ORAI1 channel gating and selectivity is differentially altered by natural mutations in the first or third transmembrane domain

J Physiol. 2019 Jan;597(2):561-582. doi: 10.1113/JP277079. Epub 2018 Nov 28.


Key points: Gain-of-function mutations in the highly selective Ca2+ channel ORAI1 cause tubular aggregate myopathy (TAM) characterized by muscular pain, weakness and cramping. TAM-associated mutations in ORAI1 first and third transmembrane domain facilitate channel opening by STIM1, causing constitutive Ca2+ influx and increasing the currents evoked by Ca2+ store depletion. Mutation V107M additionally decreases the channel selectivity for Ca2+ ions and its inhibition by acidic pH, while mutation T184M does not alter the channel sensitivity to pH or to reactive oxygen species. The ORAI blocker GSK-7975A prevents the constitutive activity of TAM-associated channels and might be used in therapy for patients suffering from TAM.

Abstract: Skeletal muscle differentiation relies on store-operated Ca2+ entry (SOCE) mediated by STIM proteins linking the depletion of endoplasmic/sarcoplasmic reticulum Ca2+ stores to the activation of membrane Ca2+ -permeable ORAI channels. Gain-of-function mutations in STIM1 or ORAI1 isoforms cause tubular aggregate myopathy (TAM), a skeletal muscle disorder with muscular pain, weakness and cramping. Here, we characterize two overactive ORAI1 mutants from patients with TAM: V107M and T184M, located in the first and third transmembrane domain of the channel. When ectopically expressed in HEK-293T cells or human primary myoblasts, the mutated channels increased basal and store-operated Ca2+ entry. The constitutive activity of V107M, L138F, T184M and P245L mutants was prevented by low concentrations of GSK-7975A while the G98S mutant was resistant to inhibition. Electrophysiological recordings confirmed ORAI1-V107M constitutive activity and revealed larger STIM1-gated V107M- and T184M-mediated currents with conserved fast and slow Ca2+ -dependent inactivation. Mutation V107M altered the channel selectivity for Ca2+ ions and conferred resistance to acidic inhibition. Ca2+ imaging and molecular dynamics simulations showed a preserved sensitivity of T184M to the negative regulation by reactive oxygen species. Both mutants were able to mediate SOCE in Stim1-/- /Stim2-/- mouse embryonic fibroblasts expressing the binding-deficient STIM1-F394H mutant, indicating a higher sensitivity for STIM1-mediated gating, with ORAI1-T184M gain-of-function being strictly dependent on STIM1. These findings provide new insights into the permeation and regulatory properties of ORAI1 mutants that might translate into therapies against diseases with gain-of-function mutations in ORAI1.

Keywords: CRAC; GSK-7975A; ORAI1; SOCE; STIM1; TAM; calcium.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Benzamides / pharmacology
  • Calcium Channel Blockers / pharmacology
  • Calcium Signaling
  • Fibroblasts / physiology
  • Gain of Function Mutation
  • HEK293 Cells
  • Humans
  • Ion Channel Gating*
  • Mice, Knockout
  • Myoblasts / physiology
  • Myopathies, Structural, Congenital / genetics*
  • Myopathies, Structural, Congenital / physiopathology
  • ORAI1 Protein / antagonists & inhibitors
  • ORAI1 Protein / chemistry
  • ORAI1 Protein / genetics*
  • ORAI1 Protein / physiology
  • Protein Domains
  • Pyrazoles / pharmacology
  • Stromal Interaction Molecule 1 / genetics
  • Stromal Interaction Molecule 2 / genetics


  • 2,6-difluoro-N-(1-(4-hydroxy-2-(trifluoromethyl)benzyl)-1H-pyrazol-3-yl)benzamide
  • Benzamides
  • Calcium Channel Blockers
  • ORAI1 Protein
  • ORAI1 protein, human
  • Pyrazoles
  • Stim1 protein, mouse
  • Stim2 protein, mouse
  • Stromal Interaction Molecule 1
  • Stromal Interaction Molecule 2