title: Nanomolar potency and selectivity of a Ca²⁺ release-activated Ca²⁺ channel inhibitor against store-operated Ca²⁺ entry and migration of vascular smooth muscle cells.
abstract: OBJECTIVE
  The aim was to advance the understanding of Orai proteins and identify a specific inhibitor of the associated calcium entry mechanism in vascular smooth muscle cells (VSMCs).
  
  
  METHODS
  Proliferating VSMCs were cultured from human saphenous veins. Intracellular calcium was measured using fura-2, whole-cell current was recorded using patch-clamp and cell migration quantified in modified Boyden chambers. Subcellular protein localization was determined by microscopy. Isometric tension was recorded from mouse aortic rings.
  
  
  RESULTS
  Molecular disruption and rescue experiments indicated the importance of Orai1 in calcium entry caused by store depletion evoked passively or by platelet-derived growth factor (PDGF), suggesting the presence of Ca(2+) release-activated Ca(2+) (CRAC) channels like those of the immune system. The CRAC channel blocker, S66, was a potent inhibitor of the VSMC signals, IC(50) 26 nM, which was almost two orders of magnitude greater than with leucocytes. S66 had no effect on PDGF- and ATP-evoked calcium release, overexpressed transient receptor potential canonical (TRPC)5 channels, native TRPC1/5-containing channels, stromal interaction molecule 1 clustering, non-selective cationic current evoked by store depletion and phenylephrine-evoked aortic contraction. S66 reduced PDGF-evoked VSMC migration while having only modest effects on cell proliferation and no effect on cell viability.
  
  
  CONCLUSIONS
  The data suggest that Orai1 has a role in human VSMC migration, and that a CRAC channel inhibitor has high potency and selectivity for the associated calcium entry, suggesting a distinct characteristic of vascular CRAC channels and the potential for selective chemical suppression of vascular remodelling.
authors.affiliation: Multidisciplinary Cardiovascular Research Centre, University of Leeds, Leeds, UK.
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Lynn
authors.initials: L
authors.lastname: McKeown
authors.forename: Ogooluwa
authors.initials: O
authors.lastname: Ojelabi
authors.forename: Martin
authors.initials: M
authors.lastname: Stacey
authors.forename: Richard
authors.initials: R
authors.lastname: Foster
authors.forename: David
authors.initials: D
authors.lastname: O'Regan
authors.forename: Karen E
authors.initials: KE
authors.lastname: Porter
authors.forename: David J
authors.initials: DJ
authors.lastname: Beech
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D015220:Calcium Channels / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D049109:Cell Proliferation; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000502:physiology; D000071740:ORAI1 Protein; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism
chemical_list: D002121:Calcium Channel Blockers; D015220:Calcium Channels; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; D050052:TRPC Cation Channels; C491780:TRPC5 protein, human; D002118:Calcium