1887

Abstract

Modulation of calcium homeostasis plays a key role in regulating fundamental cellular processes, including gene transcription, cell proliferation, differentiation, and apoptosis. Store-operated calcium entry (SOCE) is the major pathway in non-excitable cells for extracellular Ca2+ influx across the plasma membrane and is regulated by the content of the intracellular Ca2+ stores. Following store depletion, the Ca2+ sensor in ER, stromal interaction molecule 1 (STIM1) clusters in ER regions close to the plasma membrane and recruits Orai1, which is a Ca2+ selective channel resulting in SOCE. Despite the significant knowledge in understanding STIM1 subcellular distribution dynamics, little is known about the trafficking of Orai1. Our laboratory previously showed that in Xenopus leavis oocytes Orai1 shuttles between plasma membrane and endosomal compartments, and that it internalizes during meiosis. However, the subcellular distribution and trafficking of Orai1 in mammalian cells is not fully understood. In this study we show that at steady state around 46% of Orai1 is on the surface of the plasma membrane of CHO cells, while the remaining 54% localizes intracellularly, suggesting that Orai1 constitutively recycles between the two compartments. Our time lapse imaging shows continuous shuttling of Orai1 to and from the PM with an exocytosis rate T1/2 of around 5 minutes. We also measured the endocytic rate of Orai1 at 5% min-1. To identify the domain within Orai1 required for its trafficking, we generated deletions of either the N- or C-terminus of Orai1. Deletion of N-terminus does not affect Orai1 trafficking to the cell membrane in CHO cells. In contrast deletion of the whole C-terminus (257-301) significantly altered Orai1 trafficking to the cell membrane. Deletion of amino acids 285-301 of Orai1 C-terminus does not have any impact on Orai1 trafficking to PM, suggesting that the information necessary for Orai1 trafficking to PM is located in the segment 257-285 of Orai1 C-terminus. In this study we determined, for the first time, the percentage of Orai1 on PM of mammalian cells and showed through time lapse-imaging that it constitutively recycles between PM and intracellular compartments. Our results also suggest, for the first time, that the amino acid region (257-285) of Orai1 C-terminus is essential for its plasma membrane trafficking.

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/content/papers/10.5339/qfarf.2013.BIOP-0131
2013-11-20
2020-10-25
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