The overall objective of this work is to establish how a high-conductance, ligand-gated Ca2+ release channel in sarcoplasmic reticulum (SR) regulates the intracellular Ca2+ ion concentration of skeletal muscle. The SR Ca2+ release channel has been recently purified, reconstituted into lipid bilayers, and shown to be identical with the large protein bridges (""""""""feet"""""""") that span the transverse (T-) tubule-sarcoplasmic reticulum junction. The proposed research is directed toward the further characterization of the structure and function of the SR Ca2+ release channel. T-tubule/SR junctional complexes (triads) and T-tubule detached (heavy) SR vesicles will be isolated to analyze T-tubule depolarization-induced and ligand- regulated SR Ca2+ release. Regulation of the T-tubule linked, T-tubule detached and purified Ca2+ release channel will be determined, on a time scale of milliseconds to minutes, in vesicles and/or at the single channel level. Effectors to be studied include the endogenous ligands Ca2+, Mg2+, H+, adenine nucleotides an calmodulin, T-tubule membrane potentials, membrane phosphorylation and drugs such as ryanodine and methylxanthines. These studies will test the hypothesis that T-tubule linked and T-tubule detached SR CA2+ release channels respond differently under identical physiological and pharmacological conditions. The domain structure of the SR CA2+ release channel will be determined with the use of proteases and site-directed antibodies. The effects of controlled proteolysis and antibodies will be assessed by immunoblot analysis, radiolabeling of regulatory ligand binding sites and peptide sequencing, sedimentation analysis of soluble channel complexes, and vesicle-ion flux and single channel measurements. Ca2+ release channel complementary DNAs will be expressed to localize the channel domain and/or regulatory site domains. Expressed proteins will be characterized as outlined above using cellular, microsomal and purified channel protein preparations.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AR018687-22
Application #
2413963
Study Section
Special Emphasis Panel (NSS)
Project Start
1976-05-01
Project End
2000-04-30
Budget Start
1997-05-01
Budget End
1998-04-30
Support Year
22
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Biochemistry
Type
Schools of Medicine
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
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Manno, Carlo; Figueroa, Lourdes; Royer, Leandro et al. (2013) Altered Ca2+ concentration, permeability and buffering in the myofibre Ca2+ store of a mouse model of malignant hyperthermia. J Physiol 591:4439-57
Mei, Yingwu; Xu, Le; Kramer, Henning F et al. (2013) Stabilization of the skeletal muscle ryanodine receptor ion channel-FKBP12 complex by the 1,4-benzothiazepine derivative S107. PLoS One 8:e54208

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