Structure-function analysis of KCNQ1 and KCNE1 C-terminal interactions
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The cardiac potassium channel KCNQ1-KCNE1 carries the IKs current that plays a critical role in the regulation of the heart beat. Congenital mutations associated with long QT syndrome (LQTS) can reduce potassium current and delay action potential repolarization, which leaves patients at increased risk for potentially deadly cardiac arrhythmias. The channel has pore-forming alpha-subunits encoded by the gene KCNQ1, and regulatory beta-subunits encoded by the gene KCNE1. Previous work had identified interactions between critical residues in the transmembrane domains of the subunits that control the kinetics of channel activation. The experiments presented in this thesis identify the C-terminal regions of subunit interaction that control channel deactivation kinetics, and additionally impact the channel's relative stability in the closed versus open states.;A KCNE1 C-terminal truncation mutant revealed that the C-terminus plays a role in controlling channel deactivation kinetics, in determining the relative stability of the channel's closed versus open states, and in facilitating proper association of KCNE1 with KCNQ1. The KCNE1 C-terminus can physically bind to KCNQ1 and impart functional changes, even in the absence of the KCNE1 transmembrane domain. To determine the specific residues of KCNE1 and KCNQ1 that interact to impart such control, hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) was first used to narrow down the region of interaction. The results indicated that the most proximal segments of both the KCNE1 C-terminus (amino acids 70-81) and the KCNQ1 C-terminus (amino acids 352-374) physically interact. Residue-specific interactions were then identified by double mutant cycle analysis. The data revealed that this region is extremely sensitive to perturbation, with most tested mutations resulting in significant shifts in voltage-dependence of activation and deactivation kinetics. The pair of residues that showed strongest coupling during the channel closed-to-open transition (KCNQ1-P369 and KCNE1-S74) was different from the pair that showed strongest coupling during the open-to-closed transition (KCNQ1-1368 and KCNE1-H73), indicating that the region may be mobile, and likely moves during channel opening and closing. LQTS-associated mutations in this region may contribute to the disease phenotype of delayed repolarization by disrupting these important interactions that contribute to deactivation rate and channel stability.