investigator's application): Organizing multimeric ion channel complexes through specific protein-protein interaction is a fundamental event in channel expression. Such protein-protein interaction is essential in determining the physiological function of the assembled channel complex, including kinetic properties, expression, and subcellular distribution. The proposed experiments in this application address questions critical for understanding subunit assembly: What molecular determinants mediate the specific subunit interactions? How the various forms of interactions may be coupled to channel assembly, expression, and properties? A large number of genes encoding potassium (K+) channels have been isolated. Vast diversity is created by homo- and hetero- multimeric assembly of the pore-forming alpha-subunits and modulatory beta-subunits. The proposed experiments in this application are focused on homo- and hetero- multimeric interactions among the Shaker -like alpha-subunits and two hydrophilic beta-subunits, Kvbeta1 and Kvbeta2. As indicated in our preliminary studies, the interactions to be studied are essential for alpha-alpha, alpha-beta, and beta-beta assembly, which are directly coupled to the K+ channel functions including kinetic properties and expression. Experiments in three areas will be performed: (1) to identify the regions and/or residues in the alpha-subunit that mediate the specific alpha-alpha, and alpha-beta interactions; (2) to identify regions in the beta-subunits that mediate the specific interaction of beta-beta and alpha-beta interactions; (3) to investigate role(s) of alpha-beta interaction in K+ channel expression. These experiments will employ a combination of molecular, biochemical, and electrophysiological methods that have been developed in the laboratory. The protein-protein interaction will be tested by biochemical binding, yeast two hybrid system, and coimmunoprecipitation. The expression of the channel protein will be assessed by immunoblot analysis and whole cell voltage clamp recording. K+ channels play critical roles in a variety of biological processes. Defects in their subunit interaction have been implicated in human diseases, such as cardiac tachyarrhythmia. The molecular understanding of the events central to their expression is essential for developing our knowledge in the area of nerve function both in health and in disease.
