Project Summary/Abstract The IKS channel is a voltage-gated potassium channel found in the heart. After a cardiac muscle cell is depolarized, the IKS channel opens and allows a potassium current to leave the cell, returning it to resting state. Dysfunction in this channel is associated with numerous acquired and inherited arrhythmias, including long QT syndome, a leading cause of sudden cardiac death. The channel itself is made of two protein components: KCNQ1 (Q1), the pore-forming subunit; and KCNE1 (E1), a single transmembrane accessory protein. When Q1 is expressed alone, the channel opens at a negative voltage and conducts a small potassium current, but with E1 bound, the channel opens at a high positive voltage and conducts a large potassium current. Thus E1 acts as an intrinsic gating regulator of the channel. The IKS channel is also regulated transiently by phosphorylation in response to signaling by the sympathetic nervous system. When phosphorylated, the channel opens more quickly and more often, which leads to an increaed potassium current. This leads to faster cell repolarization and facilitates increased heart rate. However, the molecular mechanisms of both intrinsic regulation by E1 and transient regulation by phosphorylation are not currently understood. The goal of this research is to gain a high- resolution understanding of these mechanisms of regulation. Intrinsically, phenylalanine (Phe) has been shown to play a role in many ion channel mechanisms because despite being overall nonpolar and largely hydrophobic, it has a quadropole moment that creates a negative charge in the center of its aromatic ring. This means that it can behave like an anion and form strong charge-charge interactions with other charged residues, despite being in the hydrophobic core of the channel. Preliminary data has been found that shows a particular Phe residue in the IKS channel participates in a previously uncharacterized charge interaction that slows IKS channel response to voltage, and is thus a key player in intrinsic regulation. This work will characterize this interaction, determine how E1 binding alters it, and determine the specifc role this interaction plays in regulating IKS channel function. As well as intrinsic regulation by E1, Q1 is transiently regulated by phosphorylation at two sites on the N-terminus. Cumulatively, the channel response to phosphorylation is an increase in current, but the mechanism of this increase is unknown. Additionally, the individual roles of the two phosphorylation sites are not understood. This research will use caged serine, a modified serine residue that can only be phosphorylated after photolysis of a bound caging moiety. Encoding caged serine into each of the sites individually and observing the effects of phosphorylation at each site on the channel will provide this key information about phosphoregulation of the IKS channel. This work, which will provide mechanistic explanations of intrinsic and transient r...