THIS IS A SHANNON AWARD PROVIDING PARTIAL SUPPORT FOR THE RESEARCH PROJECTS THAT FALL SHORT OF THE ASSIGNED INSTITUTE'S FUNDING RANGE BUT ARE IN THE MARGIN OF EXCELLENCE. THE SHANNON AWARD IS INTENDED TO PROVIDE SUPPORT TO TEST THE FEASIBILITY OF THE APPROACH; DEVELOP FURTHER TESTS AND REFINE RESEARCH TECHNIQUES; PERFORM SECONDARY ANALYSIS OF AVAILABLE DATA SETS; OR CONDUCT DISCRETE PROJECTS THAT CAN DEMONSTRATE THE PI'S RESEARCH CAPABILITIES OR LEAD ADDITIONAL WEIGHT TO AN ALREADY MERITORIOUS APPLICATION. THE APPLICATION BELOW IS TAKEN FROM THE ORIGINAL DOCUMENT SUBMITTED BY THE PRINCIPAL INVESTIGATOR. Voltage-gated K (Kv) channels regulate the behavior of diverse cell types. An extended family of 19 genes encode these important proteins. Delineation of the tertiary structure of any one of these channels will provide the conceptual framework for understanding ion permeation, activation, inactivation and deactivation, and be the basis for developing models of related ion channels. Such structural information could also guide the rational design of novel ion channel modulating drugs that could be used for the therapy for a wide range of conditions including diabetes, cardiac arrhythmias, stroke, autoimmune disorders, hypertension, urinary incontinence and male pattern baldness. We propose to perform a detailed structural analysis of Kv1.3, a critical modulator of T-lymphocyte function. The Kv 1.3 channel is one of the best characterized Kv channels and hundreds of micrograms of tetrameric, functional purified Kv1.3 protein are now available for structural studies. Combining complementary site-specific mutagenesis, electrophysiology, computer modeling, protein chemistry and electron crystallography into two complementary strategies, we will probe the structure of Kv1.3. The first will extend our use of scorpion toxins as molecular calipers to identify new toxin:channel interactions and refine our model of the Kv1.3 pore and surrounding regions. Using this model as a guide, we will determine Kv1.3's interactions with a new class of high- affinity organic blockers of Kv1.3, which could then serve as a template for the design of more selective and potent blockers of the channel. Second, we will generate coherent two-dimensional Kv1.3 crystals in lipid membranes and determine the structure of Kv1.3 using electron crystallography. A density map will be generated on the basis of electron and optical diffraction data collected at multiple tilt angles. The Kv1.3 polypeptide will be fitted to this map, the toxin-mapping information being valuable in its interpretation. Our goal will be to reconstruct a three-dimensional image of the channel from the 2D information.
