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 localise 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 Rab7-positive late endosomes for lysosomal degradation. Orai1-SNPs escape the degradation pathway and instead enter Rab11-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.
Hum Mol Genet