Sudden cardiac death remains one of the nation's leading unresolved health problems. This proposal focuses on the molecular structure, function and regulation of cellular channels that may play a role in controlling cell-cell communication in cardiac myocytes and thus the potential for developing arrhythmias or conduction block. Receptors for inositol-1,4,5-triosphate (IP3 receptors) serve as intracellular second messenger activated Ca2 release channels. In cardiac myocytes these receptors are localized in regions suggesting they may influence the behavior of gap junctions which provide electrical connectivity between neighboring myocytes. In sympathetic neurons, activity of the inositol phosphate cascade, of which IP3 receptors are central effectors, may affect neural activity and sympathetic tone. Important advances in understanding the molecular structure, diversity, functions and regulation of IP3 receptors have come from studies of isoforms expressed at unusually high abundance in the central nervous system. In preliminary feasibility studies we have established both technical approaches and new characterizations of Type IIP3 cerebellar receptors at the biochemical, biophysical, molecular biological and cell biological level. This proposal seeks support for more advanced characterization if IP3 receptors cloned from mammalian cerebellum and expressed in cultured cells, and receptors to be cloned and expressed from cardiac myocytes.
The aims of the studies proposed for the cerebellar receptors are I.1 to complete preparation of structural variants of the cloned Type I receptor for expression and characterization, I.2 to characterize novel aspects of ligand-receptor interaction with the expressed receptor variants. I.3 to optimize conditions for functional reconstitution of expressed receptors in planar lipid bilayers for electrophysiological measurements of structure and function.
The aims for studies proposed for cardiac receptors are II.1 to survey the ligand binding and immunochemical properties of receptors from cardiac myocytes. II.2 to clone cDNA's encoding cardiac receptor isoforms in order to identify the expressed genes, to identify possible specialized structural attributes, to search for alternatively spliced isoforms, and to facilitate the generation of Type-specific antibodies, II.3 to prepare full length cDNA constructs for overexpression in mammalian cells to permit structure-function studies in parallel with those proposed for the cerebellar receptors, and II.4 to use biochemical, immunochemical and molecular biological probes to determine what changes occur in the types and levels of IP3 receptors expressed in failing heart tissues, using materials provided by the pacing dog model for heart failure outlined in Project 7. These studies are likely to provide important new insights, both into fundamental properties of these exceptionally complex intracellular ligand-activated ion channels, and into the possible unique physiological roles for these channels in regulating performance in normal and pathological cardiac tissues.
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