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.
Highlights
Store-operated Ca2+ entry generates cytosolic Ca2+ signals that control diverse cellular functions, such as exocytosis, energy production, gene expression and growth and differentiation [29]
Transfection of S218G–Orai1, N223S–Orai1 or the double variant S218G–N223S–Orai1 plasmids all resulted in an increase in Orai1 protein levels above that seen with wild type (WT) Orai1; the increase was ∼ 2-fold at 24 hours and up to 3-fold at 48 hours (Figures 1A and 1B)
Our data show that WT human Orai1, two common human Orai1–single-nucleotide polymorphisms (SNPs) and the double variant are endocytosed in a dynamin- and flotillin-sensitive process and initially share the same intracellular vesicles
Summary
Store-operated Ca2+ entry generates cytosolic Ca2+ signals that control diverse cellular functions, such as exocytosis, energy production, gene expression and growth and differentiation [29]. Store-operated channels activate following depletion of the endoplasmic reticulum (ER) Ca2+ store This occurs upon stimulation of either G-protein or tyrosine kinase coupled cell-surface receptors that generate the second messenger inositol trisphosphate, which releases Ca2+. A few autosomal dominant gain-of-function mutations in Orai have been found and result either in constitutive Ca2+ inf lux in the absence of store depletion (G98S–Orai and L138F–Orai1) or enhanced channel activity by a reduction in Ca2+-dependent slow inactivation of the channels (P245L–Orai1;(24)). These mutations cause tubular associated myopathy and Stormorken syndrome
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