Project Summary This project is to reveal the mechanism of how voltage, phosphatidylinositol 4,5-bisphosphate (PIP2) and calmodulin (CaM) integrate to activate the IKs potassium ion channels in the heart. The IKs channel is important for repolarization of cardiac action potentials and the control of heart rhythm. CaM and the membrane lipid PIP2 are important cell signals, and meanwhile these molecules are cofactors of the IKs channel required for channel opening. These enable the IKs channel to play a critical role in the adaptation of heart rhythm to various physiological conditions. Congenital and drug induced IKs malfunction is associated with cardiac arrhythmias. Previous studies and our preliminary data indicate that the IKs channel is a novel target for antiarrhythmic therapy. The significance of this study is to improve the understanding of molecular basis of cardiac electrophysiology and antiarrhythmic drug development. At present how CaM and PIP2 interact with the IKs channel is not clear. This proposal is motivated by the recently published structural data of the channel protein. Combining these data with other published and our preliminary studies lead to an exciting hypothesis for how CaM, PIP2 and voltage integrate to activate the IKs channels. Our specific aims are designed to examine three key aspects of this hypothesis. We will use electrophysiological approaches, fluorescence spectroscopy, structure-informed mutagenesis and molecular dynamic simulations to study the IKs channels expressed in exogenous expression cells including Xenopus oocytes and mammalian cell lines. These studies will reveal the binding of PIP2 and CaM to the channel protein and subsequent conformational changes that open the channel. These results will provide a molecular basis to understand how heart rhythm is regulated by cell signals and multiple mechanisms for drugs to target and modify, which may lead to the development of more effective and safe antiarrhythmic drugs.