A severe defect in CRAC Ca2+ channel activation and altered K+ channel gating in T cells from immunodeficient patients

Engagement of the TCR triggers sustained Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels, which helps drive gene expression underlying the T cell response to pathogens. The identity and activation mechanism of CRAC channels at a molecular level are unknown. We have analyzed ion channel expression and function in T cells from SCID patients which display 1–2% of the normal level of Ca2+ influx and severely impaired T cell activation. The lack of Ca2+ influx is not due to deficient regulation of Ca2+ stores or expression of several genes implicated in controlling Ca2+ entry in lymphocytes (kcna3/Kv1.3, kcnn4/IKCa1, trpc1, trpc3, trpv6, stim1). Instead, electrophysiologic measurements show that the influx defect is due to a nearly complete absence of functional CRAC channels. The lack of CRAC channel activity is correlated with diminished voltage sensitivity and slowed activation kinetics of the voltage-dependent Kv1.3 channel. These results demonstrate that CRAC channels provide the major, if not sole, pathway for Ca2+ entry activated by the TCR in human T cells. They also offer evidence for a functional link between CRAC and Kv1.3 channels, and establish a model system for molecular genetic studies of the CRAC channel

Defective nuclear translocation of nuclear factor of activated T cells and extracellular signal-regulated kinase underlies deficient IL-2 gene expression in Wiskott-Aldrich syndrome

Producción CientíficaBackground: Proliferation and IL-2 production in response to T-cell receptor ligation are impaired in patients with Wiskott- Aldrich syndrome (WAS). The transcription factors nuclear factor-kB (NF-kB), nuclear factor of activated T cells (NF-AT), and activating protein-1 (AP-1) play a critical role in IL-2 gene expression. Objective: To investigate the mechanisms of impaired IL-2 production after T-cell receptor ligation in T cells deficient in WAS protein (WASP). Methods: T cells from WASP2/2 mice were stimulated with anti-CD3 and anti-CD28. Nuclear NF-kB, NF-AT, and AP-1 DNA-binding activity was examined by electroshift mobility assay. NF-ATp dephosphorylation and nuclear localization were examined by Western blot and indirect immunofluorescence. Phosphorylation of the mitogen-activated protein kinases Erk and Jnk, and of their nuclear substrates Elk-1 and c-Jun, was examined by Western blot. Expression of mRNA for IL-2 and the NF-kB–dependent gene A20 and of the AP-1 components c-fos and c-Jun was examined by quantitative RT-PCR. Results: Nuclear translocation and activity of NF-kB were normal in T cells from WASP2/2 mice. In contrast, NF-ATp dephosphorylation and nuclear localization, nuclear AP-1 binding activity, and expression of c-fos, but not c-Jun, were all impaired. Phosphorylation of Jnk, c-Jun, and Erk were normal. However, nuclear translocation of phosphorylated Erk and phosphorylation of its nuclear substrate Elk1, which activates the c-fos promoter, were impaired. Conclusion: These results suggest that WASP is essential for NF-ATp activation, and for nuclear translocation of p-Erk, Elk1 phosphorylation, and c-fos gene expression in T cells. These defects underlie defective IL-2 expression and T-cell proliferation in WAS

The boron-oxygen core of borinate esters is responsible for the store-operated calcium entry potentiation ability

International audienceBACKGROUND: Store-Operated Calcium Entry (SOCE) is the major Ca2+ ion entry pathway in lymphocytes and is responsible of a severe combined immunodeficiency (SCID) when deficient. It has recently been observed or highlighted in other cell types such as myoblasts and neurons, suggesting a wider physiological role of this pathway. Whereas Orai1 protein is considered to be the channel allowing the SOCE in T cells, it is hypothesized that other proteins like TRPC could associate with Orai1 to form SOCE with different pharmacology and kinetics in other cell types. Unraveling SOCE cell functions requires specific effectors to be identified, just as dihydropyridines were crucial for the study of Ca2+ voltage-gated channels, or spider/snake toxins for other ion channel classes. To identify novel SOCE effectors, we analyzed the effects of 2-aminoethyl diphenylborinate (2-APB) and its analogues. 2-APB is a molecule known to both potentiate and inhibit T cell SOCE, but it is also an effector of TRP channels and endoplasmic reticulum Ca2+-ATPase. RESULTS: A structure-function analysis allowed to discover that the boron-oxygen core present in 2-APB and in the borinate ester analogues is absolutely required for the dual effects on SOCE. Indeed, a 2-APB analogue where the boron-oxygen core is replaced by a carbon-phosphorus core is devoid of potentiating capacity (while retaining inhibition capacity), highlighting the key role of the boron-oxygen core present in borinate esters for the potentiation function. However, dimesityl borinate ester, a 2-APB analogue with a terminal B-OH group showed an efficient inhibitory ability, without any potentiating capacity. The removal or addition of phenyl groups respectively decrease or increase the efficiency of the borinate esters to potentiate and inhibit the SOCE. mRNA expression revealed that Jurkat T cells mainly expressed Orai1, and were the more sensitive to 2-APB modulation of SOCE. CONCLUSIONS: This study allows the discovery of new boron-oxygen core containing compounds with the same ability as 2-APB to both potentiate and inhibit the SOCE of different leukocyte cell lines. These compounds could represent new tools to characterize the different types of SOCE and the first step in the development of new immunomodulators

Alternative splicing converts STIM2 from an activator to an inhibitor of store-operated calcium channels

Store-operated calcium entry (SOCE) regulates a wide variety of essential cellular functions. SOCE is mediated by STIM1 and STIM2, which sense depletion of ER Ca2+ stores and activate Orai channels in the plasma membrane. Although the amplitude and dynamics of SOCE are considered important determinants of Ca2+-dependent responses, the underlying modulatory mechanisms are unclear. In this paper, we identify STIM2??, a highly conserved alternatively spliced isoform of STIM2, which, in contrast to all known STIM isoforms, is a potent inhibitor of SOCE. Although STIM2?? does not by itself strongly bind Orai1, it is recruited to Orai1 channels by forming heterodimers with other STIM isoforms. Analysis of STIM2?? mutants and Orai1-STIM2?? chimeras suggested that it actively inhibits SOCE through a sequence-specific allosteric interaction with Orai1. Our results reveal a previously unrecognized functional flexibility in the STIM protein family by which alternative splicing creates negative and positive regulators of SOCE to shape the amplitude and dynamics of Ca2+ signals.open

Relative contributions of stromal interaction molecule 1 and CalDAG-GEFI to calcium-dependent platelet activation and thrombosis: STIM1 and CalDAG-GEFI in platelet activation

Stromal interaction molecule 1 (STIM1) was recently identified as a critical component of store-operated calcium entry (SOCE) in platelets. We previously reported the Ca2+-sensing guanine nucleotide exchange factor CalDAG-GEFI as critical molecule in Ca2+ signaling in platelets

Sex-specific differences in white matter microvascular integrity after ischaemic stroke

Background and purpose Functional outcomes after ischaemic stroke are worse in women, despite adjusting for differences in comorbidities and treatment approaches. White matter microvascular integrity represents one risk factor for poor long-term functional outcomes after ischaemic stroke. The aim of the study is to characterise sex-specific differences in microvascular integrity in individuals with acute ischaemic stroke.Methods A retrospective analysis of subjects with acute ischaemic stroke and brain MRI with diffusion-weighted (DWI) and dynamic-susceptibility contrast-enhanced (DSC) perfusion-weighted imaging obtained within 9 hours of last known well was performed. In the hemisphere contralateral to the acute infarct, normal-appearing white matter (NAWM) microvascular integrity was measured using the K-2 coefficient and apparent diffusion coefficient (ADC) values. Regression analyses for predictors of K-2 coefficient, DWI volume and good outcome (90-day modified Rankin scale (mRS) score <2) were performed.Results 105 men and 79 women met inclusion criteria for analysis. Despite no difference in age, women had increased NAWM K-2 coefficient (1027.4 vs 692.7x10(-6)/s; p=0.006). In women, atrial fibrillation (beta=583.6; p=0.04) and increasing NAWM ADC (beta=4.4; p=0.02) were associated with increased NAWM K-2 coefficient. In multivariable regression analysis, the K-2 coefficient was an independent predictor of DWI volume in women (beta=0.007; p=0.01) but not men.Conclusions In women with acute ischaemic stroke, increased NAWM K-2 coefficient is associated with increased infarct volume and chronic white matter structural integrity. Prospective studies investigating sex-specific differences in white matter microvascular integrity are needed

A multiscale hybrid model for pro-angiogenic calcium signals in a vascular endothelial cell

Cytosolic calcium machinery is one of the principal signaling mechanisms by which endothelial cells (ECs) respond to external stimuli during several biological processes, including vascular progression in both physiological and pathological conditions. Low concentrations of angiogenic factors (such as VEGF) activate in fact complex pathways involving, among others, second messengers arachidonic acid (AA) and nitric oxide (NO), which in turn control the activity of plasma membrane calcium channels. The subsequent increase in the intracellular level of the ion regulates fundamental biophysical properties of ECs (such as elasticity, intrinsic motility, and chemical strength), enhancing their migratory capacity. Previously, a number of continuous models have represented cytosolic calcium dynamics, while EC migration in angiogenesis has been separately approached with discrete, lattice-based techniques. These two components are here integrated and interfaced to provide a multiscale and hybrid Cellular Potts Model (CPM), where the phenomenology of a motile EC is realistically mediated by its calcium-dependent subcellular events. The model, based on a realistic 3-D cell morphology with a nuclear and a cytosolic region, is set with known biochemical and electrophysiological data. In particular, the resulting simulations are able to reproduce and describe the polarization process, typical of stimulated vascular cells, in various experimental conditions.Moreover, by analyzing the mutual interactions between multilevel biochemical and biomechanical aspects, our study investigates ways to inhibit cell migration: such strategies have in fact the potential to result in pharmacological interventions useful to disrupt malignant vascular progressio