T cell receptor-mediated Ca2+ signaling: Release and influx are independent events linked to different Ca2+ entry pathways in the plasma membrane

In this study, we showed that cross-linking CD3 molecules on the T cell surface resulted in Ca²⁺ release from the intracellular stores followed by a sustained Ca²⁺ influx. Inhibition of release with TMB-8 did not block the influx. However, inhibition of phospholipase C activity suppressed both Ca²⁺ release and influx. Once activated, the influx pathway remained open in the absence of further hydrolysis of PIP2. Thapsigargin, a microsomal Ca²⁺ -ATPase inhibitor, stimulated Ca²⁺ entry into the cells by a mechanism other than emptying Ca²⁺ stores. In addition, Ca²⁺ entry into the Ca²⁺ -depleted cells was stimulated by low basal level of cytosolic Ca²⁺, not by the emptying of intracellular Ca²⁺ stores. Both the Ca²⁺ release and influx were dependent on high and low concentrations of extracellular Ca²⁺. At low concentrations, Mn²⁺ entered the cell through the Ca²⁺ influx pathway and quenched the sustained phase of fluorescence; whereas, at higher Mn²⁺ concentration both the transient and the sustained phases of fluorescence were quenched. Moreover, Ca²⁺ release was inhibited by low concentrations of Ni²⁺, La³⁺, and EGTA, while Ca²⁺ influx was inhibited by high concentrations. Thus, in T cells Ca²⁺ influx occurs independently of IP3-dependent Ca²⁺ release. However, some other PIP2 hydrolysis-dependent event was involved in prolonged activation of Ca²⁺ influx. Extracellular Ca²⁺ influenced Ca²⁺ release and influx through the action of two plasma membrane Ca²⁺ entry pathways with different pharmacological and biochemical properties.     

Lymphotoxin and an associated 33-kDa glycoprotein are expressed on the surface of an activated human T cell hybridoma.

A human T cell hybridoma, II-23.D7, was induced with phorbol ester to express a surface form of lymphotoxin (LT, TNF-beta) and an associated 33-kDa glycoprotein. The LT epitopes were detected by surface immunofluorescence staining and by immunoprecipitation from radioiodinated or biosynthetically labeled cells with the use of anti-rLT polyclonal and monoclonal antibodies. The epitopes detected by the antibody were related to LT because adsorption of the anti-rLT with PMA-activated II-23.D7 cells resulted in the removal of the neutralizing titer of the anti-rLT antiserum. Immunoprecipitation of surface radioiodinated II-23.D7 cells revealed two bands of 25 kDa and 33 kDa that were specifically precipitated with anti-rLT, but not anti-rTNF antibodies. Enzymatic digestion with glycanases showed both proteins to have N-linked carbohydrate, with O-linked sugar limited to the 25-kDa protein. To determine the biochemical relationship between these proteins, the two LT-like forms were purified from detergent-solubilized II-23.D7 cells by immunoaffinity chromatography. Peptide mapping using CNBr cleavage showed the 25-kDa surface form to be identical to rLT, whereas the 33-kDa protein was different. Biosynthetic labeling studies showed that p33 contained both methionine and cysteine, whereas the p25 contained only methionine. Thus, the surface LT form lacks a leader peptide indicating an anchoring mechanism distinct from that described for membrane TNF. The nature of the attachment of this LT form to the membrane surface is not clear, however, neither TNF receptor binding nor lipid linkages appear to be involved. The accessory protein, p33, may anchor LT to the surface. These findings identify a new characteristic of LT and point toward an additional pathway by which T lymphocytes may mediate cytolytic activity and regulate inflammatory processes.     

Chymotryptic-type protease inhibitors block the increase in Ca2+ and Il-2 production in activated Jurkat T cells

Several chymotryptic-type protease inhibitors were found to inhibit both anti-CD3 mAb- and PHA-induced rise in Ca2+ and IL-2 production in Jurkat T cells. The magnitude of inhibition was a function of the effectors used to stimulate Ca2+ entry and depended on the concentration of the inhibitors. Neither tryptic-type protease inhibitors nor an elastase substrate prevented anti-CD3 mAb- or PHA-induced Ca2+ rise in Jurkat cells. The inhibitory effect of N-alpha-p-tosyl-L-phenylalanine chloromethyl-ketone on anti-CD3 mAb- and PHA-induced rise in Ca2+ resulted from a rapid increase in Ca2+ efflux. The inhibitors which were effective on Ca2+ mobilization also inhibited IL-2 production initiated by an anti-CD3 mAb in the presence of 12-O-tetradecanoylphorbol-13-acetate, and to a lesser extent by PHA or the calcium ionophore A23187. No inhibition of IL-2 production was observed when tryptic-type protease inhibitors or the elastase inhibitor were used. In addition, membrane preparations from Jurkat cells were found to hydrolyze the chymotryptic substrate Suc-Ala-Ala-Phe-paranitroaniline, an effect markedly inhibited by N-alpha-p-tosyl-L-phenylalanine chloromethylketone. Moreover, this inhibitor protected one potential endogenous substrate (Mr 38 kDa) from proteolysis. Taken together, these observations show that chymotryptic-type protease inhibitors block the responses generated by the binding of anti-CD3 mAb to Jurkat cells, and suggest that a chymotryptic-like membrane protease contributes to T cell activation.     

The role of Ca2+ in activation of mature cytotoxic T lymphocytes for lysis

We carried out a detailed analysis of the requirement for Ca2+ in the lysis of target cells by cloned cytotoxic T lymphocytes (CTL). In direct, antigen-specific lysis we always observed an influx of Ca2+ into the CTL concomitant with target cell binding. However, we never observed an increase in CTL Ca2+ content during lectin-mediated lysis, or nonspecific lysis by phorbol myristate acetate-induced CTL. We found that in all three types of lysis (direct, lectin-mediated lysis, C or phorbol myristate acetate-induced) the requirement for Ca2+ in lysis was dictated by the target cell used; the same CTL can kill one target cell in the absence of detectable Ca2+, and absolutely require Ca2+ for the lysis of another target cell. Target cell killing, when it occurred in the absence of Ca2+, was accompanied by microtubule organizing center reorientation in the CTL, showing that this function is not uniformly Ca2+ dependent. These results provide further evidence that Ca2+ is not always required for activation of the lytic pathway in CTL, although Ca2+ may be absolutely required for other CTL functions such as interleukin production or expression of the interleukin 2 receptor.     

The EF-hand Ca2+-binding protein p22 plays a role in microtubule and endoplasmic reticulum organization and dynamics with distinct Ca2+-binding requirements

We have reported that p22, an N-myristoylated EF-hand Ca(2+)-binding protein, associates with microtubules and plays a role in membrane trafficking. Here, we show that p22 also associates with membranes of the early secretory pathway membranes, in particular endoplasmic reticulum (ER). On binding of Ca(2+), p22's ability to associate with membranes increases in an N-myristoylation-dependent manner, which is suggestive of a nonclassical Ca(2+)-myristoyl switch mechanism. To address the intracellular functions of p22, a digitonin-based "bulk microinjection" assay was developed to load cells with anti-p22, wild-type, or mutant p22 proteins. Antibodies against a p22 peptide induce microtubule depolymerization and ER fragmentation; this antibody-mediated effect is overcome by preincubation with the respective p22 peptide. In contrast, N-myristoylated p22 induces the formation of microtubule bundles, the accumulation of ER structures along the bundles as well as an increase in ER network formation. An N-myristoylated Ca(2+)-binding p22 mutant, which is unable to undergo Ca(2+)-mediated conformational changes, induces microtubule bundling and accumulation of ER structures along the bundles but does not increase ER network formation. Together, these data strongly suggest that p22 modulates the organization and dynamics of microtubule cytoskeleton in a Ca(2+)-independent manner and affects ER network assembly in a Ca(2+)-dependent manner.     

Lymphotoxin is expressed as a heteromeric complex with a distinct 33-kDa glycoprotein on the surface of an activated human T cell hybridoma.

We characterized the membrane-associated form of lymphotoxin (surface LT) on the activated II-23.D7 T cell hybridoma. Antibodies to rLT precipitated both surface LT and a distinct 33-kDa glycoprotein (p33). Because p33 and surface LT were antigenically unrelated, their coprecipitation suggested a physical association of p33 and surface LT on the membrane. Pulse-chase analysis indicated that LT and p33 associate with each other early in the LT biosynthetic pathway, precluding the possibility that LT is secreted and bound to p33 or a surface receptor. Furthermore, no p33 was associated with the secreted form of LT. Isoelectric focusing of surface LT and p33 under nondenaturing and denaturing conditions confirmed that surface LT and p33 existed as a complex. Treatment of cells with a high concentration of salt or with acid indicated that surface LT is a peripheral membrane protein. Although secreted LT is a homologous trimer, protein cross-linking studies revealed that surface LT existed as a monomer associated with a dimer of p33. Together the results demonstrate a novel mechanism for stable membrane expression of LT by activated T cells.     

Structural requirements for differential sensitivity of KCNQ K+ channels to modulation by Ca2+/calmodulin

Calmodulin modulation of ion channels has emerged as a prominent theme in biology. The sensitivity of KCNQ1-5 K+ channels to modulation by Ca2+/calmodulin (CaM) was studied using patch-clamp, Ca2+ imaging, and biochemical and pharmacological approaches. Coexpression of CaM in Chinese hamster ovary (CHO) cells strongly reduced currents of KCNQ2, KCNQ4, and KCNQ5, but not KCNQ1 or KCNQ3. In simultaneous current recording/Ca2+ imaging experiments, CaM conferred Ca2+ sensitivity to KCNQ4 and KCNQ5, but not to KCNQ1, KCNQ3, or KCNQ1/KCNE1 channels. A chimera constructed from the carboxy terminus of KCNQ4 and the rest KCNQ1 displayed Ca2+ sensitivity similar to KCNQ4. Chimeras constructed from different lengths of the KCNQ4 carboxy terminal and the rest KCNQ3 localized a region that confers sensitivity to Ca2+/CaM. Lobe-specific mutations of CaM revealed that its amino-terminal lobe mediates the Ca2+ sensitivity of the KCNQ/CaM complex. The site of CaM action within the channel carboxy terminus overlaps with that of the KCNQ opener N-ethylmaleimide (NEM). We found that CaM overexpression reduced NEM augmentation of KCNQ2, KCNQ4, and KCNQ5, and NEM pretreatment reduced Ca2+/CaM-mediated suppression of M current in sympathetic neurons by bradykinin. We propose that two functionally distinct types of carboxy termini underlie the observed differences among this channel family.     

Two distinct Ca2+ compartments show differential sensitivity to thrombin, ADP and vasopressin in human platelets

Recent studies propose the existence of two distinct Ca2+ compartments in human platelets based on the expression of different SERCA isoforms with distinct sensitivity to thapsigargin and 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). Using fura-2-loaded human platelets we have found that depletion of the TBHQ sensitive store reduces thrombin--but not ADP--or vasopressin (AVP)-induced Ca2+ release. Redistribution of cytosolic Ca2+ after thrombin stimulation resulted in overloading of the TBHQ-sensitive store. This phenomenon was not observed with ADP or AVP. We found that NAADP decreases the Ca2+ concentration into the stores in permeabilized platelets, which is prevented by depletion of the TBHQ-sensitive store. Nimodipine, an inhibitor of the NAADP receptor, reduced thrombin-induced Ca2+ release from the TBHQ-sensitive stores, without having any effect on the responses elicited by ADP or AVP. Finally, the phospholipase C inhibitor, U-73122, abolished ADP- and AVP-induced Ca2+ release, suggesting that their responses are entirely dependent on IP3 generation. In contrast, treatment with both U-73122 and nimodipine was required to abolish thrombin-induced Ca2+ release. We suggest that thrombin evokes Ca2+ release from TBHQ-sensitive and insensitive stores, which requires both NAADP and IP3, respectively, while ADP and AVP exert an IP3-dependent release of Ca2+ from the TBHQ-insensitive compartment in human platelets.     

Cell shape-dependent Control of Ca2+ influx and cell cycle progression in Swiss 3T3 fibroblasts

The ability of adherent cells such as fibroblasts to enter the cell cycle and progress to S phase is strictly dependent on the extent to which individual cells can attach to and spread on a substratum. Here we have used microengineered adhesive islands of 22 and 45 mum diameter surrounded by a nonadhesive substratum of polyhydroxyl methacrylate to accurately control the extent to which individual Swiss 3T3 fibroblasts may spread. The effect of cell shape on mitogen-evoked Ca2+ signaling events that accompany entry into the cell cycle was investigated. In unrestricted cells, the mitogens bombesin and fetal calf serum evoked a typical biphasic change in the cytoplasmic free Ca2+ concentration. However, when the spreading of individual cells was restricted, such that progression to S phase was substantially reduced, both bombesin and fetal calf serum caused a rapid transient rise in the cytoplasmic free Ca2+ concentration but failed to elicit the normal sustained influx of Ca2+ that follows Ca2+ release. As expected, restricting cell spreading led to the loss of actin stress fibers and the formation of a ring of cortical actin. Restricting cell shape did not appear to influence mitogen-receptor interactions, nor did it influence the presence of focal adhesions. Because Ca2+ signaling is an essential component of mitogen responses, these findings implicate Ca2+ influx as a necessary component of cell shape-dependent control of the cell cycle.     

CRACM1, CRACM2, and CRACM3 are store-operated Ca2+ channels with distinct functional properties

STIM1 in the endoplasmic reticulum and CRACM1 in the plasma membrane are essential molecular components for controlling the store-operated CRAC current. CRACM1 proteins multimerize and bind STIM1, and the combined overexpression of STIM1 and CRACM1 reconstitutes amplified CRAC currents. Mutations in CRACM1 determine the selectivity of CRAC currents, demonstrating that CRACM1 forms the CRAC channel's ion-selective pore, but the CRACM1 homologs CRACM2 and CRACM3 are less well characterized. Here, we show that both CRACM2 and CRACM3, when overexpressed in HEK293 cells stably expressing STIM1, potentiate I(CRAC) to current amplitudes 15-20 times larger than native I(CRAC). A nonconducting mutation of CRACM1 (E106Q) acts as a dominant negative for all three CRACM homologs, suggesting that they can form heteromultimeric channel complexes. All three CRACM homologs exhibit distinct properties in terms of selectivity for Ca(2+) and Na(+), differential pharmacological effects in response to 2-APB, and strikingly different feedback regulation by intracellular Ca(2+). Each of the CRAC channel proteins' specific functional features and the potential heteromerization provide for flexibility in shaping Ca(2+) signals, and their characteristic biophysical and pharmacological properties will aid in identifying CRAC-channel species in native cells that express them.     

Curcumin suppresses T cell activation by blocking Ca2+ mobilization and nuclear factor of activated T cells (NFAT) activation

Curcumin is the active ingredient of the spice turmeric and has been shown to have a number of pharmacologic and therapeutic activities including antioxidant, anti-microbial, anti-inflammatory, and anti-carcinogenic properties. The anti-inflammatory effects of curcumin have primarily been attributed to its inhibitory effect on NF-κB activity due to redox regulation. In this study, we show that curcumin is an immunosuppressive phytochemical that blocks T cell-activation-induced Ca(2+) mobilization with IC(50) = ～12.5 μM and thereby prevents NFAT activation and NFAT-regulated cytokine expression. This finding provides a new mechanism for curcumin-mediated anti-inflammatory and immunosuppressive function. We also show that curcumin can synergize with CsA to enhance immunosuppressive activity because of different inhibitory mechanisms. Furthermore, because Ca(2+) is also the secondary messenger crucial for the TCR-induced NF-κB signaling pathway, our finding also provides another mechanism by which curcumin suppresses NF-κB activation.     

The coexistence in rat muscle cells of two distinct classes of Ca2+-dependent K+ channels with different pharmacological properties and different physiological functions

An Arthrobacter sp. has been shown to dehalogenate 4-chlorobenzoate yielding 4-hydroxybenzoate. Experiments with 18O indicate that, in the presence of cell-free extracts, the hydroxyl group which is substituted onto the aromatic nucleus during dehalogenation is derived from water and not from molecular oxygen. Dehalogenation therefore is not catalysed by a mixed-function oxidase; instead a novel aromatic hydroxylase is implicated in the reaction.     

Comparative studies of the cytotoxic T lymphocyte-mediated cytotoxicity and of extracellular ATP-induced cell lysis. Different requirements in extracellular Mg2+ and pH

We recently proposed that extracellular ATP (ATPo) may be involved in CTL-mediated cytotoxicity by acting in concert with yet unidentified cellular components (ATPo receptors/ATPo-binding proteins, ectoprotein kinases). The TCR-triggered ATPo accumulation by CTL has been demonstrated, whereas the resistance of CTL to ATPo was explained by the action of highly active ecto-ATPases or by the absence of relevant ATP-binding proteins. However, no data were available to discriminate between the possibilities of: i) ATPo acting alone as a "hit" molecule because of the cell-permeabilizing properties of ATP4- or ii) ATPo acting as a "messenger" (as MgATP2-) in concert with other molecules. Comparing ATPo-induced and CTL-mediated cell lysis, we found that ATPo-induced lysis of some target cells is greatly decreased at neutral and acidic pH, whereas Ca(2+)-dependent CTL-mediated lysis of the same cells is barely affected. In agreement with the observed pH dependency, at low Mg2+ concentrations, which favor ATP4- over MgATP2-, maximal ATPo-induced lysis was observed. However, CTL-mediated cytotoxicity in both Ag-specific and retargeting assays was markedly reduced at low Mg2+ concentrations. These results suggest that ATPo acting alone as a "hit" molecule cannot fully account for the extracellular Ca(2+)-dependent lethal hit delivery by CTL or that ATP4- is active at very low concentrations. This conclusion was further supported by studying the lytic effect of ATPo and CTL on the anti-TCR mAb-coupled SRBC. CTL were efficient in the SRBC lysis, whereas no lysis of SRBC by ATPo was detected. The resistance of SRBC to ATPo is not caused by a high ATPo degradation, because the ecto-ATPase activity of SRBC was much lower than in ATPo-resistant CTL OE4 cells and comparable with EL4 tumor cells, which were easily lysed by ATPo. These data suggested the need for careful consideration of the pH and cation composition of the media used for studying ATPo effects. The caveats in the use of ATP-degrading enzymes to implicate the role of extracellular ATPo in the CTL-mediated cytotoxicity are described here. A clarification of the previously described cytotoxicity inhibition by hexokinase, which is caused by an inhibitory salt effect, is presented. It is suggested that if Ca(2+)-dependent lysis of SRBC and of other target cells by CTL does involve extracellular ATP, it may function as a "messenger" in concert with other extracellular molecules.(ABSTRACT TRUNCATED AT 400 WORDS)     

Coupling of K+-gating and permeation with Ca2+ block in the Ca2+-Activated K+ channel inChara australis

Summary The patch-clamp technique is used here to investigate the kinetics of Ca²⁺ block in single high-conductance Ca²⁺-activated K⁺ channels. These channels are detected in the membrane surounding cytoplasmic drops fromChara australis, a membrane which originates from the tonoplast of the parent cell. The amplitudes and durations of single channel events are measured over a wide range of membrane potential (–300 to 200 mV). Ca²⁺ on either side of the channel reduces its K⁺ conductance and alters its ion-gating characteristics in a voltage-dependent manner. This Ca²⁺-induced attenuation of conductance is analyzed using the theory of diffusion-limited ion flow through pores. Interaction of external Ca²⁺ with the channel's ion-gating mechanism is examined in terms of a kinetic model for ion-gating that includes two voltage-dependent gating mechanisms. The kinetics of channel block by external Ca²⁺ indicates that (i) external Ca²⁺ binds at two sites, a superficial site and a deep site, located at 8 and 40% along the trans-pore potential difference, (ii) the external vestibule cannot be occupied by more than one Ca²⁺ or K⁺, and (iii) the kinetics of Ca²⁺ binding at the deep site is coupled with that of a voltage-dependent gate on the external side of the channel. Kinetics of channel block by internal Ca²⁺ indicates that more than one Ca²⁺ is involved.     

The single pore residue Asp542 determines Ca2+ permeation and Mg2+ block of the epithelial Ca2+ channel

The epithelial Ca(2+) channel (ECaC), which was recently cloned from rabbit kidney, exhibits distinctive properties that support a facilitating role in transcellular Ca(2+) (re)absorption. ECaC is structurally related to the family of six transmembrane-spanning ion channels with a pore-forming region between S5 and S6. Using point mutants of the conserved negatively charged amino acids present in the putative pore, we have identified a single aspartate residue that determines Ca(2+) permeation of ECaC and modulation by extracellular Mg(2+). Mutation of the aspartate residue, D542A, abolishes Ca(2+) permeation and Ca(2+)-dependent current decay as well as block by extracellular Mg(2+), whereas monovalent cations still permeate the mutant channel. Variation of the side chain length in mutations D542N, D542E, and D542M attenuated Ca(2+) permeability and Ca(2+)-dependent current decay. Block of monovalent currents through ECaC by Mg(2+) was decreased. Exchanging the aspartate residue for a positively charged amino acid, D542K, resulted in a nonfunctional channel. Mutations of two neighboring negatively charged residues, i.e. Glu(535) and Asp(550), had only minor effects on Ca(2+) permeation properties.     

Novel function of phosphoinositide 3-kinase in T cell Ca2+ signaling. A phosphatidylinositol 3,4,5-trisphosphate-mediated Ca2+ entry mechanism

This study presents evidence that phosphoinositide (PI) 3-kinase is involved in T cell Ca(2+) signaling via a phosphatidylinositol 3,4, 5-trisphosphate PI(3,4,5)P(3)-sensitive Ca(2+) entry pathway. First, exogenous PI(3,4,5)P(3) at concentrations close to its physiological levels induces Ca(2+) influx in T cells, whereas PI(3,4)P(2), PI(4, 5)P(2), and PI(3)P have no effect on [Ca(2+)](i). This Ca(2+) entry mechanism is cell type-specific as B cells and a number of cell lines examined do not respond to PI(3,4,5)P(3) stimulation. Second, inhibition of PI 3-kinase by wortmannin and by overexpression of the dominant negative inhibitor Deltap85 suppresses anti-CD3-induced Ca(2+) response, which could be reversed by subsequent exposure to PI(3,4,5)P(3). Third, PI(3,4,5)P(3) is capable of stimulating Ca(2+) efflux from Ca(2+)-loaded plasma membrane vesicles prepared from Jurkat T cells, suggesting that PI(3,4,5)P(3) interacts with a Ca(2+) entry system directly or via a membrane-bound protein. Fourth, although D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4, 5)P(4)) mimics PI(3,4,5)P(3) in many aspects of biochemical functions such as membrane binding and Ca(2+) transport, we raise evidence that Ins(1,3,4,5)P(4) does not play a role in anti-CD3- or PI(3,4,5)P(3)-mediated Ca(2+) entry. This PI(3,4,5)P(3)-stimulated Ca(2+) influx connotes physiological significance, considering the pivotal role of PI 3-kinase in the regulation of T cell function. Given that PI 3-kinase and phospholipase C-gamma form multifunctional complexes downstream of many receptor signaling pathways, we hypothesize that PI(3,4,5)P(3)-induced Ca(2+) entry acts concertedly with Ins(1,4,5)P(3)-induced Ca(2+) release in initiating T cell Ca(2+) signaling. By using a biotinylated analog of PI(3,4,5)P(3) as the affinity probe, we have detected several putative PI(3,4,5)P(3)-binding proteins in T cell plasma membranes.