The pro-and antiapoptotic proteins belonging to the B-cell lymphoma-2 (Bcl-2) family exert a critical control over cell-death processes by enabling or counteracting mitochondrial outer membrane permeabilization. Beyond this mitochondrial function, several Bcl-2 family members have emerged as critical modulators of intracellular Ca 2+ homeostasis and dynamics, showing proapoptotic and antiapoptotic functions. Bcl-2 family proteins specifically target several intracellular Ca 2+ -transport systems, including organellar Ca 2+ channels: inositol 1,4,5-trisphosphate receptors (IP 3 Rs) and ryanodine receptors (RyRs), Ca 2+ -release channels mediating Ca 2+ flux from the endoplasmic reticulum, as well as voltage-dependent anion channels (VDACs), which mediate Ca 2+ flux across the mitochondrial outer membrane into the mitochondria. Although the formation of protein complexes between Bcl-2 proteins and these channels has been extensively studied, a major advance during recent years has been elucidating the complex interaction of Bcl-2 proteins with IP 3 Rs. Distinct interaction sites for different Bcl-2 family members were identified in the primary structure of IP 3 Rs. The unique molecular profiles of these Bcl-2 proteins may account for their distinct functional outcomes when bound to IP 3 Rs. Furthermore, Bcl-2 inhibitors used in cancer therapy may affect IP 3 R function as part of their proapoptotic effect and/or as an adverse effect in healthy cells. B-CELL LYMPHOMA-2 (Bcl-2) FAMILY OF PROTEINST he Bcl-2 family of proteins consists of proand antiapoptotic members, which are characterized by the presence of at least one of the four highly conserved α-helical motifs, termed Bcl-2 homology (BH) domains (Adams and Cory 1998). The antiapoptotic family members, such as Bcl-2, Bcl-Xl, and Mcl-1, contain all four BH domains where the BH1, BH2, and BH3 domains form a hydrophobic cleft (Fig. 1A). The hydrophobic cleft is separated from the amino-terminal BH4 domain by an unstructured

Summary Calcium release into the cytosol via the inositol 1,4,5-trisphosphate receptor (IP3R) calcium channel is important for a variety of cellular processes. As a result, impairment or inhibition of this release can result in disease. Recently, mutations in all four domains of the IP3R have been suggested to cause diseases such as ataxia, cancer, and anhidrosis; however, most of these mutations have not been functionally characterized. In this review we summarize the reported mutations, as well as the associated symptoms. Additionally, we use clues from transgenic animals, receptor stoichiometry, and domain location of mutations to speculate on the effects of individual mutations on receptor structure and function and the overall mechanism of disease.

Stromal interaction molecules, STIM1 and STIM2, sense decreases in the endoplasmic reticulum (ER) [Ca2+] ([Ca2+]ER) and cluster in ER–plasma membrane (ER–PM) junctions where they recruit and activate Orai1. While STIM1 responds when [Ca2+]ER is relatively low, STIM2 displays constitutive clustering in the junctions and is suggested to regulate basal Ca2+ entry. The cellular cues that determine STIM2 clustering under basal conditions is not known. By using gene editing to fluorescently tag endogenous STIM2, we report that endogenous STIM2 is constitutively localized in mobile and immobile clusters. The latter associate with ER–PM junctions and recruit Orai1 under basal conditions. Agonist stimulation increases immobile STIM2 clusters, which coordinate recruitment of Orai1 and STIM1 to the junctions. Extended synaptotagmin (E-Syt)2/3 are required for forming the ER–PM junctions, but are not sufficient for STIM2 clustering. Importantly, inositol 1,4,5-triphosphate receptor (IP3R) function and local [Ca2+]ER are the main drivers of immobile STIM2 clusters. Enhancing, or decreasing, IP3R function at ambient [IP3] causes corresponding increase, or attenuation, of immobile STIM2 clusters. We show that immobile STIM2 clusters denote decreases in local [Ca2+]ER mediated by IP3R that is sensed by the STIM2 N terminus. Finally, under basal conditions, ambient PIP2-PLC activity of the cell determines IP3R function, immobilization of STIM2, and basal Ca2+ entry while agonist stimulation augments these processes. Together, our findings reveal that immobilization of STIM2 clusters within ER–PM junctions, a first response to ER-Ca2+ store depletion, is facilitated by the juxtaposition of IP3R and marks a checkpoint for initiation of Ca2+ entry.

Calcium signaling is essential for lymphocyte activation, with genetic disruptions of store-operated calcium (Ca2+) entry resulting in severe immunodeficiency. The inositol 1,4,5-trisphosphate receptor (IP3R), a homo- or heterotetramer of the IP3R1-3 isoforms, amplifies lymphocyte signaling by releasing Ca2+ from endoplasmic reticulum stores following antigen stimulation. Although knockout of all IP3R isoforms in mice causes immunodeficiency, the seeming redundancy of the isoforms is thought to explain the absence of variants in human immunodeficiency. In this study, we identified compound heterozygous variants of ITPR3 (a gene encoding IP3R subtype 3) in two unrelated Caucasian patients presenting with immunodeficiency. To determine whether ITPR3 variants act in a nonredundant manner and disrupt human immune responses, we characterized the Ca2+ signaling capacity, the lymphocyte response, and the clinical phenotype of these patients. We observed disrupted Ca2+ signaling in patient-derived fibroblasts and immune cells, with abnormal proliferation and activation responses following T-cell receptor stimulation. Reconstitution of IP3R3 in IP3R knockout cell lines led to the identification of variants as functional hypomorphs that showed reduced ability to discriminate between homeostatic and induced states, validating a genotype–phenotype link. These results demonstrate a functional link between defective endoplasmic reticulum Ca2+ channels and immunodeficiency and identify IP3Rs as diagnostic targets for patients with specific inborn errors of immunity. These results also extend the known cause of Ca2+-associated immunodeficiency from store-operated entry to impaired Ca2+ mobilization from the endoplasmic reticulum, revealing a broad sensitivity of lymphocytes to genetic defects in Ca2+ signaling.

The G protein-coupled estrogen receptor 1 (GPER, formerly also known as GPR30) modulates many Ca-dependent activities in endothelial cells. However, the underlying mechanisms are poorly understood. We recently reported that GPER acts to prolong cytoplasmic Ca signals by interacting with and promoting inhibitory phosphorylation of the plasma membrane Ca-ATPase. In the present study, we examined the role of GPER activation in modulating store-operated Ca entry (SOCE) via effects on the stromal interaction molecule 1 (STIM1). GPER activation by agonist G-1 reduces the peak but prolongs the plateau of bradykinin-induced Ca signals in primary endothelial cells. G-1 dose-dependently inhibits thapsigargin-induced SOCE measured by the Mn quenching method. GPER heterologous expression reduces SOCE, which is further pronounced by G-1 treatment. Consistently, GPER gene silencing in endothelial cells is associated with an increase in SOCE. Treatment with G-1 reduces puncta formation by STIM1 triggered by the activation of SOCE. The effect of GPER activation to inhibit SOCE is not affected by combined nonphosphorylatable substitutions at serines 486 and 668 on STIM1, but is substantially reduced by similar substitutions at serines 575, 608 and 621. Taken together with our recently reported inhibitory actions of GPER on Ca efflux, the current data contribute to a model in which GPER acts to clamp agonist-induced cytoplasmic Ca signals. Kinetic modeling based on current and reported data is used to estimate the overall effect of GPER activation on point activity of endothelial nitric oxide synthase during the time course of agonist-induced total Ca signals.