oa Molecular Determinants Of The Store-operated Ca2+ Entry Channel Orai1 Trafficking In Mammalian Cells

Intracellular calcium ion is a universal messenger that plays a critical role in wide range of physiological functions and failure to maintain calcium homeostasis is linked to cellular dysfunctions in several diseases. Store operated calcium entry (SOCE) is a fundamental Ca2+ influx pathway at the cell membrane that is activated in response to intracellular store depletion. The SOCE machinery consists of the ER Ca2+ sensor STIM1, and Orai1 a Ca2+ selective channel at the plasma membrane (PM). Despite advancements in our understanding of the regulation of STIM1 and Orai1 very little is known about the biogenesis and trafficking of Orai1 and how that affects SOCE in mammalian cells. Here we describe the trafficking dynamics underlying Orai1subcelluar distribution at steady state and after store depletion. We show that at steady state 40% of total cellular Orai1 localizes to the PM of CHO cells. The other 60% of Orai1 localizes intracellularly. Time lapse imaging shows continuous shuttling of Orai1-positive vesicles between the PM and intracellular compartments. Quantitative measurements of the exocytosis and endocytosis rates at steady state show a rapid recycling rate of the protein between the intracellular pool and the plasma membrane. Following store depletion, ~80% of Orai1 is localized at PM. Interestingly, we localized Orai1 to novel sub-plasma membrane vesicular compartment of unknown molecular composition. To study the domain of Orai1 involved in its trafficking, we generated deletions of the N-(1-90) or C-(257-301)-terminus. Deletion of N-terminus did not change the surface to total distribution of Orai1, while C-terminus deletion significantly altered Orai1 distribution, with majority of Orai1-∆C localizes intracellularly. Similarly, deletion of residues 267-301 and residues 276-301 showed significantly decreased surface/total distribution compared to wild type (WT), but a less pronounced defect than Orai1-∆C. In contrast, 1-285 behaved like WT Orai1 showing that residues 286-301 are not required for Orai1 biogenesis and localization to the cell membrane. In conclusion, our data suggest that Orai1 recycles at high rate between PM and intracellular compartments at steady state and is enriched at PM after store depletion. Our results also suggest that the amino acid region (257-285) of Orai1 C-terminus is essential for its biogenesis and PM targeting.