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Published on: July 26, 2017
The Orai1 severe combined immune deficiency mutation and calcium release-activated Ca2+ channel function in the
Jill L Thompson1, Olivier Mignen, Trevor J Shuttleworth
1Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York 14642, USA.
Insights
The R91W mutation in Orai1 channels impairs store-operated calcium entry. Even with one copy of the mutation, calcium entry is reduced, impacting T cell function and potentially causing immune deficiency.
Area of Science:
- Molecular biology
- Immunology
- Channelopathies
Background:
- The R91W mutation in Orai1 causes severe combined immune deficiency (SCID) by completely blocking store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels.
- Heterozygous carriers of the R91W mutation exhibit impaired T cell calcium entry, suggesting a gene-dosage effect.
Purpose of the Study:
- To quantify the impact of the SCID-associated R91W Orai1 mutation in heterozygous carriers.
- To investigate the relationship between the number of mutant Orai1 subunits and CRAC channel function.
Main Methods:
- Generation of concatenated tetramers of Orai1 with varying numbers and arrangements of R91W mutant subunits.
- Electrophysiological recordings to measure CRAC channel currents and activation kinetics.
- Analysis of macroscopic biophysical properties of the channels.
Main Results:
- Increasing numbers of R91W mutant Orai1 subunits in tetramers progressively reduced CRAC channel currents.
- The reduction in current was independent of the spatial arrangement of mutant subunits within the tetramer.
- While macroscopic channel properties remained unchanged, the rate of current activation upon store depletion was slowed.
Conclusions:
- Incorporation of R91W mutant Orai1 subunits into the CRAC channel pore directly impacts its conductance magnitude.
- The observed graded reduction in CRAC channel function is solely dependent on the number of incorporated mutant subunits.
- These findings quantitatively support the impaired calcium entry observed in heterozygous carriers, consistent with a gene-dosage effect.
Abstract:
Homozygous expression of Orai1 bearing the R91W mutation results in the complete abrogation of currents through the store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels, resulting in a form of hereditary severe combined immune deficiency (SCID) syndrome (Feske, S., Gwack, Y., Prakriya, M., Srikanth, S., Puppel, S. H., Tanasa, B., Hogan, P. G., Lewis, R. S., Daly, M., and Rao, A. (2006) Nature 441, 179-185). Although heterozygous carriers of the mutation show no clinical symptoms of immunodeficiency, store-operated Ca(2+) entry in their T cells is impaired, suggesting a gene-dosage effect of the mutation. We have recently demonstrated that the functional CRAC channel pore is composed of a tetrameric assembly of Orai1 subunits (Mignen, O., Thompson, J. L., and Shuttleworth, T. J. (2008) J. Physiol. 586, 419-425). Therefore, to directly quantify the effect of the SCID mutant in the heterozygous situation, we generated a series of concatenated tetramers of Orai1 that included different numbers and arrangements of the R91W Orai1 subunits. The data obtained show that inclusion of increasing numbers of mutant subunits results in a graded reduction in CRAC channel currents and that this effect is independent of the spatial arrangement or order of the mutant subunits in the tetramer. Macroscopic biophysical properties of the channels were unchanged by inclusion of the mutant subunits, although the rate at which the current activates on store depletion was slowed. We conclude that incorporation of R91W mutant Orai1 subunits in the CRAC channel pore affects the overall magnitude of its conductance and that this effect is related solely to the number of mutant subunits incorporated. Predictions based on the tetrameric channel structure indicate that the graded effect of incorporation of SCID mutant subunits into such an assembly is quantitatively consistent with the previously demonstrated impaired effects on Ca(2+) entry recorded in the heterozygous carriers.
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