Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB222

Poster Communications

T-type Ca2+ channel blocker mibefradil inhibits ORAI store-operated channels

P. Li1,2, H. N. Rubaiy1, G. Chen2, T. Hallett1, S. Xu1

1. Centre for Atherothrombosis and Metabolic Research, Hull York Medical School, University of HULL, Hull, United Kingdom. 2. Institute of Cardiovascular Research, Southwest Medical University, Luzhou, China.

Introduction ORAI channels have been regarded as the molecular basis of Ca2+-release activated Ca2+ (CRAC) channels, the highly Ca2+ selective store-operated channels (SOCs). The channels can be activated by the depletion of endoplasmic reticulum (ER) Ca2+ stores via the activation of G protein-coupled receptors (GPCR) and/or protein tyrosine kinase (PTK) coupled receptors (1). There are three isoforms of ORAI channels (ORAI1-3). These channels may act as therapeutic targets for immune disorders, cardiovascular diseases and diabetic complications (2, 3). However, potent and selective inhibitors or activators for these channels are still lacking. Here we examined the effect of mibefradil (Mib), a T-type calcium channel blocker(4), on ORAI channels overexpressing in T-REx cells. Methods The plasmid cDNAs of ORAIs tagged with cyan (CFP) or red (mCherry) fluorescent protein genes were stably co-expressed with STIM1-EYFP in HEK-293 T-REx cells. The expression of ORAIs was induced by 1 µg ml−1 tetracycline for 24-72 h before patch clamp recording or Ca2+ imaging. The non-induced cells without addition of tetracycline or the non-transfected cells were used as controls. Cells were grown in DMEM-F12 medium containing 10% fetal calf serum (FCS). Cells were maintained at 37°C under 95% air and 5% CO2 and seeded on coverslips for experiments. The whole cell or excised membrane patch current was amplified using Axopatch 200B amplifier and the software pClamp 10. Ca2+ release and influx was measured by FlexStation 3 using the Ca2+ dye Fura-PE3AM. Values are means ± S.E.M., compared by ANOVA or t-test where appropriate. Results Mibefradil (Mib, 0.1∼100 µM) inhibited ORAI1, ORAI2 and ORAI3 currents in a dose-dependent manner. The IC50 for ORAI1, ORAI2 and ORAI3 was 39.3 µM, 24.8 µM and 4.2 µM, respectively (n = 2-10). Mib at 10 µM inhibited ORAI1, ORAI2 and ORAI3 currents by 21.82±2.49 (n = 10), 37.10±3.51 (n = 8) and 62.05±3.49% (n = 8), respectively. To investigate whether Mib might act on the channels directly via an intracellular or extracellular site, higher concentrations (10∼100 µM) of Mib were applied in patch pipette, and no significant inhibition was found on 2-APB (100 µM) evoked currents (the fold change of 2-APB activated current in pipette without Mib and with Mib were 11.75±2.47 vs. 10.83±2.11, respectively, n = 5, P>0.05). Outside-out patch on ORAI3 currents further demonstrated that bath application of 10 µM of Mib (n = 4) to the extracellular surface completely blocked the 2-APB (100 µM) activated currents. Additionally, Mib at lower concentration (<50 µM) has no significant effect on intracellular Ca2+ release from endoplasmic reticulum (ER), while higher concentrations of Mib (>50 µM) inhibited Ca2+ release from ER store by 35.0±4.9%. Conclusions We concluded that Mib not only acts as a T-type calcium channel blocker, but also shows a potent inhibitory effect on ORAI channels, which provides a new pharmacological profile for ORAI channel blockers.

Where applicable, experiments conform with Society ethical requirements