STIM1 (stromal interaction molecule 1) and Orai proteins are the essential components of Ca(2+) release-activated Ca(2+) (CRAC) channels. We focused on the role of cholesterol in the regulation of STIM1-mediated Orai1 currents. Chemically induced cholesterol depletion enhanced store-operated Ca(2+) entry (SOCE) and Orai1 currents. Furthermore, cholesterol depletion in mucosal-type mast cells augmented endogenous CRAC currents, which were associated with increased degranulation, a process that requires calcium influx. Single point mutations in the Orai1 amino terminus that would be expected to abolish cholesterol binding enhanced SOCE to a similar extent as did cholesterol depletion. The increase in Orai1 activity in cells expressing these cholesterol-binding-deficient mutants occurred without affecting the amount in the plasma membrane or the coupling of STIM1 to Orai1. We detected cholesterol binding to an Orai1 amino-terminal fragment in vitro and to full-length Orai1 in cells. Thus, our data showed that Orai1 senses the amount of cholesterol in the plasma membrane and that the interaction of Orai1 with cholesterol inhibits its activity, thereby limiting SOCE.

Center for Nanobiology and Structural Biology Institute of Microbiology Academy of Sciences of the Czech Republic CZ 373 33 Nové Hrady Czech Republic

Department of Physiology and Pharmacology Schulich School of Medicine and Dentistry University of Western Ontario London Ontario N6A 5C1 Canada

Institute of Biophysics Johannes Kepler University of Linz Gruberstrasse 40 4020 Linz Austria

Institute of Biophysics Medical University of Graz Harrachgasse 21 4 8010 Graz Austria

Princess Margaret Cancer Centre and Department of Medical Biophysics University of Toronto Toronto Ontario M5G 1L7 Canada

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