Dantrolene, a drug which inhibits intracellular Ca/2+ release is the only therapy of malignant hyperthermia, a genetic sensitivity to volatile anesthetics. The drug also shows therapeutic promise in models of neural excitotoxicity and sepsis. However, the molecular mechanism of action of this compound is not known. This application proposes to use a radiolabeled, pharmacologically active, photoaffinity analog of dantrolene, to identify, purify and molecularly characterize the putative dantrolene receptor from skeletal muscle. The results of these experiments will serve as the basis for future experiments leading to an understanding of the role of the dantrolene receptor in regulating intracellular Ca/2+ release in health and disease. Since [3H]dantrolene binding is greatest in the sarcoplasmic reticulum of skeletal muscle, this tissue will be used as a source of putative receptor. We present data that [3H]azidodantrolene, a custom synthesized, photoaffinity analog of dantrolene, specifically photolabels a protein of approximately 140 kDa, as determined by SDS-PAGE, electroblotting to PVDF membranes, and subsequent fluorography. By using this as a method of molecular tagging, we propose to purify the putative dantrolene receptor by standard methods of membrane protein purification, which includes preparative gel electrophoresis, electroelution, detergent solubilization, ion-exchange, hydrophobic interaction and/or affinity chromatography techniques. Once purified, the receptor will be subjected to protein microsequencing. Sequence data will be compared with known sequences from protein databases to determine whether this protein shows homology to any other previously described protein. Polyclonal antibodies to the purified protein or unique synthetic peptides constructed after sequencing will be produced by standard techniques. These antibodies will be used in Western blots to detect the putative dantrolene receptor in tissues other than skeletal muscle since it has been reported that dantrolene is effective in inhibiting Ca/2+ release in a variety of cell types. Immunocytochemistry will be used to characterize the subcellular localization of this protein with respect to other known proteins involved in calcium regulation. The results from these experiments will help in designing future experiments that would help elucidate the role of the dantrolene receptor in excitation- contraction coupling and, ultimately, in Ca/2+ regulation in other tissues.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR045593-02
Application #
6149717
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Program Officer
Lymn, Richard W
Project Start
1999-02-01
Project End
2002-01-31
Budget Start
2000-02-01
Budget End
2001-01-31
Support Year
2
Fiscal Year
2000
Total Cost
$213,213
Indirect Cost
Name
University of Medicine & Dentistry of NJ
Department
Anesthesiology
Type
Schools of Medicine
DUNS #
622146454
City
Piscataway
State
NJ
Country
United States
Zip Code
08854
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Liang, Xin; Chen, Keying; Fruen, Bradley et al. (2009) Impaired interaction between skeletal ryanodine receptors in malignant hyperthermia. Integr Biol (Camb) 1:533-9
Parness, Jerome; Herlich, Andrew; Torp, Klaus D et al. (2008) Nonmalignant hyperthermia and malignant hyperthermia confused. J Clin Anesth 20:313-4;author reply 316
Luty, Winifred H; Rodeberg, David; Parness, Jerome et al. (2007) Antiparallel segregation of notch components in the immunological synapse directs reciprocal signaling in allogeneic Th:DC conjugates. J Immunol 179:819-29
Weisleder, Noah; Brotto, Marco; Komazaki, Shinji et al. (2006) Muscle aging is associated with compromised Ca2+ spark signaling and segregated intracellular Ca2+ release. J Cell Biol 174:639-45
Zhao, Xiaoli; Weisleder, Noah; Han, Xuehai et al. (2006) Azumolene inhibits a component of store-operated calcium entry coupled to the skeletal muscle ryanodine receptor. J Biol Chem 281:33477-86
Kobayashi, Shigeki; Bannister, Mark L; Gangopadhyay, Jaya P et al. (2005) Dantrolene stabilizes domain interactions within the ryanodine receptor. J Biol Chem 280:6580-7
Paul-Pletzer, Kalanethee; Yamamoto, Takeshi; Ikemoto, Noriaki et al. (2005) Probing a putative dantrolene-binding site on the cardiac ryanodine receptor. Biochem J 387:905-9
Kobayashi, Shigeki; Yamamoto, Takeshi; Parness, Jerome et al. (2004) Antibody probe study of Ca2+ channel regulation by interdomain interaction within the ryanodine receptor. Biochem J 380:561-9
Shin, Dong Wook; Pan, Zui; Kim, Eun Kyung et al. (2003) A retrograde signal from calsequestrin for the regulation of store-operated Ca2+ entry in skeletal muscle. J Biol Chem 278:3286-92

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