The Ca(2+) release-activated Ca(2+) channel mediates Ca(2+) influx in a plethora of cell types, thereby controlling diverse cellular functions. The channel complex is composed of stromal interaction molecule 1 (STIM1), an endoplasmic reticulum Ca(2+)-sensing protein, and Orai1, a plasma membrane Ca(2+) channel. Channels composed of STIM1 and Orai1 mediate Ca(2+) influx even at low extracellular Ca(2+) concentrations. We investigated whether the activity of Orai1 adapted to different environmental Ca(2+) concentrations. We used homology modeling and molecular dynamics simulations to predict the presence of an extracellular Ca(2+)-accumulating region (CAR) at the pore entrance of Orai1. Furthermore, simulations of Orai1 proteins with mutations in CAR, along with live-cell experiments, or simulations and electrophysiological recordings of the channel with transient, electrostatic loop3 interacting with loop1 (the site of CAR) determined that CAR enhanced Ca(2+) permeation most efficiently at low external Ca(2+) concentrations. Consistent with these results, cells expressing Orai1 CAR mutants exhibited impaired gene expression stimulated by the Ca(2+)-activated transcription factor nuclear factor of activated T cells (NFAT). We propose that the Orai1 channel architecture with a close proximity of CAR to the selectivity filter, which enables Ca(2+)-selective ion permeation, enhances the local extracellular Ca(2+) concentration to maintain Ca(2+)-dependent gene regulation even in environments with relatively low Ca(2+)concentrations.

Center for Nanobiology and Structural Biology Institute of Microbiology Academy of Sciences of the Czech Republic Zamek 136 CZ 373 33 Nove Hrady Czech Republic

Department of Biophysics School of Medicine University of Saarland D 66421 Homburg Germany

Faculty of Sciences University of South Bohemia Zamek 136 CZ 373 33 Nove Hrady Czech Republic

Institute for Biophysics of Medical University Graz A 8010 Graz Austria

Institute for Experimental and Clinical Cell Therapy Paracelsus Medical University A 5020 Salzburg Austria

Institute of Biophysics JKU Life Science Center Johannes Kepler University Linz Gruberstrasse 40 4020 Linz Austria

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