Roles of Cholesterol and PtdIns(4,5)P2 in the Regulation of STIM1-Orai1 Channel Function.

Calcium is one of the most prominent second messengers. It is involved in a wide range of functions at the single-cell level but also in modulating regulatory mechanisms in the entire organism. One process mediating calcium signaling involves hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by the phospholipase-C (PLC). Thus, calcium and PtdIns(4,5)P2 are intimately intertwined two second-messenger cascades that often depend on each other. Another relevant lipid associated with calcium signaling is cholesterol. Both PtdIns(4,5)P2 and cholesterol play key roles in the formation and maintenance of specialized signaling nanodomains known as lipid rafts. Lipid rafts are particularly important in calcium signaling by concentrating and localizing calcium channels such as the Orai1 channel. Depletion of internal calcium stores is initiated by the production of inositol-1,4,5-trisphosphate (IP3). Calcium depletion from the ER induces the oligomerization of STIM1, which binds Orai1 and initiates calcium influx into the cell. In the present review, we analyzed the complex interactions between cholesterol, PtdIns(4,5)P2, and the complex formed by the Orai1 channel and the signaling molecule STIM1. We explore some of the complex mechanisms governing calcium homeostasis and phospholipid metabolism, as well as the interaction between these two apparently independent signaling cascades.

Cholesterol modulates the cellular localization of Orai1 channels and its disposition among membrane domains.

Store Operated Calcium Entry (SOCE) is one of the most important mechanisms for calcium mobilization in to the cell. Two main proteins sustain SOCE: STIM1 that acts as the calcium sensor in the endoplasmic reticulum (ER) and Orai1 responsible for calcium influx upon depletion of ER. There are many studies indicating that SOCE is modulated by the cholesterol content of the plasma membrane (PM). However, a myriad of questions remain unanswered concerning the precise molecular mechanism by which cholesterol modulates SOCE. In the present study we found that reducing PM cholesterol results in the internalization of Orai1 channels, which can be prevented by overexpressing caveolin 1 (Cav1). Furthermore, Cav1 and Orai1 associate upon SOCE activation as revealed by FRET and coimmunoprecipitation assays. The effects of reducing cholesterol were not limited to an increased rate of Orai1 internalization, but also, affects the lateral movement of Orai1, inducing movement in a linear pattern (unobstructed diffusion) opposite to basal cholesterol conditions were most of Orai1 channels moves in a confined space, as assessed by Fluorescence Correlation Spectroscopy, Cav1 overexpression inhibited these alterations maintaining Orai1 into a confined and partially confined movement. These results not only highlight the complex effect of cholesterol regulation on SOCE, but also indicate a direct regulatory effect on Orai1 localization and compartmentalization by this lipid.

A cholesterol-binding domain in STIM1 modulates STIM1-Orai1 physical and functional interactions.

STIM1 and Orai1 are the main components of a widely conserved Calcium influx pathway known as store-operated calcium entry (SOCE). STIM1 is a calcium sensor, which oligomerizes and activates Orai channels when calcium levels drop inside the endoplasmic reticulum (ER). The series of molecular rearrangements that STIM1 undergoes until final activation of Orai1 require the direct exposure of the STIM1 domain known as SOAR (Stim Orai Activating Region). In addition to these complex molecular rearrangements, other constituents like lipids at the plasma membrane, play critical roles orchestrating SOCE. PI(4,5)P2 and enriched cholesterol microdomains have been shown as important signaling platforms that recruit the SOCE machinery in steps previous to Orai1 activation. However, little is known about the molecular role of cholesterol once SOCE is activated. In this study we provide clear evidence that STIM1 has a cholesterol-binding domain located inside the SOAR region and modulates Orai1 channels. We demonstrate a functional association of STIM1 and SOAR to cholesterol, indicating a close proximity of SOAR to the inner layer of the plasma membrane. In contrast, the depletion of cholesterol induces the SOAR detachment from the plasma membrane and enhances its association to Orai1. These results are recapitulated with full length STIM1.