The Ca²⁺ sensor STIM1 and the Ca²⁺ channel Orai1 that form the store-operated Ca²⁺ (SOC) channel complex are key targets for drug development. Selective SOC inhibitors are currently undergoing clinical evaluation for the treatment of auto-immune and inflammatory responses and are also deemed promising anti-neoplastic agents since SOC channels are linked with enhanced cancer cell progression. Here, we describe an investigation of the site of binding of the selective inhibitor Synta66 to the SOC channel Orai1 using docking and molecular dynamics simulations, and live cell recordings. Synta66 binding was localized to the extracellular site close to the transmembrane (TM)1 and TM3 helices and the extracellular loop segments, which, importantly, are adjacent to the Orai1-selectivity filter. Synta66-sensitivity of the Orai1 pore was, in fact, diminished by both Orai1 mutations affecting Ca²⁺ selectivity and permeation of Na⁺ in the absence of Ca²⁺. Synta66 also efficiently blocked SOC in three glioblastoma cell lines but failed to interfere with cell viability, division and migration. These experiments provide new structural and functional insights into selective drug inhibition of the Orai1 Ca²⁺ channel by a high-affinity pore blocker.

Cell survival and normal cell function require a highly coordinated and precise regulation of basal cytosolic Ca²⁺ concentrations. The primary source of Ca²⁺ entry into the cell is mediated by the Ca²⁺ release-activated Ca²⁺ (CRAC) channel. Its action is stimulated in response to internal Ca²⁺ store depletion. The fundamental constituents of CRAC channels are the Ca²⁺ sensor, stromal interaction molecule 1 (STIM1) anchored in the endoplasmic reticulum, and a highly Ca²⁺-selective pore-forming subunit Orai1 in the plasma membrane. The precise nature of the Orai1 pore opening is currently a topic of intensive research. This review describes how Orai1 gating checkpoints in the middle and cytosolic extended transmembrane regions act together in a concerted manner to ensure an opening-permissive Orai1 channel conformation. In this context, we highlight the effects of the currently known multitude of Orai1 mutations, which led to the identification of a series of gating checkpoints and the determination of their role in diverse steps of the Orai1 activation cascade. The synergistic action of these gating checkpoints maintains an intact pore geometry, settles STIM1 coupling, and governs pore opening. We describe the current knowledge on Orai1 channel gating mechanisms and summarize still open questions of the STIM1–Orai1 machinery.

The initial activation step in the gating of ubiquitously expressed Orai1 calcium (Ca²⁺) ion channels represents the activation of the Ca²⁺-sensor protein STIM1 upon Ca²⁺ store depletion of the endoplasmic reticulum. Previous studies using constitutively active Orai1 mutants gave rise to, but did not directly test, the hypothesis that STIM1-mediated Orai1 pore opening is accompanied by a global conformational change of all Orai transmembrane domain (TM) helices within the channel complex. We prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that these locally induced global, opening-permissive TM motions are indispensable for pore opening and require clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in the middle and cytosolic extended TM domain regions. Our findings are based on a library of double point mutants that contain each one loss-of-function with one gain-of-function point mutation in a series of possible combinations. We demonstrated that an array of loss-of-function mutations are dominant over most gain-of-function mutations within the same as well as of an adjacent Orai subunit. We further identified inter- and intramolecular salt-bridge interactions of Orai subunits as a core element of an opening-permissive Orai channel architecture. Collectively, clearance and synergistic action of all these gating checkpoints are required to allow STIM1 coupling and Orai1 pore opening. Our results unravel novel insights in the preconditions of the unique fingerprint of CRAC channel activation, provide a valuable source for future structural resolutions, and help to understand the molecular basis of disease-causing mutations.

Stromal interaction molecule 1 (STIM1) is a ubiquitously expressed Ca²⁺ sensor protein that induces permeation of Orai Ca²⁺ channels upon endoplasmic reticulum Ca²⁺-store depletion. A drop in luminal Ca²⁺ causes partial unfolding of the N-terminal STIM1 domains and thus initial STIM1 activation. We compared the STIM1 structure upon Ca²⁺ depletion from our molecular dynamics (MD) simulations with a recent 2D NMR structure. Simulation- and structure-based results showed unfolding of two α-helices in the canonical and in the non-canonical EF-hand. Further, we structurally and functionally evaluated mutations in the non-canonical EF-hand that have been shown to cause tubular aggregate myopathy. We found these mutations to cause full constitutive activation of Ca²⁺-release-activated Ca²⁺ currents (I_CRAC) and to promote autophagic processes. Specifically, heterologously expressed STIM1 mutations in the non-canonical EF-hand promoted translocation of the autophagy transcription factors microphthalmia-associated transcription factor (MITF) and transcription factor EB (TFEB) into the nucleus. These STIM1 mutations additionally stimulated an enhanced production of autophagosomes. In summary, mutations in STIM1 that cause structural unfolding promoted Ca²⁺ down-stream activation of autophagic processes.

Ca²⁺ ion channels are critical in a variety of physiological events, including cell growth, differentiation, gene transcription and apoptosis. One such essential entry pathway for calcium into the cell is the Ca²⁺ release-activated Ca²⁺ (CRAC) channel. It consists of the Ca²⁺ sensing protein, stromal interaction molecule 1 (STIM1) located in the endoplasmic reticulum (ER) and a Ca²⁺ ion channel Orai in the plasma membrane. The Orai channel family includes three homologues Orai1, Orai2 and Orai3. While Orai1 is the “classical” Ca²⁺ ion channel within the CRAC channel complex and plays a universal role in the human body, there is increasing evidence that Orai2 and Orai3 are important in specific physiological and pathophysiological processes. This makes them an attractive target in drug discovery, but requires a detailed understanding of the three Orai channels and, in particular, their differences. Orai channel activation is initiated via Ca²⁺ store depletion, which is sensed by STIM1 proteins, and induces their conformational change and oligomerization. Upon STIM1 coupling, Orai channels activate to allow Ca²⁺ permeation into the cell. While this activation mechanism is comparable among the isoforms, they differ by a number of functional and structural properties due to non-conserved regions in their sequences. In this review, we summarize the knowledge as well as open questions in our current understanding of the three isoforms in terms of their structure/function relationship, downstream signaling and physiology as well as pathophysiology.

Ca²⁺ ions play a variety of roles in the human body as well as within a single cell. Cellular Ca²⁺ signal transduction processes are governed by Ca²⁺ sensing and Ca²⁺ transporting proteins. In this review, we discuss the Ca²⁺ and the Ca²⁺-sensing ion channels with particular focus on the structure-function relationship of the Ca²⁺ release-activated Ca²⁺ (CRAC) ion channel, the Ca²⁺-activated K⁺ (K_Ca2+) ion channels, and their modulation via other cellular components. Moreover, we highlight their roles in healthy signaling processes as well as in disease with a special focus on cancer. As K_Ca2+ channels are activated via elevations of intracellular Ca²⁺ levels, we summarize the current knowledge on the action mechanisms of the interplay of CRAC and K_Ca2+ ion channels and their role in cancer cell development.