The long-term goal of the Investigators is to define the mechanisms of excitation-contraction coupling reticulum (SR) calcium release channel (RyR-1) is the focus of this pursuit. RyR-1 is important not only because it acts as the calcium release channel but also because it makes up a major part of the junctional foot complex. RyR-1 either directly or indirectly transduces the charge movement created by t-tubule depolarization, which is thought to induce the release of Ca2+ into the myoplasm. The studies proposed below represent a crucial first step in the analysis of the molecular mechanisms of E-C coupling and are unique because they will be done in genetically altered muscle cells in which all of the elements required for E-C coupling except RyR-1 are present (dyspedic muscle cells). Dyspedic muscle cells will allow us to overcome the problems experienced by other investigators who are studying the channel in non-muscle expression systems. When RyR-1 is expressed in non muscle cells, conductance and gating is abnormal because these cells lack expression of other essential proteins such as DHP receptor, FKBP12 and triadin needed for normal E-C coupling. We will first verify in our dyspedic muscle cells that the protein encoded by the RyR-1 cDNA replicates the function of the native RyR-1 protein. We will then mutate specific regions of the RyR1 cDNA to study the relationship between the structure of the expressed mutated RyR-1 protein and its function.
Three specific aims are addressed in detail: A) To express the full length cDNA encoding RyR-1 and characterize its function. We will transfect the RyR-1 deficient myoblasts with the cDNA encoding the wild type RyR-1 protein and reconstitute E-C coupling. The biochemical and physiological properties of expressed RyR-1 will be studied in intact cells using 1) ratio fluorescence imaging techniques, 2) radioligand receptor binding analysis, 3) macroscopic measurements of Ca2+ transport across SR membrane vesicles, and 4) single channel gating kinetics under voltage clamp. This step is necessary to demonstrate that the normal phenotype can be restored by the RyR-1 cDNA and serves as a base line control for structure function studies B) To express a cDNA containing the Arg615/Cys615 mutation in RyR-1 deficient myoblasts. Altered sensitivity of RyR-1 to know antagonists and antagonists will be determined at the cellular and subcellular levels. C) To express cDNA constructs possessing site-directed mutations at putative Ca2+ and FKBP12-binding domains to reveal now structure-function information with molecular detail. A mutation strategy targeting regions of primary sequence with partial """"""""E-F"""""""" structure will be implemented to identify elements important for channel activation and inhibition. A novel strategy utilizing radiolabeled bastadin 5 will permit identification of structural elements essential to the formation of a functional FKBP12/RyR-1 complex. Complementary studies using site-directed mutation analysis will verify which elements of RyR-1 structure are necessary for recognition and functional modulation by FKBP12. The proposed studies will be the first to establish the relationships between RyR-1 protein structure and SR Ca2+ channel function and provide powerful new tools to better understand E-C coupling in striated muscle.
Showing the most recent 10 out of 45 publications
