LAUSR

BACKGROUND AND PURPOSE Mibefradil (Mib), a T-type Ca2+ channel blocker, has been investigated for treating solid tumours. However, its underlying mechanisms are still unclear. Here we aimed to investigate the pharmacological aspect of Mib on ORAI store-operated Ca2+ channels. EXPERIMENTAL APPROACH Human ORAI1-3 in tetracycline-regulated pcDNA4/TO vectors was transfected into HEK293 T-REx cells with STIM1 stable expression. The ORAI currents were recorded by whole-cell and excised-membrane patch clamp. Ca2+ influx or release was measured by Fura-PE3/AM. Cell growth and death were monitored by WST-1, LDH assays and flow cytometry. KEY RESULTS Mib inhibited ORAI1, ORAI2 and ORAI3 currents in a dose-dependent manner. The IC50 for ORAI1, ORAI2 and ORAI3 was 52.6 μM, 14.1 μM and 3.8 μM, respectively. Outside-out patch demonstrated that perfusion of 10 μM Mib to the extracellular surface completely blocked ORAI3 currents and single channel activity evoked by 2-APB. Intracellular application of Mib did not alter ORAI3 channel activity. Mib at higher concentrations (>50 μM) inhibited Ca2+ release, but had no effect on cytosolic STIM1 translocation evoked by thapsigargin. The inhibition of Mib on ORAI channels is structure-related, since other T-type Ca2+ channel blockers with different structures, such as ethosuximide and ML218, had no or very small effect on ORAI channels. Moreover, Mib inhibited cell proliferation, induced apoptosis and arrested cell cycle progression. CONCLUSIONS AND IMPLICATIONS Our results suggest that Mib is a potent extracellular ORAI channel blocker, which provides a new pharmacological profile for the compound in regulating cell growth and death as an anti-cancer drug.