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Published on: July 26, 2017
Single-nucleotide polymorphisms in Orai1 associated with atopic dermatitis inhibit protein turnover, decrease calcium
Yi-Chun Yeh1, Yu-Ping Lin1, Holger Kramer2
1Department of Physiology, Anatomy and Genetics, Parks Road, Oxford, OX1 3PT UK.
Abstract:
Loss-of function mutations in Orai1 Ca2+ channels lead to a form of severe combined immunodeficiency, auto-immunity, muscle hypotonia and defects in dental enamel production and sweat gland function. Two single-nucleotide polymorphisms (SNPs) in Orai1 have been found and localize to the second extracellular loop. These polymorphisms associate with atopic dermatitis but how they affect Ca2+ signalling and cell function is unknown. Here, we find that Orai1-SNPs turnover considerably more slowly than wild type Orai1 and are more abundantly expressed in the plasma membrane. We show a central role for flotillin in the endocytotic recycling of Orai1 channels and that endocytosed wild type Orai1 is trafficked to Rab 7-positive late endosomes for lysosomal degradation. Orai1-SNPs escape the degradation pathway and instead enter Rab 11-positive recycling endosomes, where they are returned to the surface membrane through Arf6-dependent exocytosis. We find that Orai1-SNPs escape late endosomes through endosomal pH regulation of interaction between the channel and flotillin. We identify a pH-sensitive electrostatic interaction between positively charged arginine in extracellular loop 2 (K210) and a negatively charged aspartate (D112) in extracellular loop 1 that helps determine Orai1 turnover. The increase in membrane Orai1-SNP leads to a mis-match in Orai1-STIM stoichiometry, resulting in inhibition of Ca2+ entry and Ca2+-dependent gene expression. Our results identify new strategies for targeting atopic dermatitis.
Insights
Single-nucleotide polymorphisms (SNPs) in Orai1 channels are linked to atopic dermatitis. These Orai1-SNPs exhibit slower turnover and altered recycling, impacting calcium signaling and gene expression, offering new therapeutic targets.
Area of Science:
- Cell Biology
- Immunology
- Biochemistry
Background:
- Loss-of-function mutations in Orai1 Ca2+ channels cause severe combined immunodeficiency, autoimmunity, and developmental defects.
- Two single-nucleotide polymorphisms (SNPs) in Orai1, located in the second extracellular loop, are associated with atopic dermatitis, but their functional impact remains unclear.
Purpose of the Study:
- To investigate the functional consequences of Orai1-SNPs on Orai1 channel trafficking, calcium signaling, and cellular function.
- To elucidate the mechanisms underlying the altered turnover and localization of Orai1-SNPs.
Main Methods:
- Utilized cell-based assays to compare the turnover and localization of wild-type Orai1 and Orai1-SNPs.
- Investigated the role of flotillin, Rab proteins (Rab 7 and Rab 11), and Arf6 in Orai1 channel trafficking.
- Analyzed the impact of altered Orai1 localization on calcium influx and gene expression.
Main Results:
- Orai1-SNPs exhibit significantly slower turnover and increased abundance in the plasma membrane compared to wild-type Orai1.
- Flotillin mediates the endocytotic recycling of Orai1 channels.
- Wild-type Orai1 undergoes lysosomal degradation via late endosomes, while Orai1-SNPs are directed to recycling endosomes and escape degradation through pH-sensitive interactions with flotillin.
- Increased membrane Orai1-SNP levels disrupt Orai1-STIM stoichiometry, inhibiting Ca2+ entry and Ca2+-dependent gene expression.
Conclusions:
- Orai1-SNPs escape normal degradation pathways, leading to altered calcium channel function.
- pH-sensitive interactions involving extracellular loops of Orai1 regulate its turnover and trafficking.
- The aberrant trafficking and signaling of Orai1-SNPs provide novel therapeutic targets for atopic dermatitis.
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