Faculty Bibliography

It is widely assumed that inositol trisphosphate (IP₃) and ryanodine (Ry) receptors share the same Ca²⁺ pool in central mammalian neurons. We now demonstrate that in hippocampal CA1 pyramidal neurons IP₃- and Ry-receptors are associated with two functionally distinct intracellular Ca²⁺ stores, respectively. While the IP₃-sensitive Ca²⁺ store refilling requires Orai2 channels, Ry-sensitive Ca²⁺ store refilling involves voltage-gated Ca²⁺ channels (VGCCs). Our findings have direct implications for the understanding of function and plasticity in these central mammalian neurons.

Pathogenic Th17 cells play important roles in many autoimmune and inflammatory diseases. Their function depends on T cell receptor (TCR) signaling and cytokines that activate signal transducer and activator of transcription 3 (STAT3). TCR engagement activates stromal interaction molecule 1 (STIM1) and calcium (Ca²⁺) influx through Ca²⁺-release-activated Ca²⁺ (CRAC) channels. Here, we show that abolishing STIM1 and Ca²⁺ influx in T cells expressing a hyperactive form of STAT3 (STAT3C) attenuates pathogenic Th17 cell function and inflammation associated with STAT3C expression. Deletion of STIM1 in pathogenic Th17 cells reduces the expression of genes required for mitochondrial function and oxidative phosphorylation (OXPHOS) but enhances reactive oxygen species (ROS) production. STIM1 deletion or inhibition of OXPHOS is associated with a non-pathogenic Th17 gene expression signature and impaired pathogenic Th17 cell function. Our findings establish Ca²⁺ influx as a critical regulator of mitochondrial function and oxidative stress in pathogenic Th17 cell-mediated multiorgan inflammation.

Store-operated Ca²⁺ entry (SOCE) channels are highly selective Ca²⁺ channels activated by the endoplasmic reticulum (ER) sensors STIM1 and STIM2. Their direct interaction with the pore-forming plasma membrane ORAI proteins (ORAI1, ORAI2, and ORAI3) leads to sustained Ca²⁺ fluxes that are critical for many cellular functions. Mutations in the human ORAI1 gene result in immunodeficiency, anhidrotic ectodermal dysplasia, and enamel defects. In our investigation of the role of ORAI proteins in enamel, we identified enamel defects in a patient with an ORAI1 null mutation. Targeted deletion of the Orai1 gene in mice showed enamel defects and reduced SOCE in isolated enamel cells. However, Orai2^-/- mice showed normal enamel despite having increased SOCE in the enamel cells. Knockdown experiments in the enamel cell line LS8 suggested that ORAI2 and ORAI3 modulated ORAI1 function, with ORAI1 and ORAI2 being the main contributors to SOCE. ORAI1-deficient LS8 cells showed altered mitochondrial respiration with increased oxygen consumption rate and ATP, which was associated with altered redox status and enhanced ER Ca²⁺ uptake, likely due to S-glutathionylation of SERCA pumps. Our findings demonstrate an important role of ORAI1 in Ca²⁺ influx in enamel cells and establish a link between SOCE, mitochondrial function, and redox homeostasis.

T regulatory (Treg) cells maintain immunological tolerance and organ homeostasis. Activated Treg cells differentiate into effector Treg subsets that acquire tissue-specific functions. Ca²⁺ influx via Ca²⁺ release-activated Ca²⁺ (CRAC) channels formed by STIM and ORAI proteins is required for the thymic development of Treg cells, but its function in mature Treg cells remains unclear. Here we show that deletion of Stim1 and Stim2 genes in mature Treg cells abolishes Ca²⁺ signaling and prevents their differentiation into follicular Treg and tissue-resident Treg cells. Transcriptional profiling of STIM1/STIM2-deficient Treg cells reveals that Ca²⁺ signaling regulates transcription factors and signaling pathways that control the identity and effector differentiation of Treg cells. In the absence of STIM1/STIM2 in Treg cells, mice develop a broad spectrum of autoantibodies and fatal multiorgan inflammation. Our findings establish a critical role of CRAC channels in controlling lineage identity and effector functions of Treg cells.

Ion channels facilitate the movement of ions across the plasma and organellar membranes. A recent symposium brought together scientists who study ion channels and transporters in immune cells, which highlighted advances in this emerging field and served to chart new avenues for investigating the roles of ion channels in immunity.

Ca²⁺ release-activated Ca²⁺ (CRAC) channels are intimately linked with health and disease. The gene encoding the CRAC channel, ORAI1, was discovered in part by genetic analysis of patients with abolished CRAC channel function. And patients with autosomal recessive loss-of-function (LOF) mutations in ORAI1 and its activator stromal interaction molecule 1 (STIM1) that abolish CRAC channel function and store-operated Ca²⁺ entry (SOCE) define essential functions of CRAC channels in health and disease. Conversely, gain-of-function (GOF) mutations in ORAI1 and STIM1 are associated with tubular aggregate myopathy (TAM) and Stormorken syndrome due to constitutive CRAC channel activation. In addition, genetically engineered animal models of ORAI and STIM function have provided important insights into the physiological and pathophysiological roles of CRAC channels in cell types and organs beyond those affected in human patients. The picture emerging from this body of work shows CRAC channels as important regulators of cell function in many tissues, and as potential drug targets for the treatment of autoimmune and inflammatory disorders.

The earliest intracellular signals that occur after T cell activation are local, subsecond Ca²⁺ microdomains. Here, we identified a Ca²⁺ entry component involved in Ca²⁺ microdomain formation in both unstimulated and stimulated T cells. In unstimulated T cells, spontaneously generated small Ca²⁺ microdomains required ORAI1, STIM1, and STIM2. Super-resolution microscopy of unstimulated T cells identified a circular subplasmalemmal region with a diameter of about 300 nm with preformed patches of colocalized ORAI1, ryanodine receptors (RYRs), and STIM1. Preformed complexes of STIM1 and ORAI1 in unstimulated cells were confirmed by coimmunoprecipitation and Förster resonance energy transfer studies. Furthermore, within the first second after T cell receptor (TCR) stimulation, the number of Ca²⁺ microdomains increased in the subplasmalemmal space, an effect that required ORAI1, STIM2, RYR1, and the Ca²⁺ mobilizing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate). These results indicate that preformed clusters of STIM and ORAI1 enable local Ca²⁺ entry events in unstimulated cells. Upon TCR activation, NAADP-evoked Ca²⁺ release through RYR1, in coordination with Ca²⁺ entry through ORAI1 and STIM, rapidly increases the number of Ca²⁺ microdomains, thereby initiating spread of Ca²⁺ signals deeper into the cytoplasm to promote full T cell activation.

BACKGROUND: Store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels is an essential signaling pathway in many cell types. CRAC channels are formed by ORAI1, ORAI2 and ORAI3 proteins and activated by stromal interaction molecule 1 (STIM1) and STIM2. Mutations in ORAI1 and STIM1 genes that abolish SOCE cause a combined immunodeficiency (CID) syndrome that is accompanied by autoimmunity and non-immunological symptoms. OBJECTIVE: Molecular and immunological analysis of patients with CID, anhidrosis and ectodermal dysplasia of unknown etiology. METHODS: DNA sequencing of ORAI1 gene, modeling of mutations on ORAI1 crystal structure, analysis of ORAI1 mRNA and protein expression, measurements of SOCE, immunological analysis of peripheral blood lymphocyte populations by flow cytometry, histological and ultrastructural analysis of patient tissues. RESULTS: We identified 3 novel autosomal recessive mutations in ORAI1 in unrelated kindreds with CID, autoimmunity, ectodermal dysplasia with anhidrosis (EDA) and muscular dysplasia. The patients were homozygous for p.V181SfsX8, p.L194P and p.G98R mutations in the ORAI1 gene that suppressed ORAI1 protein expression and SOCE in the patients' lymphocytes and fibroblasts. Besides impaired T cell cytokine production, ORAI1 mutations were associated with strongly reduced numbers of invariant natural killer (iNKT) and regulatory T (Treg) cells, and altered composition of gammadelta T cell and NK cell subsets. CONCLUSION: ORAI1 null mutations are associated with reduced numbers of iNKT and Treg cells that likely contribute to the patients' immunodeficiency and autoimmunity. ORAI1 deficient patients suffer from dental enamel defects and anhidrosis representing a new form of anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) that is distinct from previously reported patients with EDA-ID due to mutations in the NF-kB signaling pathway (IKBKG and NFKBIA).