Heterozygous mutations in the NPT2a gene may be responsible for hypophosphatemia and urinary phosphate loss in persons with urolithiasis or bone demineralization.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) release calcium ions, Ca2+, from intracellular stores, but their roles in mediating Ca2+ entry are unclear. IP3 stimulated opening of very few (1.9 +/- 0.2 per cell) Ca2+-permeable channels in whole-cell patch-clamp recording of DT40 chicken or mouse B cells. Activation of the B cell receptor (BCR) in perforated-patch recordings evoked the same response. IP3 failed to stimulate intracellular or plasma membrane (PM) channels in cells lacking IP3R. Expression of IP3R restored both responses. Mutations within the pore affected the conductances of IP3-activated PM and intracellular channels similarly. An impermeant pore mutant abolished BCR-evoked Ca2+ signals, and PM IP3Rs were undetectable. After introduction of an alpha-bungarotoxin binding site near the pore, PM IP3Rs were modulated by extracellular alpha-bungarotoxin. IP(3)Rs are unusual among endoplasmic reticulum proteins in being also functionally expressed at the PM, where very few IP3Rs contribute substantially to the Ca2+ entry evoked by the BCR.

Inositol 1,4,5-trisphosphate receptors (IP3R) are ubiquitous intracellular Ca2+ channels. IP3binding to the IP3-binding core (IBC) near the N-terminal initiates conformational changes that lead to opening of a pore. The mechanisms are unresolved. We synthesized 2-O-modified IP3 analogues that are partial agonists of IP3R. These are like IP3 in their interactions with the IBC, but they are less effective than IP3 in rearranging the relationship between the IBC and N-terminal suppressor domain (SD), and they open the channel at slower rates. IP3R with a mutation in the SD occupying a position similar to the 2-O-substituent of the partial agonists has a reduced open probability that is similar for full and partial agonists. Bulky or charged substituents from either the ligand or SD therefore block obligatory coupling of the IBC and SD. Analysis of ΔG for ligand binding shows that IP3 is recognised by the IBC and conformational changes then propagate entirely via the SD to the pore.

In chronic kidney disease (CKD), proteinuria results in severe tubulointerstitial lesions, which ultimately lead to end-stage renal disease. Here we identify 4-phenylbutyric acid (PBA), a chemical chaperone already used in humans, as a novel therapeutic strategy capable to counteract the toxic effect of proteinuria. Mechanistically, we show that albumin induces tubular unfolded protein response via cytosolic calcium rise, which leads to tubular apoptosis by Lipocalin 2 (LCN2) modulation through ATF4. Consistent with the key role of LCN2 in CKD progression, Lcn2 gene inactivation decreases ER stress-induced apoptosis, tubulointerstitial lesions and mortality in proteinuric mice. More importantly, the inhibition of this pathway by PBA protects kidneys from morphological and functional degradation in proteinuric mice. These results are relevant to human CKD, as LCN2 is increased in proteinuric patients. In conclusion, our study identifies a therapeutic strategy susceptible to improve the benefit of RAS inhibitors in proteinuria-induced CKD progression.

In Arabidopsis suspension cells a rapid plasma membrane depolarization is triggered by abscisic acid (ABA). Activation of anion channels was shown to be a component leading to this ABA-induced plasma membrane depolarization. Using experiments employing combined voltage clamping, continuous measurement of extracellular pH, we examined whether plasma membrane H 1 -ATPases could also be involved in the depolarization. We found that ABA causes simultaneously cell depolarization and medium alkalinization, the second effect being abolished when ABA is added in the presence of H 1 pump inhibitors. Inhibition of the proton pump by ABA is thus a second component leading to the plasma membrane depolarization. The ABA-induced depolarization is therefore the result of two different processes: activation of anion channels and inhibition of H 1 -ATPases. These two processes are independent because impairing one did not suppress the depolarization. Both processes are however dependent on the [Ca 21 ] cyt increase induced by ABA since increase in [Ca 21 ] cyt enhanced anion channels and impaired H 1 -ATPases.Abscisic acid (ABA) induces the depolarization of the plasma membrane (Thiel et al., 1992). This depolarization has been interpreted as the consequence of the activation of anion channels in stomatal guard cells of Vicia faba (Blatt, 1990;Schroeder and Keller, 1992;Thiel et al., 1992;Ward et al., 1995), Nicotiana benthamiana and Commelina communis (Schwartz et al., 1995;Leonhardt et al., 1999). In addition, we demonstrated that the extracellular perception of ABA in Arabidopsis suspension cells was necessary for the activation of anion channels inducing the plasma membrane depolarization (Ghelis et al., 2000a), and recently we showed that this anion channel stimulation induced by extracellular ABA perception was dependent on phospholipase D activities (Hallouin et al., 2002). In guard cells that are the most studied plant cell model used for the dissection of ABA signaling pathways (Assmann, 1993;Schroeder et al., 2001), two distinct anion channels, rapid anion channels (R-type) and slow anion channels (S-type), were proposed to participate in the plasma membrane depolarization (Schroeder and Keller, 1992;. Both R-type and S-type channels have been suggested to contribute to an initial phase of the depolarization, while maintenance of the depolarized state of the plasma membrane was only attributed to the S-type anion channels (Schroeder and Keller, 1992). The mechanisms by which ABA activates anion channels are not entirely understood . In V. faba guard cells, activation of anion channels by ABA can be observed without variation of the cytoplasm calcium concentration, suggesting that the ABA-induced anion efflux is calcium-independent (Schwarz and Schroeder, 1998). However, numerous data support the calcium dependence of the anion channel activation in response to ABA. Some of the anion channels involved in a long-term plasma membrane depolarization are Ca 21 -sensitive and therefore are activated by an increase in cyto...

BackgroundCalcium signaling plays an important role in B lymphocyte survival and activation, and is critically dependent on the inositol-1,4,5-tris-phosphate-induced release of calcium stored in the endoplasmic reticulum (ER). Calcium is accumulated in the ER by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes), and therefore these enzymes play an important role in ER calcium homeostasis and in the control of B of cell activation. Because Epstein-Barr virus (EBV) can immortalize B cells and contributes to lymphomagenesis, in this work the effects of the virus on SERCA-type calcium pump expression and calcium accumulation in the endoplasmic reticulum of B cells was investigated.ResultsTwo Sarco-Endoplasmic Reticulum Calcium transport ATPase isoforms, the low Ca2+-affinity SERCA3, and the high Ca2+-affinity SERCA2 enzymes are simultaneously expressed in B cells. Latency type III infection of Burkitt's lymphoma cell lines with immortalization-competent virus expressing the full set of latency genes selectively decreased the expression of SERCA3 protein, whereas infection with immortalization-deficient virus that does not express the EBNA2 or LMP-1 viral genes was without effect. Down-modulation of SERCA3 expression could be observed upon LMP-1, but not EBNA2 expression in cells carrying inducible transgenes, and LMP-1 expression was associated with enhanced resting cytosolic calcium levels and increased calcium storage in the endoplasmic reticulum. Similarly to virus-induced B cell immortalisation, SERCA3 expression was also decreased in normal B cells undergoing activation and blastic transformation in germinal centers of lymph node follicles.ConclusionThe data presented in this work indicate that EBV-induced immortalization leads to the remodelling of ER calcium homeostasis of B cells by LMP-1 that copies a previously unknown normal phenomenon taking place during antigen driven B cell activation. The functional remodelling of ER calcium homeostasis by down-regulation of SERCA3 expression constitutes a previously unknown mechanism involved in EBV-induced B cell immortalisation.

Inositol 1,4,5-trisphosphate receptors (IP 3 R) within the endoplasmic reticulum mediate release of Ca 2؉ from intracellular stores. Different channels usually mediate Ca 2؉ entry across the plasma membrane. In B lymphocytes and a cell line derived from them (DT40 cells), very few functional IP 3 R (ϳ2/cell) are invariably expressed in the plasma membrane, where they mediate about half the Ca 2؉ entry evoked by activation of the B-cell receptor. We show that cells reliably count ϳ2 functional IP 3 R into the plasma membrane even when their conductance and ability to bind IP 3 are massively attenuated. We conclude that very small numbers of functional IP 3 R can be reliably counted into a specific membrane compartment in the absence of feedback signals from the active protein.Inositol 1,4,5-trisphosphate receptors (IP 3 R) 2 belong to a family of intracellular Ca 2ϩ channels that mediate release of Ca 2ϩ from the intracellular stores of most animal cells (1, 2). Most IP 3 R in most cells are expressed in the membranes of the endoplasmic reticulum (ER), but smaller numbers of IP 3 R may also be targeted to additional intracellular organelles, including secretory vesicles (3, 4), the Golgi apparatus (5), and the nucleoplasm (6). IP 3 R can also be expressed in the plasma membrane (PM) (7-9), and we recently demonstrated that in B lymphocytes these IP 3 R mediate about half the Ca 2ϩ entry evoked by activation of the B-cell receptor (10). Remarkably, both native mouse B lymphocytes and avian DT40 cells, which are derived from B lymphocytes (11), reliably express just two or three functional IP 3 R in the PM, which are nevertheless sufficient to contribute substantially to the Ca 2ϩ signals evoked by a physiological stimulus (10).Most ion channels (12), indeed most proteins (13), are expressed in cells in sufficiently large numbers (typically thousands/cell) that it is easy to envisage how their expression levels can be regulated by appropriate feedback signals (14): the law of large numbers provides stability (15). However, some ion channels are expressed at the PM in much smaller numbers: two or three ryanodine receptor-like channels in portal vein myocytes (16) and ϳ10 Ca 2ϩ -activated K ϩ channels (IKCa1) in a resting T cell (17), for example. For such rare events as these and the expression of a very small number of IP 3 R in the PM (10), the law of large numbers cannot apply and the stability must be provided by additional regulatory mechanisms. Here we show that cells can reliably count very small numbers of IP 3 R into the PM in the absence of feedback signals. EXPERIMENTAL PROCEDURESExpression of IP 3 R in DT40 Cells-DT40 cells were cultured at 37°C in humidified air containing 5% CO 2 in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% chicken serum, 2 mM L-glutamine, and 10 M 2-mercaptoethanol. QuikChange II XL (Stratagene) was used to introduce point mutations into rat IP 3 R1 without the S1 splice site (GenBank TM accession number JO5510). Constructs were verified by sequencing. Stable cell ...

Background: Signals triggered by galectin-9 in human T cells are poorly understood. Results: Impairment of Lck or T cell receptor-CD3 complex inhibits calcium mobilization and cytokine production but not apoptosis induced by galectin-9. Conclusion: Galectin-9 triggers two independent pathways in T cells, with one mimicking the antigen-specific activation. Significance: We discovered a novel mechanism involved in galectin-9 immunomodulatory effects.