Effect of Azumolene on RyR1-Dependent Store Operated Calcium Entry in RyR1-Expressing, Non Excitable Cells

Abstract

Store-Operated Calcium Entry (SOCE) restores Ca2+ to depleted endoplasmic reticulum (ER) from the extracellular space via a multiprotein complex involving plasma membrane Orai1 and TRPC1, and ER membrane resident STIM1. Dantrolene and azumolene suppress the rise in intracellular Ca2+ seen during skeletal muscle during excitation-contraction coupling and in malignant hyperthermia, a hypermetabolic pharmacogenetic sensitivity to volatile anesthetics. Azumolene inhibits a component of SOCE coupled to activation of RyR1, the skeletal muscle sarcoplasmic reticulum Ca2+ release channel, but not Ca2+ release itself. Classical SOCE, activated by SR Ca2+-ATPase inhibitors, is unaffected. Thus, azumolene distinguishes between two mechanisms of cellular signaling to SOCE in skeletal muscle, one that is coupled to and one independent from RyR1. We used CHO cells stably transfected with RyR1 (C1148) and wild type (CHO-wt) to determine whether these distinguishable mechanisms of Ca2+entry are present universally, or only in excitable cells. SOCE was measured using Mn2+ quenching of Fura-2 fluorescence. C1148 cells expressing RyR1, but not CHO-wt, had high intrinsic Mn2+-quenching that is inhibited by azumolene and by the specific SOCE inhibitor BTP2, while low intrinsic Ca2+ entry of CHO-wt was unaffected by these drugs. On contrast, SOCE stimulated by the SR Ca2+-ATPase inhibitor, CPA (10μM), was inhibited by BTP2, but not by azumolene. Knockdown of STIM1 levels using shRNA demonstrates inhibition of RyR1-coupled SOCE. Immunocytochemistry of C1148 cells shows colocalization of RyR1 and STIM1 proteins in the presence of the RyR1 agonists, caffeine and ryanodine, and these proteins co-immunoprecipitate, suggesting they are interacting proteins. Thus, in RyR1-expressing, non-excitable cells, azumolene inhibits RyR1-dependent SOCE, but not Ca2+-ATPase-dependent SOCE, and suggests that STIM1, as one of the components of the SOCE machinery, may need to interact with RyR1 in this pathway.