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J Physiol. 2018 Nov 1. doi: 10.1113/JP277079. [Epub ahead of print]

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

Author information

1
Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland.
2
Institute of Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland.
3
Departments of Physiology and Global Medical Science, Yonsei University Wonju College of Medicine, Wonju, Republic of Korea.
4
Mitohormesis Research Center, Yonsei University Wonju College of Medicine, Wonju, Republic of Korea.

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 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 ROS. 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 STIM. 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. This article is protected by copyright. All rights reserved.

KEYWORDS:

CRAC; GSK-7975A; ORAI1; SOCE; STIM1; TAM; calcium

PMID:
30382595
DOI:
10.1113/JP277079

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