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.
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