The long-term objectives of this research project are to provide information on the molecular basis for the heterogeneity of voltage-gated potassium (Kv) channels in normal and in diseased hearts, and the mechanisms/sites of actions of currently available Kv channel modulators that may provide therapeutic benefits for problems in the heart and elsewhere. Our focus here is the slow delayed rectifier (IKs) channel, an important determinant of action potential duration in human heart. The IKs channel consists of at least two components: KCNQ1 channel and KCNE1 auxiliary subunit. Mutations in kcnq1 &kcne1 genes have been linked to abnormalities in cardiac repolarization and increased risk for arrhythmias (long &short QT syndromes, LQT &SQT, and familial atrial fibrillation, fAF). Recent data from our lab and from others have suggested that the subunit composition of cardiac IKs channels may be more complex than previously believed. Transcripts of other members of the KCNE family (KCNE2 - KCNE5) have been detected in human heart, and in heterologous expression systems these KCNE subunits can all associate with the KCNQ1 channel to confer distinct channel phenotypes. We are particularly interested in KCNE2, because we have confirmed the presence of KCNE2 protein in human heart, and mutations in the kcne2 gene have been linked to LQT6 or fAF. We have shown that in heterologous expression systems KCNE2 can associate with the IKs (KCNQ1/KCNE1) channel complex to reduce its current amplitude without changing its gating kinetics. Furthermore, our preliminary pulse-chase experiments suggest that the partnership between KCNQ1 and KCNE subunits is not permanent: KCNE subunits can dissociate from KCNQ1/KCNE complexes to be replaced by new ones. These observations suggest the intriguing possibility that the subunit composition of cardiac IKs channels is dynamic: KCNE1 functions as the major auxiliary subunit to set the IKs gating kinetics, while KCNE2 functions as a dynamic regulator to fine tune the IKs current amplitude. In this proposal, we will seek direct evidence for the role of native KCNE2 in cardiac IKs channel function. We also want to quantify the relationship between KCNE1 &KCNE2 expression levels, their KCNQ1 binding affinity, and the IKs subunit composition (Aim 1), to apply the above information to the study of mechanisms for IKs remodeling in aging hearts (Aim 2), and to determine the structural basis for the dynamic interactions between KCNQ1 and the two KCNE subunits (Aim 3). To achieve these Aims, we will use a multidisciplinary approach of electrophysiology, molecular biology, protein biochemistry, confocal microscopy and molecular modeling. Importantly, we will study not only channels expressed in heterologous systems but also native channels in cardiac myocytes.
Our data will provide novel insights into the dynamic nature of cardiac IKs channel subunit composition. We believe this is one of the mechanisms by which cardiac myocytes fine tune the IKs amplitude in response to stress. We will apply this knowledge to the study of IKs remodeling during physiological and pathological aging. Finally, we will obtain structural information on IKs channel subunit interactions, and use this information to refine 3-D models of the IKs channel in different gating states. These models will be useful in structure-based design of IKs activators that can combat acquired &congenital LQT syndromes.
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