Research ArticleChannel Biology

A calcium-accumulating region, CAR, in the channel Orai1 enhances Ca2+ permeation and SOCE-induced gene transcription

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Sci. Signal.  22 Dec 2015:
Vol. 8, Issue 408, pp. ra131
DOI: 10.1126/scisignal.aab1901

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Enhancing Ca2+ concentration at the pore

Store-operated calcium entry (SOCE) produces local calcium signals that not only refill intracellular calcium stores but also regulate specific downstream signaling events, such as activation of the calcium-dependent transcription factor NFAT. The channel complexes that mediate the calcium influx include the calcium-sensing proteins of the STIM family and the pore-forming subunits of the Orai family. These channels must function in cells that are part of tissues, as well as cells that circulate in the blood stream, thus cells that are exposed to very different concentrations of extracellular calcium. Using molecular dynamics simulations and analysis of mutant proteins in cells, Frischauf et al. identified a region, the calcium-accumulating region (CAR), in Orai1 that enhanced the local concentration of calcium at the entrance to the pore. The importance of CAR was most evident under conditions of low extracellular calcium, as would occur in cells of the skin. NFAT activity was impaired in cells expressing STIM1 and Orai channel proteins with mutations in CAR or that disrupted CAR function. Thus, CAR enables Orai to mediate SOCE-induced calcium signaling even under diverse extracellular calcium concentrations.

Abstract

The Ca2+ release–activated Ca2+ channel mediates Ca2+ 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 Ca2+-sensing protein, and Orai1, a plasma membrane Ca2+ channel. Channels composed of STIM1 and Orai1 mediate Ca2+ influx even at low extracellular Ca2+ concentrations. We investigated whether the activity of Orai1 adapted to different environmental Ca2+ concentrations. We used homology modeling and molecular dynamics simulations to predict the presence of an extracellular Ca2+-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 Ca2+ permeation most efficiently at low external Ca2+ concentrations. Consistent with these results, cells expressing Orai1 CAR mutants exhibited impaired gene expression stimulated by the Ca2+-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 Ca2+-selective ion permeation, enhances the local extracellular Ca2+ concentration to maintain Ca2+-dependent gene regulation even in environments with relatively low Ca2+concentrations.

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