3H]Azidodantrolene: synthesis and use in identification of a putative skeletal muscle dantrolene binding site in sarcoplasmic reticulum.

Abstract

Dantrolene sodium is a medically important hydantoin derivative that interferes with release of Ca2+ from intracellular stores of skeletal muscle by an unknown mechanism. Identification of the molecular target of dantrolene would greatly aid in understanding both the mechanism of action of the drug and the dynamics of intracellular Ca2+ release in muscle. [3H]Azidodantrolene was designed and synthesized as a photoaffinity analogue in order to identify a putative dantrolene receptor in skeletal muscle. Introduction of 1 mole-atom of tritium into aldehyde 5b was required during radioligand synthesis in order to ensure high enough specific activity for detection of photo-cross-linked proteins by fluorographic methods. This was accomplished by reduction of ester 3 with custom synthesized, 100% tritium-labeled lithium triethylborotritide, followed by oxidation to 5b by manganese(IV) oxide. Compound 6b was demonstrated to be >/=95% tritium-labeled at the imine position by NMR spectroscopy, and the specific radioactivity of [3H]azidodantrolene sodium was empirically determined by HPLC and liquid scintillation counting to be 24.4 Ci/mmol, approximately 85% of theoretical maximum. [3H]Azidodantrolene was found to be pharmacologically active in ligand-receptor binding studies with skeletal muscle sarcoplasmic reticulum membranes. Photo-cross-linking experiments analyzed by SDS-PAGE and tritium fluorography have identified a approximately 160-kDa specifically labeled protein as the putative, intracellular, skeletal muscle dantrolene receptor. This photolabeled protein comigrates with a protein in Western blots immunologically cross-reactive to a polyclonal anti-rabbit skeletal muscle ryanodine receptor antibody. Thus, the putative dantrolene receptor may be related to the skeletal muscle ryanodine receptor.