Mibefradil, a T-type Ca2+ channel blocker also blocks Orai channels by action at the extracellular surface

Li, Pengyun; Rubaiy, Hussein N; Chen, Gui-Lan; Hallett, Thomas; Zaibi, Nawel; Zeng, Bo; Saurabh, Rahul; Xu, Shang-Zhong

doi:10.1111/bph.14788

Mibefradil, a T-type Ca2+ channel blocker also blocks Orai channels by action at the extracellular surface

Li, Pengyun; Rubaiy, Hussein N; Chen, Gui-Lan; Hallett, Thomas; Zaibi, Nawel; Zeng, Bo; Saurabh, Rahul; Xu, Shang-Zhong

Authors

Pengyun Li

Hussein N Rubaiy

Gui-Lan Chen

Thomas Hallett

Nawel Zaibi

Bo Zeng

Rahul Saurabh

Dr Sam Xu S.Xu@hull.ac.uk
Reader

Abstract

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.

Citation

Li, P., Rubaiy, H. N., Chen, G.-L., Hallett, T., Zaibi, N., Zeng, B., Saurabh, R., & Xu, S.-Z. (2019). Mibefradil, a T-type Ca2+ channel blocker also blocks Orai channels by action at the extracellular surface. British Journal of Pharmacology, 176, 3845–3856. https://doi.org/10.1111/bph.14788

Journal Article Type	Article
Acceptance Date	Jun 28, 2019
Online Publication Date	Aug 19, 2019
Publication Date	Oct 8, 2019
Deposit Date	Jul 5, 2019
Publicly Available Date	Aug 20, 2020
Journal	British Journal of Pharmacology
Print ISSN	0007-1188
Publisher	Wiley
Peer Reviewed	Peer Reviewed
Volume	176
Pages	3845–3856
DOI	https://doi.org/10.1111/bph.14788
Public URL	https://hull-repository.worktribe.com/output/2113279
Contract Date	Jul 5, 2019

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