Bidirectional regulation of calcium release–activated calcium (CRAC) channel by SARAF

Store-operated calcium entry (SOCE) through the Ca2+ release–activated Ca2+ (CRAC) channel is a central mechanism by which cells generate Ca2+ signals and mediate Ca2+-dependent gene expression. The molecular basis for CRAC channel regulation by the SOCE-associated regulatory factor (SARAF) remained insufficiently understood. Here we found that following ER Ca2+ depletion, SARAF facilitates a conformational change in the ER Ca2+ sensor STIM1 that relieves an activation constraint enforced by the STIM1 inactivation domain (ID; aa 475–483) and promotes initial activation of STIM1, its translocation to ER–plasma membrane junctions, and coupling to Orai1 channels. Following intracellular Ca2+ rise, cooperation between SARAF and the STIM1 ID controls CRAC channel slow Ca2+-dependent inactivation. We further show that in T lymphocytes, SARAF is required for proper T cell receptor evoked transcription. Taking all these data together, we uncover a dual regulatory role for SARAF during both activation and inactivation of CRAC channels and show that SARAF fine-tunes intracellular Ca2+ responses and downstream gene expression in cells.

Physiological Functions of CRAC Channels

Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ signaling pathway that is evolutionarily conserved across eukaryotes. SOCE is triggered physiologically when the endoplasmic reticulum (ER) Ca2+ stores are emptied through activation of inositol-1,4,5-trisphosphate receptors. SOCE is mediated by the Ca2+ release-activated Ca2+ (CRAC) channels, which are highly Ca2+ selective. Upon store depletion, the ER Ca2+-sensing STIM proteins aggregate and gain extended conformations spanning the ER-plasma membrane junctional space to bind and activate Orai, the pore-forming proteins of hexameric CRAC channels. In recent years, studies on STIM and Orai tissue-specific knockout mice and gain- and loss-of-function mutations in humans have shed light on the physiological functions of SOCE in various tissues. Here, we describe recent findings on the composition of native CRAC channels and their physiological functions in immune, muscle, secretory, and neuronal systems to draw lessons from transgenic mice and human diseases caused by altered CRAC channel activity. Expected final online publication date for the Annual Review of Physiology, Volume 84 is February 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Immune Checkpoint Inhibitors Regulate K+ Channel Activity in Cytotoxic T Lymphocytes of Head and Neck Cancer Patients

Programmed death receptor-1 (PD-1) and its ligand (PD-L1) interaction negatively regulates T cell function in head and neck squamous cell carcinoma (HNSCC). Overexpression of PD-1 reduces intracellular Ca2+ fluxes, and thereby T cell effector functions. In HNSCC patients, PD-1 blockade increases KCa3.1 and Kv1.3 activity along with Ca2+ signaling and mobility in CD8+ peripheral blood T cells (PBTs). The mechanism by which PD-L1/PD-1 interaction regulates ion channel function is not known. We investigated the effects of blocking PD-1 and PD-L1 on ion channel functions and intracellular Ca2+ signaling in CD8+ PBTs of HNSCC patients and healthy donors (HDs) using single-cell electrophysiology and live microscopy. Anti-PD-1 and anti-PD-L1 antibodies increase KCa3.1 and Kv1.3 function in CD8+ PBTs of HNSCC patients. Anti-PD-1 treatment increases Ca2+ fluxes in a subset of HSNCC patients. In CD8+ PBTs of HDs, exposure to PD-L1 reduces KCa3.1 activity and Ca2+ signaling, which were restored by anti-PD-1 treatment. The PD-L1-induced inhibition of KCa3.1 channels was rescued by the intracellular application of the PI3 kinase modulator phosphatidylinositol 3-phosphate (PI3P) in patch-clamp experiments. In HNSCC CD8+ PBTs, anti-PD-1 treatment did not affect the expression of KCa3.1, Kv1.3, Ca2+ release activated Ca2+ (CRAC) channels, and markers of cell activation (CD69) and exhaustion (LAG-3 and TIM-3). Our data show that immune checkpoint blockade improves T cell function by increasing KCa3.1 and Kv1.3 channel activity in HNSCC patients.

Regulation of Store-Operated Ca2+ Entry by SARAF

Calcium (Ca2+) signaling plays a dichotomous role in cellular biology, controlling cell survival and proliferation on the one hand and cellular toxicity and cell death on the other. Store-operated Ca2+ entry (SOCE) by CRAC channels represents a major pathway for Ca2+ entry in non-excitable cells. The CRAC channel has two key components, the endoplasmic reticulum Ca2+ sensor stromal interaction molecule (STIM) and the plasma-membrane Ca2+ channel Orai. Physical coupling between STIM and Orai opens the CRAC channel and the resulting Ca2+ flux is regulated by a negative feedback mechanism of slow Ca2+ dependent inactivation (SCDI). The identification of the SOCE-associated regulatory factor (SARAF) and investigations of its role in SCDI have led to new functional and molecular insights into how SOCE is controlled. In this review, we provide an overview of the functional and molecular mechanisms underlying SCDI and discuss how the interaction between SARAF, STIM1, and Orai1 shapes Ca2+ signaling in cells.

S-acylation of Orai1 regulates store-operated Ca2+ entry

ABSTRACT Store-operated Ca2+ entry is a central component of intracellular Ca2+ signaling pathways. The Ca2+ release-activated channel (CRAC) mediates store-operated Ca2+ entry in many different cell types. The CRAC channel is composed of the plasma membrane (PM)-localized Orai1 channel and endoplasmic reticulum (ER)-localized STIM1 Ca2+ sensor. Upon ER Ca2+ store depletion, Orai1 and STIM1 form complexes at ER–PM junctions, leading to the formation of activated CRAC channels. Although the importance of CRAC channels is well described, the underlying mechanisms that regulate the recruitment of Orai1 to ER–PM junctions are not fully understood. Here, we describe the rapid and transient S-acylation of Orai1. Using biochemical approaches, we show that Orai1 is rapidly S-acylated at cysteine 143 upon ER Ca2+ store depletion. Importantly, S-acylation of cysteine 143 is required for Orai1-mediated Ca2+ entry and recruitment to STIM1 puncta. We conclude that store depletion-induced S-acylation of Orai1 is necessary for recruitment to ER–PM junctions, subsequent binding to STIM1 and channel activation.

Regulation of Interorganellar Ca2+ Transfer and NFAT Activation by the Mitochondrial Ca2+ Uniporter

Mitochondrial Ca2+ uptake is crucial for coupling receptor stimulation to cellular bioenergetics. Further, Ca2+ uptake by respiring mitochondria prevents Ca2+-dependent inactivation (CDI) of store-operated Ca2+ release-activated Ca2+ (CRAC) channels and inhibits Ca2+ extrusion to sustain cytosolic Ca2+ signaling. However, how Ca2+ uptake by the mitochondrial Ca2+ uniporter (MCU) shapes receptor-evoked interorganellar Ca2+ signaling is unknown. Here, we generated several cell lines with MCU-knockout (MCU-KO) as well as tissue-specific MCU-knockdown mice. We show that mitochondrial depolarization, but not MCU-KO, inhibits store-operated Ca2+ entry (SOCE). Paradoxically, despite enhancing Ca2+ extrusion and promoting CRAC channel CDI, MCU-KO increased cytosolic Ca2+ in response to store depletion. Further, physiological agonist stimulation in MCU-KO cells led to enhanced frequency of cytosolic Ca2+ oscillations, endoplasmic reticulum Ca2+ refilling, NFAT nuclear translocation and proliferation. However, MCU-KO did not affect inositol-1,4,5-trisphosphate receptor activity. Mathematical modeling supports that MCU-KO enhances cytosolic Ca2+, despite limiting CRAC channel activity.

The Orai Pore Opening Mechanism

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.

Store-Operated Ca2+ Channels: Mechanism, Function, Pharmacology, and Therapeutic Targets

Calcium (Ca2+) release–activated Ca2+ (CRAC) channels are a major route for Ca2+ entry in eukaryotic cells. These channels are store operated, opening when the endoplasmic reticulum (ER) is depleted of Ca2+, and are composed of the ER Ca2+ sensor protein STIM and the pore-forming plasma membrane subunit Orai. Recent years have heralded major strides in our understanding of the structure, gating, and function of the channels. Loss-of-function and gain-of-function mutants combined with RNAi knockdown strategies have revealed important roles for the channel in numerous human diseases, making the channel a clinically relevant target. Drugs targeting the channels generally lack specificity or exhibit poor efficacy in animal models. However, the landscape is changing, and CRAC channel blockers are now entering clinical trials. Here, we describe the key molecular and biological features of CRAC channels, consider various diseases associated with aberrant channel activity, and discuss targeting of the channels from a therapeutic perspective.

Abstract 14278: Microglial Calcium Activity During Ischemic Stroke

Introduction: Ischemic stroke triggers waves of propagating action potentials followed by a loss of homeostatic ion gradients, known as cortical spreading depolarizations (SD). Our data indicate that microglia respond to SD by raising intracellular Ca 2+ , triggering a release of proinflammatory cytokines that may exacerbate post-stroke morbidity. Hypothesis: Inhibiting calcium release-activated calcium (CRAC) channels may block microglial Ca2+ influx and activation to potentially provide therapeutic benefit in ischemic stroke. Methods: We generated a mouse line expressing the Ca2+ indicator GCaMP5 in cortical microglia. Using two-photon microscopy, we imaged microglia in vivo following physical stroke via middle cerebral artery occlusion (MCAo) or chemical stroke via 1M KCl solution application. Controls were compared to mice treated with lipopolysaccharide (LPS) to mimic the inflammation of ischemic stroke. To study CRAC channels as a therapeutic target, we administered CM-EX-137, a CRAC channel blocker, and compared Ca2+ activity to controls. Results: We identified periodical Ca2+ waves in cortical microglia following MCAo (Fig. 1A), or localized KCl application (Fig. 1B-E). Further, when compared to controls, LPS-exposed mice expressed significantly greater microglial Ca2+ activity during KCl-triggered SD (Fig. 1C). Additionally, administration of CM-EX-137 effectively abolished the Ca2+ signals in the microglia and propagation of SD upon application of KCl (Fig. 1E). Conclusions: Blocking the Ca2+ influx into microglia after ischemic stroke may decrease the frequency of SD and reduce microglial activation, potentially leading to smaller stroke volumes and improved clinical outcomes. Figure 1: Microglial Ca 2+ after SD. (A) Ca 2+ transients in microglia after MCAo. (B) KCl-induced Ca 2+ activity in naïve microglia. (C) KCl-induced Ca 2+ activity after LPS. (D, E) KCl-induced Ca 2+ wave after (D) vehicle or (E) CM-EX-137 administration.