title: Conformation of ryanodine receptor-2 gates store-operated calcium entry in rat pulmonary arterial myocytes.
abstract: 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.
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University School of Medicine, 5501 Hopkins Bayview Circle, Baltimore, MD 21224, USA.
authors.forename: Amanda H Y
authors.initials: AH
authors.lastname: Lin
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University School of Medicine, 5501 Hopkins Bayview Circle, Baltimore, MD 21224, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Sun
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University School of Medicine, 5501 Hopkins Bayview Circle, Baltimore, MD 21224, USA.
authors.forename: Omkar
authors.initials: O
authors.lastname: Paudel
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University School of Medicine, 5501 Hopkins Bayview Circle, Baltimore, MD 21224, USA Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China.
authors.forename: Mo-Jun
authors.initials: MJ
authors.lastname: Lin
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University School of Medicine, 5501 Hopkins Bayview Circle, Baltimore, MD 21224, USA jsks@jhmi.edu.
authors.forename: James S K
authors.initials: JS
authors.lastname: Sham
mesh_terms: D000818:Animals; D002120:Calcium Channel Agonists / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D008297:Male; D008564:Membrane Potentials; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D009154:Mutation; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*; D000071740:ORAI1 Protein / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011651:Pulmonary Artery / Q000187:drug effects / Q000378:metabolism; D034622:RNA Interference; D017208:Rats, Wistar; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D000071737:Stromal Interaction Molecule 1 / Q000235:genetics / Q000378:metabolism; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
chemical_list: D002120:Calcium Channel Agonists; D002121:Calcium Channel Blockers; D000071740:ORAI1 Protein; C532493:Orai1 protein, rat; C000612605:RyR2 protein, rat; D019837:Ryanodine Receptor Calcium Release Channel; C518857:Stim1 protein, rat; D000071737:Stromal Interaction Molecule 1; D050052:TRPC Cation Channels; C096351:transient receptor potential cation channel, subfamily C, member 1
keywords: Canonical transient receptor potential 1 (TRPC1); Pulmonary arterial smooth muscle cells; Ryanodine receptor (RyR); Store-operated Ca2+ entry (SOCE); Stromal interaction molecule 1 (STIM1)