The overall goal of the research proposed in this application is to unravel the molecular determinants of beta adrenergic receptor (beta-AR) regulation of IKs channels, one of two families of cardiac delayed rectifier K+ channels instrumental in the control of action potential duration of the in the heart, particularly during beta-AR stimulation. Dysfunction in beta-AR regulation of IKs channels very likely triggers fatal arrhythmias in at least one human disease, LQTS-1, a form of the Long QT syndrome (LQTS). The experiments outlined in this proposal are designed to identify specific residues on the IKs channel protein that are substrates for protein kinase A induced phosphorylation and to determine whether or not putative phosphorylation reactions are under the control of local signaling complexes coordinated, in part, by motifs of the channel protein itself. The experimental plan combines unique in vitro and in vivo studies of the human isoforms of the two subunits that encode IKs channels: hminK and hKvLQT-1. Biochemical analysis of channel proteins or channel protein fragments and site directed mutagenesis of channel constructs will complement patch clamp analysis of transfected Chinese Hamster Ovary (CHO) cells and/or cells isolated from the hearts of mice that have been genetically engineered to overexpress IKs channels. There are three specific aims of this proposal.
Aim 1 is to determine whether specific sites on the KvLQT1 subunit are targets of phosphorylation by PKA.
Aim 2 is to determine the functional consequences of phosphorylation of identified sites on KvLQT1 subunits.
Aim 3 is to test the hypothesis that local signaling exists for IKs and that signaling complexes are coordinated by distinct motifs on the KvLQT-1 subunit. Identification of localized molecular determinants of IKS regulation by beta-AR signaling has the potential of uncovering novel targets for pharmacological intervention in the treatment and prevention of fatal cardiac rhythms triggered by dysfunction in this critical regulatory pathway.
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