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

Abstract

Aims: Store-operated Ca2+ 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 Ca2+ 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 Ca2+ stores and SOCE, and characterize the properties of RyR-gated Ca2+ entry in pulmonary artery smooth muscle cells (PASMCs). Methods and results: 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 Ca2+ entry in PASMCs. It was blocked by Gd3+ 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 Ca2+ entry. Inclusion of ryanodine, which itself did not cause Ca2+ entry, uncovered caffeine-induced SOCE in a concentration-dependent manner, suggesting binding of ryanodine to RyR is permissive for the process. This Ca2+ entry had the same molecular and pharmacological properties of 4-CmC-induced SOCE, and it persisted once activated even after caffeine washout. Measurement of Ca2+ in sarcoplasmic reticulum (SR) showed that 4-CmC and caffeine application with or without ryanodine reduced SR Ca2+ 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. Conclusions: RyR2-gated Ca2+ store contributes to SOCE in PASMCs; however, store-depletion alone is insufficient but requires a specific RyR conformation modifiable by ryanodine binding to activate Ca2+ entry.