Conformation of ryanodine receptor-2 gates store-operated calcium entry in rat pulmonary arterial myocytes

• Published in: Cardiovascular research Vol. 111; no. 1; pp. 94 - 104
• Main Authors: Lin, Amanda H Y, Sun, Hui, Paudel, Omkar, Lin, Mo-Jun, Sham, James S K
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.07.2016
• Series: Editor's choice
• Subjects: Animals; Calcium Channel Agonists - pharmacology; Calcium Channel Blockers - pharmacology; Calcium Signaling - drug effects; HEK293 Cells; Humans; Ion Channel Gating - drug effects; Male; Membrane Potentials; Muscle, Smooth, Vascular - drug effects; Muscle, Smooth, Vascular - metabolism; Mutation; Myocytes, Smooth Muscle - drug effects; Myocytes, Smooth Muscle - metabolism; ORAI1 Protein - genetics; ORAI1 Protein - metabolism; ORIGINAL ARTICLES; Protein Binding; Protein Conformation; Pulmonary Artery - drug effects; Pulmonary Artery - metabolism; Rats, Wistar; RNA Interference; Ryanodine Receptor Calcium Release Channel - chemistry; Ryanodine Receptor Calcium Release Channel - drug effects; Ryanodine Receptor Calcium Release Channel - genetics; Ryanodine Receptor Calcium Release Channel - metabolism; Stromal Interaction Molecule 1 - genetics; Stromal Interaction Molecule 1 - metabolism; Time Factors; Transfection; TRPC Cation Channels - genetics; TRPC Cation Channels - metabolism; Canonical transient receptor potential 1 (TRPC1); Stromal interaction molecule 1 (STIM1); Ryanodine receptor (RyR); Pulmonary arterial smooth muscle cells; Store-operated Ca2+ entry (SOCE)
• ISSN: 0008-6363; 1755-3245
• DOI: 10.1093/cvr/cvw067

Store-operated Ca(2+) entry (SOCE) contributes to a multitude of physiological and pathophysiological functions in pulmonary vasculatures. SOCE attributable to inositol 1,4,5-trisphosphate receptor (InsP3R)-gated Ca(2+) store has been studied extensively, but the role of ryanodine receptor (RyR)-gated store in SOCE remains unclear. The present study aims to delineate the relationship between RyR-gated Ca(2+) stores and SOCE, and characterize the properties of RyR-gated Ca(2+) entry in pulmonary artery smooth muscle cells (PASMCs). PASMCs were isolated from intralobar pulmonary arteries of male Wister rats. Application of the RyR1/2 agonist 4-chloro-m-cresol (4-CmC) activated robust Ca(2+) entry in PASMCs. It was blocked by Gd(3+) and the RyR2 modulator K201 but was unaffected by the RyR1/3 antagonist dantrolene and the InsP3R inhibitor xestospongin C, suggesting RyR2 is mainly involved in the process. siRNA knockdown of STIM1, TRPC1, and Orai1, or interruption of STIM1 translocation with ML-9 significantly attenuated the 4-CmC-induced SOCE, similar to SOCE induced by thapsigargin. However, depletion of RyR-gated store with caffeine failed to activate Ca(2+) entry. Inclusion of ryanodine, which itself did not cause Ca(2+) entry, uncovered caffeine-induced SOCE in a concentration-dependent manner, suggesting binding of ryanodine to RyR is permissive for the process. This Ca(2+) entry had the same molecular and pharmacological properties of 4-CmC-induced SOCE, and it persisted once activated even after caffeine washout. Measurement of Ca(2+) in sarcoplasmic reticulum (SR) showed that 4-CmC and caffeine application with or without ryanodine reduced SR Ca(2+) to similar extent, suggesting store-depletion was not the cause of the discrepancy. Moreover, caffeine/ryanodine and 4-CmC failed to initiate SOCE in cells transfected with the ryanodine-binding deficient mutant RyR2-I4827T. RyR2-gated Ca(2+) store contributes to SOCE in PASMCs; however, store-depletion alone is insufficient but requires a specific RyR conformation modifiable by ryanodine binding to activate Ca(2+) entry.