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

BACKGROUND AND PURPOSEThe 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 APPROACHProliferating 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 RESULTSMolecular 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 Ca2+ release-activated Ca2+ (CRAC) channels like those of the immune system. The CRAC channel blocker, S66, was a potent inhibitor of the VSMC signals, IC50 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 IMPLICATIONSThe 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.

Differential Roles of the C and N Termini of Orai1 Protein in Interacting with Stromal Interaction Molecule 1 (STIM1) for Ca2+ Release-activated Ca2+ (CRAC) Channel Activation

“…The present study aims to advance our knowledge on the gating mechanism of CRAC channel, a type of voltageindependent, highly Ca 2ϩ -selective channel with low singlechannel conductance, which is predominant in most nonexcitable cells and plays indispensable roles in Ca 2ϩ signal transduction (11). Accumulating evidence suggests that CRAC channels are also crucial in refilling the Ca 2ϩ store/sarcoplasmic reticulum in skeletal muscles during contraction (46), regulating the proliferation and migration of smooth muscle cells (47)(48)(49)(50), triggering the hypertrophic growth of cardiomyocytes (51,52), and modulating Ca 2ϩ homeostasis in neurons (53,54). Mammalian immune cells exert their biological functions through surface receptor activation by utilizing elevated intracellular Ca 2ϩ conducted by CRAC channels (55).…”

Molecular pharmacology of store-operated CRAC channels

“…139 In RBL cells, antigen-mediated syk phosphorylation was inhibited by Synta66, pointing to the role of CRAC channels in this signaling pathway. 138 In another study, anti-CD3/CD28 stimulated mononuclear cells isolated from the lamina propria of patients with Inflammatory bowel disease showed reduced cytokine production and T-bet transcription factor activation when pre-treated with Synta66, suggesting a potential role for CRAC channels in this chronic inflammatory state.…”

Evaluation of known and novel inhibitors of Orai1-mediated store operated Ca 2+ entry in MDA-MB-231 breast cancer cells using a Fluorescence Imaging Plate Reader assay

“…Finally, the extensively-studied biphenyl analogue Synta66 (13) [36][37][38] has been demonstrated to be a potent (IC 50 26 nM by patch-clamp assay) and selective Orai1 inhibitor. This was confirmed in the FLIPR assay, where it was the most potent (IC 50 1.3 µM) literature compound evaluated.…”