Cholesterol regulates Orai currents via an N-terminal cholesterol binding motif

STIM1 and Orai1 have been elucidated as sufficient to reconstitute the most prominent and best characterized Ca2+ entry into the cell. Recently, a series of additional proteins, like SARAF, junctate, CRACR2A have been reported to regulate Orai currents. As STIM1 and Orai1 have already been reported to locate in lipid rafts, which contain increased amount of cholesterol, I plan to investigate the potential role of cholesterol in Orai regulation. My initial experiments revealed an almost doubled enhancement of STIM1-mediated Orai1 currents upon chemically induced cholesterol depletion. Upon a screen of the primary sequence of Orai I discovered a cholesterol binding domain in the N-terminal conserved region that perfectly matches with the cholesterol recognition/interaction amino acid consensus (CRAC) motif and fulfills the criterion to be located near the plasma membrane. Point mutations, that have been reported to decrease cholesterol sequestration of CRAC motifs of other proteins, revealed also an increase in the Ca2+ currents of an Orai CRAC motif mutant in accordance with results obtained by cholesterol depleting drugs. The fact that CRAC motifs should form a-helices, could be confirmed by CD spectra displaying that an Orai1 N-terminal peptide (aa 71-95) forms a mixture of a -helical and random coil structure. Further preliminary intrinsic fluorescence measurements of this CRAC-motif containing peptide displayed an increased intensity as well as a shift in the maximal emission wavelength in the presence of cholesterol compared to its absence, indicating a more buried conformation in the presence of cholesterol compared to its absence. Based on these data, I aim to investigate the role of cholesterol by Ca2+ imaging and electrophysiological studies together with biochemical assays evaluating direct cholesterol binding to the N-terminus of Orai1 as well as structural resolution studies by CD and/or NMR. Moreover, these studies will be complemented by investigations of the role of cholesterol in RBL and LNCaP cell lines endogenously exhibiting CRAC currents. In summary these investigations will reveal insight into the role of cholesterol in Orai regulation and resolve the cholesterol binding pocket structurally.

Calcium (Ca2+) ions play an important role in the functions of the immune system. One main Ca2+ entry pathway are the so-called Ca2+-release-activated Ca2+ (CRAC) ion pores located in the cell membrane. These Ca2+ ion channels are constituted by the two molecular key players STIM1 and Orai1. In the course of this FWF-project a novel concept of cholesterol- mediated regulation of CRAC channels has been published in the scientific peer-reviewed journal Science Signaling. Here, it has been proposed for the first time, that cholesterol reduces the transport of calcium ions via Orai1 into the cell. These findings might explain, why patients with a defect in the synthesis of cholesterol suffer more from allergies. Cholesterol is an important building block of the cell membrane of humans and animals. The Austrian research group together with an international team, reduced cholesterol chemically in the cell membrane of immune- and kidney cells and consequently investigated the activity of the Orai1 Ca2+-ion-pores in the cell membrane. Typically, these ion pores enable only Ca2+ entry into the cell, when it runs short there. We have shown, that the reduction in cholesterol levels in the cell membrane enhances the activity of the Ca2+ pores which leads to a higher Ca2+ entry into the cell. Followingly, mast cells (these are specific immune cells responsible for allergic reactions) release allergy producing substances, such as histamines. These processes might explain, why patients with a metabolic disorder namely Smith- Lemli-Opitz syndrome tend to suffer more from allergies. These patients contain a mutated enzyme which causes a defect cholesterol synthesis. Hence, these patients have very low cholesterol in the cell membrane. Another research group has discovered that these patients display an enhanced release of 10allergy producing substances such as histamine. Cholesterol inhibits calcium entry into the cell upon direct contact with the Orai1 ion pore. Mutations within Orai1, that impair cholesterol binding, induce also an enhanced Ca2+ entry into the cell, as the researcher in Linz have discovered together with two other research groups in Graz and Toronto (Canada).