Abstract

Our long term goal is to understand the structural and functional bases of the interaction of dantrolene with the intracellular RyR/Ca release channel. We have identified a sequence domain called DP1 (a. a. 590-609) on the skeletal muscle ryanodine receptor (RyR1) as a target for dantrolene, a region tightly linked to mutations resulting in susceptibility to malignant hyperthermia. Dantrolene appears to interact with RyR1 rather than cardiac RyR2 despite an identical DP1 sequence. The weak interaction of dantrolene with RyR2, therefore, may reflect conformational constraints imparted by the rest of the channel or associated macromolecules. Key question regarding the molecular basis of dantrolene interaction with RyR isoforms are: What are the contextual requirements, both domain-domain and protein-protein interactions that determine dantrolene binding and pharmacologic activity in RyR isoforms? The specific hypothesis for this proposal is that residues 590-609 on RyRldefine the major portion of the dantrolene binding site, and that conformation of this site determines the specific interaction of an RyR isoform with dantrolene and plays important role in the regulation of this Ca release channel.
Two specific aims are designed to test this hypothesis:
Aim 1, to define the structural and biochemical basis of dantrolene interaction with RyRI. Photoaffinity labeling with photoactive dantrolene congeners combined with mass spectrometry, and mutational analysis of identified sequences and functional analysis in Ca fluorescence measurements will be used.
Aim 2, to explore the differential effects of dantrolene on RyR1 and RyR2 using pharmacological, immunological and molecular approaches. RyR1-RyR2 chimeras will be expressed in heterologous and homologous cells and their responses to dantrolene probed by Ca imaging and spectroscopy. The information gained will provide insight into the molecular mechanism by which dantrolene interferes with intracellular Ca release and lead to rational drug design for the therapy of Ca sensitive muscle diseases.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR045593-08
Application #
7268820
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Program Officer
Boyce, Amanda T
Project Start
1999-02-01
Project End
2009-07-31
Budget Start
2007-08-01
Budget End
2009-07-31
Support Year
8
Fiscal Year
2007
Total Cost
$273,999
Indirect Cost

  Institution

Name
Children's Hosp Pittsburgh/Upmc Health Sys
Department
Type
DUNS #
044304145
City
Pittsburgh
State
PA
Country
United States
Zip Code
15224

  Publications

Parness, Jerome; Bandschapp, Oliver; Girard, Thierry (2009) The myotonias and susceptibility to malignant hyperthermia. Anesth Analg 109:1054-64
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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