The physiological properties of a neuron and other cells of the body are strongly affected by the variety and abundance of potassium channels expressed in the cell. A large number of K+ channel genes are being cloned that apparently code for single subunits of multisubunit K+ channel proteins, but how are these subunit proteins targeted to form the variety of K+ channel proteins found in a cell. A specific question these experiments will address is whether subunit primary structures guide K+ channel assembly in forming the electrical properties of neurons. The Preliminary Results describe experiments that identify a domain of a Shaker Subfamily K+ channel protein (T1 domain) that appears to be a self organizing tetramerization center; tetramerization is thought to be a critical step in the formation of K+ channels by K+ channel subunit proteins. This T1 domain does not appear to interact with a K+ channel clone from another gene subfamily.
The Specific Aims of this grant are: 1) Further Characterization of the Shaker Subfamily T1 N-Terminal Tetramerization Domain, 2) Identification of Other domains Involved in K+ Channel Subunit Multimerization, 3) Characterization of the Subfamily Dependent Differences in the T1 Domain and Testing Whether T1 Subfamily Specificity is Dominant, and 4) Examination of the Role of T1 Domain Compatibility in the Formation of Functional K+ Channel Proteins. The experiments to accomplish these Specific Aims rely exclusively on techniques or methods that are presented in the Preliminary Results or are being used currently in the lab. The general approach is to clone subfragments of different K+ channel subunit proteins that are thought to encode T1 domains, and to test for their function and subfamily specificity. A series of mutagenesis experiments will identify specific regions and amino acid residues that are critical for T1 domain tetramerization and subfamily specificity. Finally, a set of chimeric K+ channel subunit proteins will be created, with swapped T1 domains, to test for the importance of T1 in determining subunit protein interaction and formation of homomultimeric and heteromultimeric K+ channels. Because K+ channel proteins are an increasingly important target for drugs to treat asthma, high blood pressure, diabetes, multiple sclerosis, epilepsy and potentially a host of other disorders, these experiments will benefit health research by providing critical information about the events and domains of K+ channel subunit proteins that are involved in forming the ion channel.
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