Human Trp3 forms both inositol trisphosphate receptor-dependent and receptor-independent store-operated cation channels in DT40 avian B lymphocytes.

Mammalian Trp proteins are candidates for plasma membrane calcium channels regulated by receptor activation or by intracellular calcium store depletion [capacitative calcium entry (CCE)]. One extensively investigated member of the Trp family, the human Trp3 (hTrp3), behaves as a receptor-activated, calcium-permeable, nonselective cation channel when expressed in cell lines and does not appear to be activated by store depletion. Nonetheless, there is good evidence that Trp3 can be regulated by interacting with inositol trisphosphate receptors (IP(3)Rs), reminiscent of the conformational coupling mode of CCE. To investigate the role of Trp3 in CCE, and its regulation by IP(3)R, we transiently expressed hTrp3 in the wild-type DT40 chicken B lymphocyte cell line and its variant lacking IP(3)R. Expression of hTrp3 in either wild-type or IP(3)R-knockout cells did not increase basal membrane permeability, but resulted in a substantially greater divalent cation entry after thapsigargin-induced store depletion. This hTrp3-dependent divalent cation entry was significantly greater in the wild type than in IP(3)R-knockout cells. Thus, it appears that in this cell line, hTrp3 forms channels that are store-operated by both IP(3)R-dependent and IP(3)R-independent mechanisms. Trp3, or one of its structural relatives, is a candidate for the store-operated, nonselective cation channels observed in smooth muscle cells and other cell types.

Mechanisms of capacitative calcium entry.

Capacitative Ca(2+) entry involves the regulation of plasma membrane Ca(2+) channels by the filling state of intracellular Ca(2+) stores in the endoplasmic reticulum (ER). Several theories have been advanced regarding the mechanism by which the stores communicate with the plasma membrane. One such mechanism, supported by recent findings, is conformational coupling: inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) receptors in the ER may sense the fall in Ca(2+) levels through Ca(2+)-binding sites on their lumenal domains, and convey this conformational information directly by physically interacting with Ca(2+) channels in the plasma membrane. In support of this idea, in some cell types, store-operated channels in excised membrane patches appear to depend on the presence of both Ins(1,4,5)P(3) and Ins(1,4,5)P(3) receptors for activity; in addition, inhibitors of Ins(1,4,5)P(3) production that either block phospholipase C or inhibit phosphatidylinositol 4-kinase can block capacitative Ca(2+) entry. However, the electrophysiological current underlying capacitative Ca(2+) entry is not blocked by an Ins(1,4,5)P(3) receptor antagonist, and the blocking effects of a phospholipase C inhibitor are not reversed by the intracellular application of Ins(1,4,5)P(3). Furthermore, cells whose Ins(1,4,5)P(3) receptor genes have been disrupted can nevertheless maintain their capability to activate capacitative Ca(2+) entry channels in response to store depletion. A tentative conclusion is that multiple mechanisms for signaling capacitative Ca(2+) entry may exist, and involve conformational coupling in some cell types and perhaps a diffusible signal in others.

Role of the phospholipase C-inositol 1,4,5-trisphosphate pathway in calcium release-activated calcium current and capacitative calcium entry.

We investigated the putative roles of phospholipase C, polyphosphoinositides, and inositol 1,4,5-trisphosphate (IP(3)) in capacitative calcium entry and calcium release-activated calcium current (I(crac)) in lacrimal acinar cells, rat basophilic leukemia cells, and DT40 B-lymphocytes. Inhibition of phospholipase C with blocked calcium entry and I(crac) activation whether in response to a phospholipase C-coupled agonist or to calcium store depletion with thapsigargin. Run-down of cellular polyphosphoinositides by concentrations of wortmannin that block phosphatidylinositol 4-kinase completely blocked calcium entry and I(crac). The membrane-permeant IP(3) receptor inhibitor, 2-aminoethoxydiphenyl borane, blocked both capacitative calcium entry and I(crac). However, it is likely that 2-aminoethoxydiphenyl borane does not inhibit through an action on the IP(3) receptor because the drug was equally effective in wild-type DT40 B-cells and in DT40 B-cells whose genes for all three IP(3) receptors had been disrupted. Intracellular application of another potent IP(3) receptor antagonist, heparin, failed to inhibit activation of I(crac). Finally, the inhibition of I(crac) activation by or wortmannin was not reversed or prevented by direct intracellular application of IP(3). These findings indicate a requirement for phospholipase C and for polyphosphoinositides for activation of capacitative calcium entry. However, the results call into question the previously suggested roles of IP(3) and IP(3) receptor in this mechanism, at least in these particular cell types.

Cloning and expression of the human transient receptor potential 4 (TRP4) gene: localization and functional expression of human TRP4 and TRP3.

Mammalian homologues of the Drosophila transient receptor potential (TRP) protein have been proposed to function as ion channels, and in some cases as store-operated or capacitative calcium entry channels. However, for each of the mammalian TRP proteins, different laboratories have reported distinct modes of cellular regulation. In the present study we describe the cloning and functional expression of the human form of TRP4 (hTRP4), and compare its activity with another well studied protein, hTRP3. When hTRP4 was transiently expressed in human embryonic kidney (HEK)-293 cells, basal bivalent cation permeability (barium) was increased. Whole-cell patch-clamp studies of hTRP4 expressed in Chinese hamster ovary cells revealed a constitutively active non-selective cation current which probably underlies the increased bivalent cation entry. Barium entry into hTRP4-transfected HEK-293 cells was not further increased by phospholipase C (PLC)-linked receptor activation, by intracellular calcium store depletion with thapsigargin, or by a synthetic diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (OAG). In contrast, transient expression of hTRP3 resulted in a bivalent cation influx that was markedly increased by PLC-linked receptor activation and by OAG, but not by thapsigargin. Despite the apparent differences in regulation of these two putative channel proteins, green fluorescent protein fusions of both molecules localized similarly to the plasma-membrane, notably in discrete punctate regions suggestive of specialized signalling complexes. Our findings indicate that while both hTRP4 and hTRP3 can apparently function as cation channels, their putative roles as components of capacitative calcium entry channels are not readily demonstrable by examining their behaviour when exogenously expressed in cells.

Nitric oxide inhibits tumor necrosis factor-alpha-induced apoptosis by reducing the generation of ceramide.

Apoptosis triggered by death receptors proceeds after defined signal-transduction pathways. Whether signaling at the receptor level is regulated by intracellular messengers is still unknown. We have investigated the role of two messengers, ceramide and nitric oxide (NO), on the apoptotic pathway activated in human monocytic U937 cells by tumor necrosis factor-alpha (TNF-alpha) working at its p55 receptor. Two transduction events, the receptor recruitment of the adapter protein, TRADD, and the activation of the initiator caspase, caspase 8, were investigated. When administered alone, neither of the messengers had any effect on these events. In combination with TNF-alpha, however, ceramide potentiated, whereas NO inhibited, TNF-alpha-induced TRADD recruitment and caspase 8 activity. The effect of NO, which was cGMP-dependent, was due to inhibition of the TNF-alpha-induced generation of ceramide. Our results identify a mechanism of regulation of a signal-transduction pathway activated by death receptors.

The p75(NTR)-induced apoptotic program develops through a ceramide-caspase pathway negatively regulated by nitric oxide.

SK-N-BE neuroblastoma cell clones transfected with p75(NTR) and lacking Trk neurotrophin receptors, previously reported to undergo extensive spontaneous apoptosis and to be protected by nerve growth factor (NGF) (Bunone, G., Mariotti, A., Compagni, A., Morandi, E., and Della Valle, G. (1997) Oncogene 14, 1463-1470), are shown to exhibit (i) increased levels of the pro-apoptotic lipid metabolite ceramide and (ii) high activity of caspases, the proteases of the cell death cascade. In the p75(NTR)-expressing cells, these parameters were partially normalized by prolonged NGF treatment, which, in addition, decreased apoptosis, similar to caspase blockers. Conversely, exogenous ceramide increased caspase activity and apoptosis in both wild-type and p75(NTR)-expressing cells. A new p75(NTR)-expressing clone characterized by low spontaneous apoptosis exhibited high endogenous ceramide and low caspase levels. A marked difference between the apoptotic and resistant clones concerned the very low and high activities of nitric-oxide (NO) synthase, respectively. Protection from apoptosis by NO was confirmed by results with the NO donor S-nitrosoacetylpenicillamine and the NO-trapping agent hemoglobin. We conclude that the p75(NTR) receptor, while free of NGF, triggers a cascade leading to apoptosis; the cascade includes generation of ceramide and increased caspase activity; and the protective role of NO occurs at step(s) in between the latter events.

BiP, a major chaperone protein of the endoplasmic reticulum lumen, plays a direct and important role in the storage of the rapidly exchanging pool of Ca2+.

The activity of BiP, the major chaperone of the endoplasmic reticulum (ER) lumen, is known to be Ca2+-regulated; however, the participation of this protein in the ER storage of the cation has not yet been investigated. Here such a role is demonstrated in human epithelial (HeLa) cells transiently transfected with the hamster BiP cDNA and incubated in Ca2+-free medium, as revealed by two different techniques. In the first, co-transfected aequorin was employed as a probe for assaying either the cytosolic of the mitochondrial free Ca2+ concentration. By this approach higher Ca2+ release responses were revealed in BiP-transfected cells by experiments in which extensive store depletion was induced either by repetitive stimulation with inositol 1,4,5-trisphosphate-generating agonists or by treatment with the Ca2+ ionophore, A23187. In the second technique the cells were loaded at the equilibrium with 45Ca, and the release of the tracer observed upon treatment with thapsigargin, a blocker of the ER Ca2+ ATPases, was larger in BiP-transfected than in control cells. The latter results were obtained also when BiP was overexpressed not via transfection but as a response to ER stress by tunicamycin. These results are sustained by increases of the ER Ca2+ storage capacity rather than by artifacts or indirect readjustments induced in the cells by the overexpression of the chaperone since (a) the exogenous and endogenous BiP were both confined to the ER, (b) the expression levels of other proteins active in the ER Ca2+ storage were not changed, and (c) effects similar to those of wild type BiP were obtained with a deletion mutant devoid of chaperone activity. The specificity of the results was confirmed by parallel 45Ca experiments carried out in HeLa cells transfected with two other Ca2+-binding proteins, calreticulin and CaBP2(ERp72), only the first of which induced increases of Ca2+ capacity. We conclude that BiP has a dual function, in addition to its chaperone role it is a bona fide ER lumenal Ca2+ storage protein contributing, under resting cell conditions, to around 25% of the store, with a stoichiometry of 1-2 moles of calcium/mole of BiP.

The properties of a subtype of the inositol 1,4,5-trisphosphate receptor resulting from alternative splicing of the mRNA in the ligand-binding domain.

Subtypes of the type-1 inositol 1,4,5-trisphosphate (InsP3) receptor differ at the mRNA level in two small variably spliced segments. One segment (SI) encodes for a sequence within the InsP3-binding domain, thus its presence or absence could affect the functions of the receptor. We have used anti-peptide antibodies to confirm the existence of different subtypes of the InsP3 receptor (InsP3R) protein. The antibody against residues 322-332, within the SI region, recognized a 260 kDa polypeptide in membranes prepared from rat cerebellum or cerebral cortex. The cerebellum contained a few percent of the InsP3R protein having the SI region, whereas the cerebral cortex contained a high proportion of receptors with the SI region. These two tissues were representative of both isoforms, SI- or SI+, and displayed the same [3H]InsP3-binding characteristics. Thus, the SI region was not involved in the basic properties of the receptor. Deletion of the peptide 316-352 containing the SI segment greatly reduced InsP3 binding [Miyawaki, Furuichi, Ryou, Yoshikawa, Nakagawa, Saitoh and Mikoshiba (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4911-4915]. The antibodies against the SI region or against residues 337-349 did not modify the binding of [3H]InsP3 in the cortical membranes rich in the SI+ isoform or in cerebellar membranes. These results suggested that the SI region was not part of the binding site. The subcellular distribution of these two isoforms was then investigated in rat liver. The two isoforms were identified in different membrane fractions and they followed the same subcellular distribution. We suggest that the domain with the SI region may be involved in a function other than InsP3-induced Ca2+ release.

Intracellular calcium stores and inositol 1,4,5-trisphosphate receptor in rat liver cells.

The D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor was localized by immunofluorescence experiments in situ in liver cryosections. Two anti-Ins(1,4,5)P3 receptor antibodies (against the 14 C-terminal residues of the type 1 receptor or against the entire cerebellar receptor) weakly decorated the whole cytoplasm, and a more intense labelling was observed at the periphery of the hepatocytes, particularly beneath the canalicular and the sinusoidal domains of the plasma membrane (PM). Antibodies against calreticulin, the Ca2+ pump (SERCA2b) or endoplasmic reticulum (ER) membranes homogeneously labelled the cytoplasm and the subplasmalemmal area. These data indicate that the ER can be divided into at least two specialized subregions: one is located throughout most of the cytoplasm and contains markers of the rough ER (RER), calreticulin, SERCA2b and a low density of Ins(1,4,5)P3 receptor, and the other is confined to the periphery of the cells and contains calreticulin, Ca2+ pump, RER markers and a high density of Ins(1,4,5)P3 receptor. A membrane fraction enriched in Ins(1,4,5)P3 receptor and in markers of the PM was immuno-adsorbed with the antibody against the C-terminal end of the Ins(1,4,5)P3 receptor and pelleted with Sepharose protein A. The immuno-isolated material was enriched in Ins(1,4,5)P3 receptor, but none of the markers of the ER or of the PM could be detected. This suggests that the Ins(1,4,5)P3 receptor is localized on discrete domains of the ER membrane beneath the canalicular and the sinusoidal membranes, where it was found at higher densities than the other markers.

The inositol 1,4,5-trisphosphate receptor is localized on specialized sub-regions of the endoplasmic reticulum in rat liver.

Inositol 1,4,5-trisphosphate (InsP3) is involved in the mobilization of Ca2+ from intracellular non-mitochondrial stores. In rat liver, it has been shown that the InsP3-binding site co-purifies with the plasma membrane. This suggests that in the liver the InsP3 receptor (InsP3R) associates with plasma membrane. We studied the subcellular distribution of the liver InsP3R by measuring the maximal binding capacity of [3H]InsP3 and using antibodies against the 14 C-terminal residues of the type 1 InsP3R. The antibodies recognized a large amount of an InsP3R protein of 260 kDa in a membrane fraction which is also enriched with [3H]InsP3-binding sites and with markers of the basal, the lateral and the bile-canalicular membrane and the plasma-membrane Ca2+ pump (PMCA). The fractions enriched in markers of the endoplasmic reticulum (ER) and the Ca2+ pump of the ER (SERCA2b) contained low levels of InsP3 receptors. The immunofluorescent labelling of cultured hepatocytes with anti-InsP3R antibodies indicated that the receptor is concentrated in the perinuclear area and in some regions near the plasma membrane. The fraction enriched with InsP3R is also contaminated with markers of the ER and with SERCA2b. It was exposed to alkaline medium (pH 10.5) to extract endogenous actin and membrane-associated proteins before being subfractionated by Percoll-gradient centrifugation. The alkaline treatment allowed partial separation of the markers of the ER from the markers of the plasma membrane. The InsP3R was recovered in the heavy subfraction, which was also enriched with markers for the ER and with the SERCA2b and contained low levels of markers of the plasma membrane. These data indicate that the InsP3R is neither localized on the plasma membrane itself nor homogeneously distributed on the ER membrane. This supports the view that part of the receptor is localized on a specialized sub-region of the ER which interacts with the plasma membrane.

The inositol 1,4,5-trisphosphate receptor: kinetic properties and regulation.

Inositol 1,4,5-triphosphate (InsP3) is a second messenger responsible for the mobilization of intracellular Ca2+ after receptor-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate. InsP3 binds to a specific receptor located on the membrane of an intracellular compartment and opens a Ca2+ channel causing the cytosolic Ca2+ concentration to increase. Measurement of radiolabelled InsP3 binding and InsP3-induced Ca2+ release in parallel experiments indicated that the liver InsP3 receptor exists in two main states: an active state (A) and an inactive one (I). The "I" form of the receptor is found in the presence of high Ca2+ concentrations (above 1 microM). The binding properties of the "A" and the "I" states of the receptor have been characterized by analysing a membrane fraction enriched in InsP3 receptors. The inactive "I" state displays a high affinity (Kd = 2 nM) and slow rates of association and dissociation. The active state "A" of the receptor displays complex kinetic properties. The rate of association and the rate of dissociation of labelled InsP3 are rapid phenomena probably involving several components. The apparent Kd for the InsP3 binding is about 40 nM in a low Ca2+ medium. The affinity of the "A" state of the receptor is increased by Ca2+ (at concentrations lower than 0.5 microM) and by thiol reagents. The increase of the affinity of the receptor is due to a decrease of the dissociation rate constants. This lowers the threshold such that Ca2+ is released at lower concentrations of InsP3. These data indicate that the binding of InsP3 to its receptor is a complex phenomenon involving the transition among several states.(ABSTRACT TRUNCATED AT 250 WORDS)