Discoidin Domain Receptor 1 (DDR1) tyrosine kinase is upregulated in PKD kidneys but does not play a role in the pathogenesis of polycystic kidney disease.

Tolvaptan is the only drug approved to slow cyst growth and preserve kidney function in patients with autosomal dominant polycystic kidney disease (ADPKD). However, its limited efficacy combined with significant side effects underscores the need to identify new and safe therapeutic drug targets to slow progression to end stage kidney disease. We identified Discoidin Domain Receptor 1 (DDR1) as receptor tyrosine kinase upregulated in vivo in 3 mouse models of ADPKD using a novel mass spectrometry approach to identify kinases upregulated in ADPKD. Previous studies demonstrating critical roles for DDR1 to cancer progression, its potential role in the pathogenesis of a variety of other kidney disease, along with the possibility that DDR1 could provide new insight into how extracellular matrix impacts cyst growth led us to study the role of DDR1 in ADPKD pathogenesis. However, genetic deletion of DDR1 using CRISPR/Cas9 failed to slow cyst growth or preserve kidney function in both a rapid and slow mouse model of ADPKD demonstrating that DDR1 does not play a role in PKD pathogenesis and is thus a not viable drug target. In spite of the negative results, our studies will be of interest to the nephrology community as it will prevent others from potentially conducting similar experiments on DDR1 and reinforces the potential of performing unbiased screens coupled with in vivo gene editing using CRISPR/Cas9 to rapidly identify and confirm new potential drug targets for ADPKD.

Glutamine metabolism via glutaminase 1 in autosomal-dominant polycystic kidney disease.

Background: Metabolism of glutamine by glutaminase 1 (GLS1) plays a key role in tumor cell proliferation via the generation of ATP and intermediates required for macromolecular synthesis. We hypothesized that glutamine metabolism also plays a role in proliferation of autosomal-dominant polycystic kidney disease (ADPKD) cells and that inhibiting GLS1 could slow cyst growth in animal models of ADPKD. Methods: Primary normal human kidney and ADPKD human cyst-lining epithelial cells were cultured in the presence or absence of two pharmacologic inhibitors of GLS1, bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide 3 (BPTES) and CB-839, and the effect on proliferation, cyst growth in collagen and activation of downstream signaling pathways were assessed. We then determined if inhibiting GLS1 in vivo with CB-839 in the Aqp2-Cre; Pkd1fl/fl and Pkhd1-Cre; Pkd1fl/fl mouse models of ADPKD slowed cyst growth. Results: We found that an isoform of GLS1 (GLS1-GAC) is upregulated in cyst-lining epithelia in human ADPKD kidneys and in mouse models of ADPKD. Both BPTES and CB-839 blocked forskolin-induced cyst formation in vitro. Inhibiting GLS1 in vivo with CB-839 led to variable outcomes in two mouse models of ADPKD. CB-839 slowed cyst growth in Aqp2-Cre; Pkd1fl/fl mice, but not in Pkhd1-Cre; Pkd1fl/fl mice. While CB-839 inhibited mammalian target of rapamycin (mTOR) and MEK activation in Aqp2-Cre; Pkd1fl/fl, it did not in Pkhd1-Cre; Pkd1fl/fl mice. Conclusion: These findings provide support that alteration in glutamine metabolism may play a role in cyst growth. However, testing in other models of PKD and identification of the compensatory metabolic changes that bypass GLS1 inhibition will be critical to validate GLS1 as a drug target either alone or when combined with inhibitors of other metabolic pathways.

Regulation of KATP Channel Trafficking in Pancreatic ß-Cells by Protein Histidine Phosphorylation.

Protein histidine phosphatase 1 (PHPT-1) is an evolutionarily conserved 14-kDa protein that dephosphorylates phosphohistidine. PHPT-1-/- mice were generated to gain insight into the role of PHPT-1 and histidine phosphorylation/dephosphorylation in mammalian biology. PHPT-1-/- mice exhibited neonatal hyperinsulinemic hypoglycemia due to impaired trafficking of KATP channels to the plasma membrane in pancreatic ß-cells in response to low glucose and leptin and resembled patients with congenital hyperinsulinism (CHI). The defect in KATP channel trafficking in PHPT-1-/- ß-cells was due to the failure of PHPT-1 to directly activate transient receptor potential channel 4 (TRPC4), resulting in decreased Ca2+ influx and impaired downstream activation of AMPK. Thus, these studies demonstrate a critical role for PHPT-1 in normal pancreatic ß-cell function and raise the possibility that mutations in PHPT-1 and/or TRPC4 may account for yet to be defined cases of CHI.

Phosphatidlyinositol-3-kinase C2 beta (PI3KC2ß) is a potential new target to treat IgE mediated disease.

Cross linking of the IgE receptor (FcεRI) on mast cells plays a critical role in IgE-dependent allergy including allergic rhinitis, asthma, anaphylaxis, and delayed type hypersensitivity reactions. The Ca2+ activated K+ channel, KCa3.1, plays a critical role in IgE-stimulated Ca2+ entry and degranulation in mast cells by helping to maintain a negative membrane potential, which provides an electrochemical gradient to drive Ca2+ influx. Of the 3 classes of PI3K, the class II PI3Ks are the least studied and little is known about the roles for class II PI3Ks in vivo in the context of the whole organism under normal and pathological conditions. Studying bone marrow derived mast cells (BMMC) isolated from PI3KC2ß-/- mice, we now show that the class II PI3KC2ß is critical for FcεRI stimulated KCa3.1 channel activation and the subsequent activation of mast cells. We found FcεRI-stimulated Ca2+ entry, cytokine production, and degranulation are decreased in BMMC isolated from PI3KC2ß-/- mice. In addition, PI3KC2ß-/- mice are markedly resistant to both passive cutaneous and passive systemic anaphylaxis. These findings identify PI3KC2ß as a new pharmacologic target to treat IgE-mediated disease.

PLCε1 regulates SDF-1α-induced lymphocyte adhesion and migration to sites of inflammation.

Regulation of integrins is critical for lymphocyte adhesion to endothelium and migration throughout the body. Inside-out signaling to integrins is mediated by the small GTPase Ras-proximate-1 (Rap1). Using an RNA-mediated interference screen, we identified phospholipase Cε 1 (PLCε1) as a crucial regulator of stromal cell-derived factor 1 alpha (SDF-1α)-induced Rap1 activation. We have shown that SDF-1α-induced activation of Rap1 is transient in comparison with the sustained level following cross-linking of the antigen receptor. We identified that PLCε1 was necessary for SDF-1α-induced adhesion using shear stress, cell morphology alterations, and crawling on intercellular adhesion molecule 1 (ICAM-1)-expressing cells. Structure-function experiments to separate the dual-enzymatic function of PLCε1 uncover necessary contributions of the CDC25, Pleckstrin homology, and Ras-associating domains, but not phospholipase activity, to this pathway. In the mouse model of delayed type hypersensitivity, we have shown an essential role for PLCε1 in T-cell migration to inflamed skin, but not for cytokine secretion and proliferation in regional lymph nodes. Our results reveal a signaling pathway where SDF-1α induces T-cell adhesion through activation of PLCε1, suggesting that PLCε1 is a specific potential target in treating conditions involving migration of T cells to inflamed organs.

Nucleoside Diphosphate Kinase-C Suppresses cAMP Formation in Human Heart Failure.

BACKGROUND: Chronic heart failure (HF) is associated with altered signal transduction via ß-adrenoceptors and G proteins and with reduced cAMP formation. Nucleoside diphosphate kinases (NDPKs) are enriched at the plasma membrane of patients with end-stage HF, but the functional consequences of this are largely unknown, particularly for NDPK-C. Here, we investigated the potential role of NDPK-C in cardiac cAMP formation and contractility. METHODS: Real-time polymerase chain reaction, (far) Western blot, immunoprecipitation, and immunocytochemistry were used to study the expression, interaction with G proteins, and localization of NDPKs. cAMP levels were determined with immunoassays or fluorescent resonance energy transfer, and contractility was determined in cardiomyocytes (cell shortening) and in vivo (fractional shortening). RESULTS: NDPK-C was essential for the formation of an NDPK-B/G protein complex. Protein and mRNA levels of NDPK-C were upregulated in end-stage human HF, in rats after long-term isoprenaline stimulation through osmotic minipumps, and after incubation of rat neonatal cardiomyocytes with isoprenaline. Isoprenaline also promoted translocation of NDPK-C to the plasma membrane. Overexpression of NDPK-C in cardiomyocytes increased cAMP levels and sensitized cardiomyocytes to isoprenaline-induced augmentation of contractility, whereas NDPK-C knockdown decreased cAMP levels. In vivo, depletion of NDPK-C in zebrafish embryos caused cardiac edema and ventricular dysfunction. NDPK-B knockout mice had unaltered NDPK-C expression but showed contractile dysfunction and exacerbated cardiac remodeling during long-term isoprenaline stimulation. In human end-stage HF, the complex formation between NDPK-C and Gαi2 was increased whereas the NDPK-C/Gαs interaction was decreased, producing a switch that may contribute to an NDPK-C-dependent cAMP reduction in HF. CONCLUSIONS: Our findings identify NDPK-C as an essential requirement for both the interaction between NDPK isoforms and between NDPK isoforms and G proteins. NDPK-C is a novel critical regulator of ß-adrenoceptor/cAMP signaling and cardiac contractility. By switching from Gαs to Gαi2 activation, NDPK-C may contribute to lower cAMP levels and the related contractile dysfunction in HF.

Histidine phosphorylation relieves copper inhibition in the mammalian potassium channel KCa3.1.

KCa2.1, KCa2.2, KCa2.3 and KCa3.1 constitute a family of mammalian small- to intermediate-conductance potassium channels that are activated by calcium-calmodulin. KCa3.1 is unique among these four channels in that activation requires, in addition to calcium, phosphorylation of a single histidine residue (His358) in the cytoplasmic region, by nucleoside diphosphate kinase-B (NDPK-B). The mechanism by which KCa3.1 is activated by histidine phosphorylation is unknown. Histidine phosphorylation is well characterized in prokaryotes but poorly understood in eukaryotes. Here, we demonstrate that phosphorylation of His358 activates KCa3.1 by antagonizing copper-mediated inhibition of the channel. Furthermore, we show that activated CD4(+) T cells deficient in intracellular copper exhibit increased KCa3.1 histidine phosphorylation and channel activity, leading to increased calcium flux and cytokine production. These findings reveal a novel regulatory mechanism for a mammalian potassium channel and for T-cell activation, and highlight a unique feature of histidine versus serine/threonine and tyrosine as a regulatory phosphorylation site.

Identification of PGAM5 as a Mammalian Protein Histidine Phosphatase that Plays a Central Role to Negatively Regulate CD4(+) T Cells.

Whereas phosphorylation of serine, threonine, and tyrosine is exceedingly well characterized, the role of histidine phosphorylation in mammalian signaling is largely unexplored. Here we show that phosphoglycerate mutase family 5 (PGAM5) functions as a phosphohistidine phosphatase that specifically associates with and dephosphorylates the catalytic histidine on nucleoside diphosphate kinase B (NDPK-B). By dephosphorylating NDPK-B, PGAM5 negatively regulates CD4(+) T cells by inhibiting NDPK-B-mediated histidine phosphorylation and activation of the K(+) channel KCa3.1, which is required for TCR-stimulated Ca(2+) influx and cytokine production. Using recently developed monoclonal antibodies that specifically recognize phosphorylation of nitrogens at the N1 (1-pHis) or N3 (3-pHis) positions of the imidazole ring, we detect for the first time phosphoisoform-specific regulation of histidine-phosphorylated proteins in vivo, and we link these modifications to TCR signaling. These results represent an important step forward in studying the role of histidine phosphorylation in mammalian biology and disease.

Tumor Anatomy Scoring and Renal Function for Nephron-Sparing Treatment Selection in Patients With Small Renal Masses: A Microsimulation-Based Decision Analysis.

OBJECTIVE: The purpose of this article is to compare the effectiveness of a treatment algorithm for small renal tumors incorporating the nephrometry score, a renal tumor anatomy scoring system developed by urologists, with the current standard of uniformly recommended partial nephrectomy in patients with mild-to-moderate chronic kidney disease (CKD). MATERIALS AND METHODS: We developed a state-transition microsimulation model to project life expectancy (LE) in hypothetic patients with baseline mild or moderate CKD undergoing treatment of small renal masses. Our model incorporated the nephrometry score, which is predictive of postsurgical renal function loss. The two tested strategies were uniform treatment with partial nephrectomy and selective treatment based on nephrometry score and CKD stage, including percutaneous ablation for CKD stages 2 or 3a and intermediate-to-high nephrometry score or stage 3b CKD and any nephrometry score; otherwise, partial nephrectomy was assumed for other CKD stages and nephrometry scores. The model accounted for benign and malignant lesions, renal function decline, recurrence, and metastatic disease rates specific to each treatment, mortality by CKD stage, and comorbidities. Sensitivity analysis tested the stability of results when varying key parameters. RESULTS: Selective treatment with partial nephrectomy resulted in an average LE benefit of 0.48 year (95% interpercentile range, 0.42-0.54 year) in 65-year-old men and 0.37 year (95% interpercentile range, 0.30-0.43 year) in 65-year-old women relative to nondiscriminatory surgery, due to worsening CKD and cardiovascular mortality associated with partial nephrectomy. Model results were most sensitive to the rate of renal function decline and CKD-related mortality. CONCLUSION: Nephron-sparing treatment selection for small renal masses based on nephrometry score may improve LE in patients with mild or moderate CKD.

Nucleoside diphosphate kinase B deficiency causes a diabetes-like vascular pathology via up-regulation of endothelial angiopoietin-2 in the retina.

AIMS: Nucleoside diphosphate kinase B (NDPKB) is capable of maintaining the cellular nucleotide triphosphate pools. It might therefore supply UTP for the formation of UDP-GlcNAc from glucose. As NDPKB contributes to vascular dysfunction, we speculate that NDPKB might play a role in microangiopathies, such as diabetic retinopathy (DR). Therefore, we investigated the impact of NDPKB on retinal vascular damage using NDPKB(-/-) mice during development of DR and its possible mechanisms. METHODS: Pericyte loss and acellular capillary (AC) formation were assessed in streptozotocin-induced diabetic NDPKB(-/-) and wild-type (WT) mice. Expression of angiopoietin-2 (Ang2) and protein N-acetylglucosamine modification (GlcNAcylation) were assessed by western blot and/or immunofluorescence in the diabetic retinas as well as in endothelial cells depleted of NDPKB by siRNA and stimulated with high glucose. RESULTS: Similar to diabetic WT retinas, non-diabetic NDPKB(-/-) retinas showed a significant decrease in pericyte coverage in comparison with non-diabetic WT retinas. Hyperglycemia further aggravates pericyte loss in diabetic NDPKB(-/-) retinas. AC formation was detected in the diabetic NDPKB(-/-) retinas. Similar to hyperglycemia, NDPKB deficiency induced Ang2 expression and protein GlcNAcylation that were not further altered in the diabetic retinas. In cultured endothelial cells, stimulation with high glucose and NDPKB depletion comparably increased Ang2 expression and protein GlcNAcylation. CONCLUSIONS: Our data identify NDPKB as a protective factor in the retina, which controls Ang2 expression and the hexosamine pathway. NDPKB-deficient mice are a suitable model for studying mechanisms underlying diabetic retinal vascular damage.

Nucleoside diphosphate kinase B-activated intermediate conductance potassium channels are critical for neointima formation in mouse carotid arteries.

OBJECTIVE: Vascular smooth muscle cells (VSMC) proliferation is a hallmark of atherosclerosis and vascular restenosis. The intermediate conductance Ca(2+)-activated K(+) (SK4) channel is required for pathological VSMC proliferation. In T lymphocytes, nucleoside diphosphate kinase B (NDPKB) has been implicated in SK4 channel activation. We thus investigated the role of NDPKB in the regulation of SK4 currents (ISK4) in proliferating VSMC and neointima formation. APPROACH AND RESULTS: Function and expression of SK4 channels in VSMC from injured mouse carotid arteries were assessed by patch-clamping and real-time polymerase chain reaction. ISK4 was detectable in VSMC from injured but not from uninjured arteries correlating with the occurrence of the proliferative phenotype. Direct application of NDPKB to the membrane of inside-out patches increased ISK4, whereas NDPKB did not alter currents in VSMC obtained from injured vessels of SK4-deficient mice. The NDPKB-induced increase in ISK4 was prevented by protein histidine phosphatase 1, but not an inactive protein histidine phosphatase 1 mutant indicating that ISK4 is regulated via histidine phosphorylation in proliferating VSMC; moreover, genetic NDPKB ablation reduced ISK4 by 50% suggesting a constitutive activation of ISK4 in proliferating VSMC. In line, neointima formation after wire injury of the carotid artery was substantially reduced in mice deficient in SK4 channels or NDPKB. CONCLUSIONS: NDPKB to SK4 signaling is required for neointima formation. Constitutive activation of SK4 by NDPKB in proliferating VSMC suggests that targeting this interaction via, for example, activation of protein histidine phosphatase 1 may provide clinically meaningful effects in vasculoproliferative diseases such as atherosclerosis and post angioplasty restenosis.

Ion channels in innate and adaptive immunity.

Ion channels and transporters mediate the transport of charged ions across hydrophobic lipid membranes. In immune cells, divalent cations such as calcium, magnesium, and zinc have important roles as second messengers to regulate intracellular signaling pathways. By contrast, monovalent cations such as sodium and potassium mainly regulate the membrane potential, which indirectly controls the influx of calcium and immune cell signaling. Studies investigating human patients with mutations in ion channels and transporters, analysis of gene-targeted mice, or pharmacological experiments with ion channel inhibitors have revealed important roles of ionic signals in lymphocyte development and in innate and adaptive immune responses. We here review the mechanisms underlying the function of ion channels and transporters in lymphocytes and innate immune cells and discuss their roles in lymphocyte development, adaptive and innate immune responses, and autoimmunity, as well as recent efforts to develop pharmacological inhibitors of ion channels for immunomodulatory therapy.

Metabolic inflexibility impairs insulin secretion and results in MODY-like diabetes in triple FoxO-deficient mice.

Pancreatic ß cell failure in type 2 diabetes is associated with functional abnormalities of insulin secretion and deficits of ß cell mass. It's unclear how one begets the other. We have shown that loss of ß cell mass can be ascribed to impaired FoxO1 function in different models of diabetes. Here we show that ablation of the three FoxO genes (1, 3a, and 4) in mature ß cells results in early-onset, maturity-onset diabetes of the young (MODY)-like diabetes, with abnormalities of the MODY networks Hnf4α, Hnf1α, and Pdx1. FoxO-deficient ß cells are metabolically inflexible, i.e., they preferentially utilize lipids rather than carbohydrates as an energy source. This results in impaired ATP generation and reduced Ca(2+)-dependent insulin secretion. The present findings demonstrate a secretory defect caused by impaired FoxO activity that antedates dedifferentiation. We propose that defects in both pancreatic ß cell function and mass arise through FoxO-dependent mechanisms during diabetes progression.

Nucleoside diphosphate kinase B regulates angiogenesis through modulation of vascular endothelial growth factor receptor type 2 and endothelial adherens junction proteins.

OBJECTIVE: Nucleoside diphosphate kinase B (NDPKB) participates in the activation of heterotrimeric and monomeric G proteins, which are pivotal mediators in angiogenic signaling. The role of NDPKB in angiogenesis has to date not been defined. Therefore, we analyzed the contribution of NDPKB to angiogenesis and its underlying mechanisms in well-characterized in vivo and in vitro models. APPROACH AND RESULTS: Zebrafish embryos were depleted of NDPKB by morpholino-mediated knockdown. These larvae displayed severe malformations specifically in vessels formed by angiogenesis. NDPKB-deficient (NDPKB(-/-)) mice were subjected to oxygen-induced retinopathy. In this model, the number of preretinal neovascularizations in NDPKB(-/-) mice was strongly reduced in comparison with wild-type littermates. In accordance, a delayed blood flow recovery was detected in the NDPKB(-/-) mice after hindlimb ligation. In in vitro studies, a small interfering RNA-mediated knockdown of NDPKB was performed in human umbilical endothelial cells. NDPKB depletion impaired vascular endothelial growth factor (VEGF)-induced sprouting and hampered the VEGF-induced spatial redistributions of the VEGF receptor type 2 and VE-cadherin at the plasma membrane. Concomitantly, NDPKB depletion increased the permeability of the human umbilical endothelial cell monolayer. CONCLUSIONS: This is the first report to show that NDPKB is required for VEGF-induced angiogenesis and contributes to the correct localization of VEGF receptor type 2 and VE-cadherin at the endothelial adherens junctions. Therefore, our data identify NDPKB as a novel molecular target to modulate VEGF-dependent angiogenesis.

Sequential breakdown of 3-phosphorylated phosphoinositides is essential for the completion of macropinocytosis.

Macropinocytosis is a highly conserved endocytic process by which extracellular fluid and solutes are internalized into cells. Macropinocytosis starts with the formation of membrane ruffles at the plasma membrane and ends with their closure. The transient and sequential emergence of phosphoinositides PI(3,4,5)P3 and PI(3,4)P2 in the membrane ruffles is essential for macropinocytosis. By making use of information in the Caenorhabditis elegans mutants defective in fluid-phase endocytosis, we found that mammalian phosphoinositide phosphatase MTMR6 that dephosphorylates PI(3)P to PI, and its binding partner MTMR9, are required for macropinocytosis. INPP4B, which dephosphorylates PI(3,4)P2 to PI(3)P, was also found to be essential for macropinocytosis. These phosphatases operate after the formation of membrane ruffles to complete macropinocytosis. Finally, we showed that KCa3.1, a Ca(2+)-activated K(+) channel that is activated by PI(3)P, is required for macropinocytosis. We propose that the sequential breakdown of PI(3,4,5)P3 → PI(3,4)P2 → PI(3)P → PI controls macropinocytosis through specific effectors of the intermediate phosphoinositides.

Regulation of the epithelial Ca²âº channel TRPV5 by reversible histidine phosphorylation mediated by NDPK-B and PHPT1.

The kidney, together with bone and intestine, plays a crucial role in maintaining whole-body calcium (Ca(2+)) homoeostasis, which is primarily mediated by altering the reabsorption of Ca(2+) filtered by the glomerulus. The transient receptor potential-vanilloid-5 (TRPV5) channel protein forms a six- transmembrane Ca(2+)-permeable channel that regulates urinary Ca(2+) excretion by mediating active Ca(2+) reabsorption in the distal convoluted tubule of the kidney. Here we show that the histidine kinase, nucleoside diphosphate kinase B (NDPK-B), activates TRPV5 channel activity and Ca(2+) flux, and this activation requires histidine 711 in the carboxy-terminal tail of TRPV5. In addition, the histidine phosphatase, protein histidine phosphatase 1, inhibits NDPK-B-activated TRPV5 in inside/out patch experiments. This is physiologically relevant to Ca(2+) reabsorption in vivo, as short hairpin RNA knockdown of NDPK-B leads to decreased TRPV5 channel activity, and urinary Ca(2+) excretion is increased in NDPK-B(-/-) mice fed a high-Ca(2+) diet. Thus these findings identify a novel mechanism by which TRPV5 and Ca(2+) reabsorption is regulated by the kidney and support the idea that histidine phosphorylation plays other, yet-uncovered roles in mammalian biology.

Coexistence of ANCA-associated glomerulonephritis and anti-phospholipase A(2) receptor antibody-positive membranous nephropathy.

Antibodies to myeloperoxidase (MPO) and proteinase 3 (PR3) have been demonstrated to mediate anti-neutrophil cytoplasmic antibody (ANCA)-associated disease. For membranous nephropathy, antibodies to the podocyte-expressed phospholipase A(2) receptor (anti-PLA(2)R) are highly associated with disease activity and have been reported in at least 70% of patients with idiopathic membranous nephropathy (IMN). We present a case of a 56-year-old male with a 1 year history of hypertension, leg edema, and proteinuria, who presented with advanced renal failure and was found to have both ANCA-associated glomerulonephritis (GN) and IMN on kidney biopsy. Consistent with the idea that this is due to the chance occurrence of two independent diseases, we found both anti-MPO and anti-PLA(2)R antibodies in the patient's sera. Treatment with methylprednisolone, plasmapheresis, and cyclophosphamide resulted in improvement in kidney function and proteinuria, together with the simultaneous decrease in both autoantibodies. This is the first demonstration of two pathogenic antibodies giving rise to ANCA-associated GN and IMN in the same patient. It confirms the importance of classifying disease based upon the underlying mechanism, in addition to renal histopathology, to both optimize therapy and predict prognosis.

Ion channels and transporters in lymphocyte function and immunity.

Lymphocyte function is regulated by a network of ion channels and transporters in the plasma membrane of B and T cells. These proteins modulate the cytoplasmic concentrations of diverse cations, such as calcium, magnesium and zinc ions, which function as second messengers to regulate crucial lymphocyte effector functions, including cytokine production, differentiation and cytotoxicity. The repertoire of ion-conducting proteins includes calcium release-activated calcium (CRAC) channels, P2X receptors, transient receptor potential (TRP) channels, potassium channels, chloride channels and magnesium and zinc transporters. This Review discusses the roles of ion conduction pathways in lymphocyte function and immunity.

Phosphatidylinositol-3-kinase C2ß and TRIM27 function to positively and negatively regulate IgE receptor activation of mast cells.

Cross-linking of the IgE receptor (FcεRI) on mast cells plays a critical role in IgE-dependent allergy, including allergic rhinitis, asthma, anaphylaxis, and immediate-type hypersensitivity reactions. Previous studies have demonstrated that the K(+) channel, KCa3.1, plays a critical role in IgE-stimulated Ca(2+) entry and degranulation in both human and mouse mast cells. We now have shown that the class II phosphatidylinositol-3-kinase C2ß (PI3KC2ß) is necessary for FcεRI-stimulated activation of KCa3.1, Ca(2+) influx, cytokine production, and degranulation of bone marrow-derived mast cells (BMMC). In addition, we found that the E3 ubiquitin ligase, tripartite motif containing protein 27 (TRIM27), negatively regulates FcεRI activation of KCa3.1 and downstream signaling by ubiquitinating and inhibiting PI3KC2ß. TRIM27(-/-) mice are also more susceptible in vivo to acute anaphylaxis. These findings identify TRIM27 as an important negative regulator of mast cells in vivo and suggest that PI3KC2ß is a potential new pharmacologic target to treat IgE-mediated disease.