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

Abstract

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.

• Authors: M Bulla, G Gyimesi, J H Kim, R Bhardwaj, M A Hediger, M Frieden, N Demaurex
• Journal: The Journal of physiology (J Physiol) Vol. 597 Issue 2 Pg. 561-582 (01 2019) ISSN: 1469-7793 [Electronic] England
• PMID: 30382595 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2018 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

Chemical References

• 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

Topics

• 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, physiopathology)
• ORAI1 Protein (antagonists & inhibitors, chemistry, genetics, physiology)
• Protein Domains
• Pyrazoles (pharmacology)
• Stromal Interaction Molecule 1 (genetics)
• Stromal Interaction Molecule 2 (genetics)