Using Voltage Clamp Fluorometry To Understand Kcnq Channel Pharmacology

With the emergence of retigabine, the first-in-class potassium channel activator used for treatment of epilepsy, KCNQ channels have entered the spotlight for the development of targeted therapeutics to treat excitability disorders. The detailed mechanisms of novel KCNQ activators are largely unexplored, and many fundamental questions regarding their effects on channel function remain unanswered. For instance - how/why do some drugs specifically modulate channel open probability, while others impact voltage sensitivity, or both? To investigate these questions we have employed voltage clamp fluorometry to measure retigabine effects on voltage sensor movements in KCNQ3 channels. Fluorescence reports from channels labelled at position Q218C (in the S3-S4 linker) indicate tight coupling between the voltage sensor domain (VSD) and pore domains (PD) in KCNQ3 channels, with both processes displaying similar voltage dependence and kinetics. In response to the prototype KCNQ activator retigabine, VSD conformational changes and pore gating are equally shifted to hyperpolarized potentials, reflecting the ability of retigabine to stabilize the channel active state. To separate possible effects of retigabine on the VSD versus PD, we have co-expressed voltage sensitive phosphatases (CiVSP and DrVSP) to deplete membrane PIP2. VSP-mediated PIP2 hydrolysis causes pore closure and rundown of KCNQ3 current, but does not alter the voltage-sensor fluorescence report, suggesting that pore gating has been dissociated from VSD motions. This experimental system will allow investigation of whether retigabine effects require intact coupling between the VSD and PD. Our findings reveal unique aspects of VSD-PD coupling in KCNQ3 channels, and highlight the role of PIP2 as an important co-factor for channel activity and drug sensitivity of KCNQ channels.