Transmembrane helix connectivity in Orai1 controls two gates for calcium-dependent transcription

Orai1 enables Ca2+ influx into cells in response to ER Ca2+ depletion, resulting in various Ca2+-dependent signaling events, including the stimulation of transcription factors such as NFAT. Frischauf et al. (see also the Focus by Muallem et al.) characterized various Orai1 mutants that they identified from cancer databases using cBioPortal and an Orai1 mutant associated with myopathy. The constitutively active mutants not only activated NFA but also stimulated mitophagy and autophagy, processes that can contribute to tumor progression. A combination of biochemical, electrophysiological, and structural analyses revealed how Orai1 is gated, how this Ca2+ channel can be in a partially active state, and how constitutively activating mutations result in increased Ca2+ influx. Because Orai1 is found in diverse cell types, these results have broad implications for Orai1-mediated Ca2+ signaling in homeostasis and disease.The channel Orai1 requires Ca2+ store depletion in the endoplasmic reticulum and an interaction with the Ca2+ sensor STIM1 to mediate Ca2+ signaling. Alterations in Orai1-mediated Ca2+ influx have been linked to several pathological conditions including immunodeficiency, tubular myopathy, and cancer. We screened large-scale cancer genomics data sets for dysfunctional Orai1 mutants. Five of the identified Orai1 mutations resulted in constitutively active gating and transcriptional activation. Our analysis showed that certain Orai1 mutations were clustered in the transmembrane 2 helix surrounding the pore, which is a trigger site for Orai1 channel gating. Analysis of the constitutively open Orai1 mutant channels revealed two fundamental gates that enabled Ca2+ influx: Arginine side chains were displaced so they no longer blocked the pore, and a chain of water molecules formed in the hydrophobic pore region. Together, these results enabled us to identify a cluster of Orai1 mutations that trigger Ca2+ permeation associated with gene transcription and provide a gating mechanism for Orai1.