Nanomolar potency and selectivity of a Ca²âº release-activated Ca²âº channel inhibitor against store-operated Ca²âº entry and migration of vascular smooth muscle cells.

ABSTRACT

BACKGROUND AND PURPOSE: 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). EXPERIMENTAL APPROACH: 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. KEY 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 AND IMPLICATIONS 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.