epilepsy manifests in diverse ways in patients, ranging from mild cognitive effects, to seizures, to the more severe epileptic encephalopathy. While many pharmacological approaches exist for the treatment of epilepsy, 30% of the epileptic population do not respond to such treatments. The resistance to treatment is perhaps due to the diverse neurological mechanisms underlying epilepsy, but also the limited molecular mechanisms by which conventional therapies target. Recent studies have made significant progress in unveiling the mechanisms of a novel class of anti-epileptic drugs, the Kv7/KCNQ activators, which targets a class of voltage-gated potassium channels involved in M-channel formation, and are implicated in neonatal seizures and epileptic encephalopathy. Retigabine (RTG) is the first-in-class clinically approved Kv7/KCNQ channel opener for the management of pharmacoresistant epilepsies. Despite its success in treating many patients, RTG was eventually removed from the market due to lack of target specificity and abundant side effects, which created concern for patient safety. Recently, another experimental compound was developed - ICA-069673 (ICA-73). ICA-73 has been shown through recent studies to exhibit greater specificity, selectivity, and state-dependence on limited members of the Kv7/KCNQ subclass. Currently, there remains a limited understanding of the function and regulation of KCNQ channels, particularly with regards to mechanisms behind subtype-selective tetrameric assembly and stability of the selectivity filter. In addition, there is little understanding of the relationship between KCNQ mutations and neurological disease. My thesis uses the improved understanding of ICA-73 to develop a pharmacological assay, which was used together with two electrode voltage clamp electrophysiology to study the relationship between genetic mutations in KCNQ channels and clinical cases of neurological disorders. Through our approach, we improved the current understanding of M-channels and determined that the calmodulin binding helices are likely involved in tetrameric channel assembly. We also determined that the selectivity filter of KCNQ channels are likely stabilized by a similar hydrogen bond previously identified in Drosophila Shaker channels, and disruption of this interaction can cause neonatal epilepsy. Finally, we made significant improvements to an optical technique, voltage clamp fluorometry, by dramatically increasing the amplitudes and consistency of signals obtainable from KCNQ3, using an approach that can be expanded to other members of the KCNQ family, and can be potentially applied to study other poorly understood KCNQ-targeted drugs.