Structure and autoregulation of a P4-ATPase lipid flippase.

Type 4 P-type ATPases (P4-ATPases) are lipid flippases that drive the active transport of phospholipids from exoplasmic or luminal leaflets to cytosolic leaflets of eukaryotic membranes. The molecular architecture of P4-ATPases and the mechanism through which they recognize and transport lipids have remained unknown. Here we describe the cryo-electron microscopy structure of the P4-ATPase Drs2p-Cdc50p, a Saccharomyces cerevisiae lipid flippase that is specific to phosphatidylserine and phosphatidylethanolamine. Drs2p-Cdc50p is autoinhibited by the C-terminal tail of Drs2p, and activated by the lipid phosphatidylinositol-4-phosphate (PtdIns4P or PI4P). We present three structures that represent the complex in an autoinhibited, an intermediate and a fully activated state. The analysis highlights specific features of P4-ATPases and reveals sites of autoinhibition and PI4P-dependent activation. We also observe a putative lipid translocation pathway in this flippase that involves a conserved PISL motif in transmembrane segment 4 and polar residues of transmembrane segments 2 and 5, in particular Lys1018, in the centre of the lipid bilayer.

Effects of Covalent Conjugates of Fullerene Derivatives with Xanthene Dyes on Activity of Ca2+-ATPase of the Sarcoplasmic Reticulum.

The effects of the newly synthesized covalent conjugates of water-soluble fullerene derivatives (WSFD) with xanthene dyes: polyanionic WSFD-fluorescein (1), polycationic WSFD-fluorescein (2), polyanionic WSFD-eosin (3), and polyanionic WSFD (4), polycationic WSFD (5), fluorescein (6) and eosin (7), on activity of the membrane-bound Ca2+-ATPase of the sarcoplasmic reticulum (SR Ca2+-ATPase) were studied. Compounds 1, 3, 4, 6, and 7 inhibit the hydrolytic function of the enzyme, the inhibition constants for these compounds are Ki=1.3×10-5 M (1), Ki=4.7×10-6 M (3), Ki=2.5×10-6 M (4), Ki=6.1×10-5 M (6), and Ki=5.8×10-6 M (7). The effects of compounds 3, 6, and 7 on the hydrolytic function of the enzyme is competitive; compounds 1 and 4 are noncompetitive. Polycationic WSFD fluorescein (2) and polycationic WSFD (5) do not affect ATP hydrolysis, but inhibit active Ca2+ transport in a concentration of 0.01 mM by 100±10 and 40±4%, respectively. Conjugates 1 and 3 completely inhibit the hydrolytic and transport functions of the enzyme in a concentration of 0.01 mM, and in a concentration of 0.001 mM inhibit active Ca2+ transport by 60±6 and 55±6% uncoupling the hydrolytic and transport functions of SR Ca2+-ATPases. The obtained results demonstrate a significant effect of the studied compounds on the active transmembrane transfer of Ca2+ and make it possible to predict the presence of antimetastatic and antiaggregatory activities of the studied compounds.

Neuroleptic Chlorpromazine Modulates Ca2+ Responses in Macrophages.

Using Fura-2AM microfluorimetry, we have shown for the first time that sigma-1 receptor antagonist neuroleptic chlorpromazine significantly inhibits glutoxim- and molixan-induced Ca2+ responses and Ca2+ responses induced by endoplasmic reticulum Са2+-ATPase inhibitors thapsigargin and cyclopiazonic acid in rat peritoneal macrophages. The results suggest the involvement of sigma-1 receptors in the signaling cascade induced by glutoxim or molixan and leading to intracellular Ca2+ concentration increase and in the regulation of store-dependent Ca2+ entry in macrophages.

Biochemical characterization and chemical inhibition of PfATP4-associated Na+-ATPase activity in Plasmodium falciparum membranes.

The antimalarial activity of chemically diverse compounds, including the clinical candidate cipargamin, has been linked to the ATPase PfATP4 in the malaria-causing parasite Plasmodium falciparum The characterization of PfATP4 has been hampered by the inability thus far to achieve its functional expression in a heterologous system. Here, we optimized a membrane ATPase assay to probe the function of PfATP4 and its chemical sensitivity. We found that cipargamin inhibited the Na+-dependent ATPase activity present in P. falciparum membranes from WT parasites and that its potency was reduced in cipargamin-resistant PfATP4-mutant parasites. The cipargamin-sensitive fraction of membrane ATPase activity was inhibited by all 28 of the compounds in the "Malaria Box" shown previously to disrupt ion regulation in P. falciparum in a cipargamin-like manner. This is consistent with PfATP4 being the direct target of these compounds. Characterization of the cipargamin-sensitive ATPase activity yielded data consistent with PfATP4 being a Na+ transporter that is sensitive to physiologically relevant perturbations of pH, but not of [K+] or [Ca2+]. With an apparent Km for ATP of 0.2 mm and an apparent Km for Na+ of 16-17 mm, the protein is predicted to operate at below its half-maximal rate under normal physiological conditions, allowing the rate of Na+ efflux to increase in response to an increase in cytosolic [Na+]. In membranes from a cipargamin-resistant PfATP4-mutant line, the apparent Km for Na+ is slightly elevated. Our study provides new insights into the biochemical properties and chemical sensitivity of an important new antimalarial drug target.

Functional characterization of the Ca2+-ATPase SMA1 from Schistosoma mansoni.

Schistosoma mansoni is a parasite that causes bilharzia, a neglected tropical disease affecting hundreds of millions of people each year worldwide. In 2012, S. mansoni had been identified as the only invertebrate possessing two SERCA-type Ca2+-ATPases, SMA1 and SMA2. However, our analysis of recent genomic data shows that the presence of two SERCA pumps is rather frequent in parasitic flatworms. To understand the reasons of this redundancy in S. mansoni, we compared SMA1 and SMA2 at different levels. In terms of sequence and organization, the genes SMA1 and SMA2 are similar, suggesting that they might be the result of a duplication event. At the protein level, SMA1 and SMA2 only slightly differ in length and in the sequence of the nucleotide-binding domain. To get functional information on SMA1, we produced it in an active form in Saccharomyces cerevisiae, as previously done for SMA2. Using phosphorylation assays from ATP, we demonstrated that like SMA2, SMA1 bound calcium in a cooperative mode with an apparent affinity in the micromolar range. We also showed that SMA1 and SMA2 had close sensitivities to cyclopiazonic acid but different sensitivities to thapsigargin, two specific inhibitors of SERCA pumps. On the basis of transcriptomic data available in GeneDB, we hypothesize that SMA1 is a housekeeping Ca2+-ATPase, whereas SMA2 might be required in particular striated-like muscles like those present the tail of the cercariae, the infecting form of the parasite.

Thapsigargin, Inhibitor of Sarco-Endoplasmic Ca2+-ATPase, Effectively Suppresses the Expression of S100A4 Protein in Human Breast Cancer Cell Line.

Thapsigargin (SERCA ATPase inhibitor) inhibited the S100A4 metastatic marker expression in MDA-MB231 breast cancer cells. We found that S100A4 gene transcription is regulated by Ca2+ signaling pathways. We found that the synthesis of S100A4 mRNA and S100A4 protein in MDA-MB231 cells was effectively suppressed by thapsigargin at a concentration of 0.4-4 µM with retaining cell viability. We assume that the change in the gene transcription in response to disturbance of Ca2+ homeostasis is directly involved in the remodeling of Ca2+ signaling pathways.

Structure/activity relationship of thapsigargin inhibition on the purified Golgi/secretory pathway Ca2+/Mn2+-transport ATPase (SPCA1a).

The Golgi/secretory pathway Ca2+/Mn2+-transport ATPase (SPCA1a) is implicated in breast cancer and Hailey-Hailey disease. Here, we purified recombinant human SPCA1a from Saccharomyces cerevisiae and measured Ca2+-dependent ATPase activity following reconstitution in proteoliposomes. The purified SPCA1a displays a higher apparent Ca2+ affinity and a lower maximal turnover rate than the purified sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1a). The lipids cholesteryl hemisuccinate, linoleamide/oleamide, and phosphatidylethanolamine inhibit and phosphatidic acid and sphingomyelin enhance SPCA1a activity. Moreover, SPCA1a is blocked by micromolar concentrations of the commonly used SERCA1a inhibitors thapsigargin (Tg), cyclopiazonic acid, and 2,5-di-tert-butylhydroquinone. Because tissue-specific targeting of SERCA2b by Tg analogues is considered for prostate cancer therapy, the inhibition of SPCA1a by Tg might represent an off-target risk. We assessed the structure-activity relationship (SAR) of Tg for SPCA1a by in silico modeling, site-directed mutagenesis, and measuring the potency of a series of Tg analogues. These indicate that Tg and the analogues are bound via the Tg scaffold but with lower affinity to the same homologous cavity as on the membrane surface of SERCA1a. The lower Tg affinity may depend on a more flexible binding cavity in SPCA1a, with low contributions of the Tg O-3, O-8, and O-10 chains to the binding energy. Conversely, the protein interaction of the Tg O-2 side chain with SPCA1a appears comparable with that of SERCA1a. These differences define a SAR of Tg for SPCA1a distinct from that of SERCA1a, indicating that Tg analogues with a higher specificity for SPCA1a can probably be developed.

Inhibition of Lithium Sensitive Orai1/ STIM1 Expression and Store Operated Ca2+ Entry in Chorea-Acanthocytosis Neurons by NF-κB Inhibitor Wogonin.

BACKGROUND/AIMS: The neurodegenerative disease Chorea-Acanthocytosis (ChAc) is caused by loss-of-function-mutations of the chorein-encoding gene VPS13A. In ChAc neurons transcript levels and protein abundance of Ca2+ release activated channel moiety (CRAC) Orai1 as well as its regulator STIM1/2 are decreased, resulting in blunted store operated Ca2+-entry (SOCE) and enhanced suicidal cell death. SOCE is up-regulated and cell death decreased by lithium. The effects of lithium are paralleled by upregulation of serum & glucocorticoid inducible kinase SGK1 and abrogated by pharmacological SGK1 inhibition. In other cell types SGK1 has been shown to be partially effective by upregulation of NFκB, a transcription factor stimulating the expression of Orai1 and STIM. The present study explored whether pharmacological inhibition of NFκB interferes with Orai1/STIM1/2 expression and SOCE and their upregulation by lithium in ChAc neurons. METHODS: Cortical neurons were differentiated from induced pluripotent stem cells generated from fibroblasts of ChAc patients and healthy volunteers. Orai1 and STIM1 transcript levels and protein abundance were estimated from qRT-PCR and Western blotting, respectively, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarco-endoplasmatic Ca2+-ATPase inhibitor thapsigargin (1µM), as well as CRAC current utilizing whole cell patch clamp recording. RESULTS: Orai1 and STIM1 transcript levels and protein abundance as well as SOCE and CRAC current were significantly enhanced by lithium treatment (2 mM, 24 hours). These effects were reversed by NFκB inhibitor wogonin (50 µM). CONCLUSION: The stimulation of expression and function of Orai1/STIM1/2 by lithium in ChAc neurons are disrupted by pharmacological NFκB inhibition.

Design, synthesis and anti-malarial activities of synthetic analogs of biselyngbyolide B, a Ca2+ pump inhibitor from marine cyanobacteria.

Biselyngbyaside, an 18-membered macrolide glycoside from marine cyanobacteria, and its derivatives are known to be sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitors. Recently, a SERCA orthologue of the malaria parasite, PfATP6, has attracted attention as a malarial drug target. To provide a novel drug lead, we designed new synthetic analogs of biselyngbyolide B, the aglycone of biselyngbyaside, based on the co-crystal structure of SERCA with biselyngbyolide B, and synthesized them using the established synthetic route for biselyngbyolide B. Their biological activities against malarial parasites were evaluated.

Nanoscale patterning of STIM1 and Orai1 during store-operated Ca2+ entry.

Stromal interacting molecule (STIM) and Orai proteins constitute the core machinery of store-operated calcium entry. We used transmission and freeze-fracture electron microscopy to visualize STIM1 and Orai1 at endoplasmic reticulum (ER)-plasma membrane (PM) junctions in HEK 293 cells. Compared with control cells, thin sections of STIM1-transfected cells possessed far more ER elements, which took the form of complex stackable cisternae and labyrinthine structures adjoining the PM at junctional couplings (JCs). JC formation required STIM1 expression but not store depletion, induced here by thapsigargin (TG). Extended molecules, indicative of STIM1, decorated the cytoplasmic surface of ER, bridged a 12-nm ER-PM gap, and showed clear rearrangement into small clusters following TG treatment. Freeze-fracture replicas of the PM of Orai1-transfected cells showed extensive domains packed with characteristic "particles"; TG treatment led to aggregation of these particles into sharply delimited "puncta" positioned upon raised membrane subdomains. The size and spacing of Orai1 channels were consistent with the Orai crystal structure, and stoichiometry was unchanged by store depletion, coexpression with STIM1, or an Orai1 mutation (L273D) affecting STIM1 association. Although the arrangement of Orai1 channels in puncta was substantially unstructured, a portion of channels were spaced at ∼15 nm. Monte Carlo analysis supported a nonrandom distribution for a portion of channels spaced at ∼15 nm. These images offer dramatic, direct views of STIM1 aggregation and Orai1 clustering in store-depleted cells and provide evidence for the interaction of a single Orai1 channel with small clusters of STIM1 molecules.

Preferential nitrite inhibition of the mitochondrial F1FO-ATPase activities when activated by Ca(2+) in replacement of the natural cofactor Mg(2+).

BACKGROUND: The mitochondrial F1FO-ATP synthase has not only the known life function in building most cellular ATP, but also, as recently hinted, an amazing involvement in cell death. Accordingly, the two-faced enzyme complex, which catalyzes both ATP synthesis and ATP hydrolysis, has been involved in the mitochondrial permeability transition, the master player in apoptosis and necrosis. Nitrite, a cellular nitric oxide reservoir, has a recognized role in cardiovascular protection, through still unclear mechanisms. METHODS: In swine heart mitochondria the effect of nitrite on the F1FO-ATPase activity activated by Ca(2+), henceforth defined as Ca-ATPase(s), or by the natural cofactor Mg(2+), was investigated by evaluating ATP hydrolysis under different assay conditions. RESULTS: Ca(2+) is far less efficient than the natural cofactor Mg(2+) in the ATPase activation. However, when activated by Ca(2+) the ATPase activity is especially responsive to nitrite, which acts as uncompetitive inhibitor and up to 2 mM inhibits the Ca2+-activated-ATPase(s), probably by promoting dytirosine formation on the enzyme proteins, leaving the Mg-ATPase(s) unaffected. Most likely these ATPases refer to the same F1FO complex, even if coexistent ATPases may overlap. CONCLUSIONS: The preferential inhibition by nitrite of the Ca-ATPase(s), due to post-translational tyrosine modifications, may prevent the calcium-dependent functionality of the mitochondrial F1FO complex and related events. GENERAL SIGNIFICANCE: In mitochondria the preferential inhibition of the Ca-ATPase activity/ies by nitrite concentrations which do not affect the coexistent Mg-ATPase(s) may quench the negative events linked to the calcium-dependent functioning mode of the F1FO complex under pathological conditions.

A pollen coat-inducible autoinhibited Ca2+-ATPase expressed in stigmatic papilla cells is required for compatible pollination in the Brassicaceae.

In the Brassicaceae, intraspecific non-self pollen (compatible pollen) can germinate and grow into stigmatic papilla cells, while self-pollen or interspecific pollen is rejected at this stage. However, the mechanisms underlying this selective acceptance of compatible pollen remain unclear. Here, using a cell-impermeant calcium indicator, we showed that the compatible pollen coat contains signaling molecules that stimulate Ca(2+) export from the papilla cells. Transcriptome analyses of stigmas suggested that autoinhibited Ca(2+)-ATPase13 (ACA13) was induced after both compatible pollination and compatible pollen coat treatment. A complementation test using a yeast Saccharomyces cerevisiae strain lacking major Ca(2+) transport systems suggested that ACA13 indeed functions as an autoinhibited Ca(2+) transporter. ACA13 transcription increased in papilla cells and in transmitting tracts after pollination. ACA13 protein localized to the plasma membrane and to vesicles near the Golgi body and accumulated at the pollen tube penetration site after pollination. The stigma of a T-DNA insertion line of ACA13 exhibited reduced Ca(2+) export, as well as defects in compatible pollen germination and seed production. These findings suggest that stigmatic ACA13 functions in the export of Ca(2+) to the compatible pollen tube, which promotes successful fertilization.

Identification of novel inhibitors of human SPCA2.

To identify specific inhibitors of the human secretary pathway Ca(2+)-ATPase 2 (hSPCA2), a recombinant hSPCA2 was expressed in Saccharomyces cerevisiae, and purified by Co(2+)-chelating chromatography. The isolated hSPCA2 catalyzed ATP hydrolysis in the presence of Ca(2+) ions. The Ca(2+) dissociation constant for ATPase activation was 25 nM. hSPCA2 activity was inhibited by thapsigargin, 2,2'-methylenebis(6-tert-butyl-p-cresol), and 4-octylphenol in the low-micromolar concentration range. Unexpectedly, the organic solvent wash from standard laboratory polypropylene microtubes showed strong inhibitory potency toward hSPCA2 activity. The extract was found to comprise mainly primary fatty acid amides (PFAAs) by NMR analysis. Individual PFAAs, especially oleamide and linoleamide, almost completely inhibited hSPCA2 activity with IC50 values of 7.5 µM and 3.8 µM, respectively.

Abnormal synaptic Ca(2+) homeostasis and morphology in cortical neurons of familial hemiplegic migraine type 1 mutant mice.

OBJECTIVE: Migraine is among the most common and debilitating neurological conditions. Familial hemiplegic migraine type 1 (FHM1), a monogenic migraine subtype, is caused by gain-of-function of voltage-gated CaV 2.1 calcium channels. FHM1 mice carry human pathogenic mutations in the α1A subunit of CaV 2.1 channels and are highly susceptible to cortical spreading depression (CSD), the electrophysiologic event underlying migraine aura. To date, however, the mechanism underlying increased CSD/migraine susceptibility remains unclear. METHODS: We employed in vivo multiphoton microscopy of the genetically encoded Ca(2+)-indicator yellow cameleon to investigate synaptic morphology and [Ca(2+)]i in FHM1 mice. To study CSD-induced cerebral oligemia, we used in vivo laser speckle flowmetry and multimodal imaging. With electrophysiologic recordings, we investigated the effect of the CaV 2.1 gating modifier tert-butyl dihydroquinone on CSD in vivo. RESULTS: FHM1 mutations elevate neuronal [Ca(2+)]i and alter synaptic morphology as a mechanism for enhanced CSD susceptibility that we were able to normalize with a CaV 2.1 gating modifier in hyperexcitable FHM1 mice. At the synaptic level, axonal boutons were larger, and dendritic spines were predominantly of the mushroom type, which both provide a structural correlate for enhanced neuronal excitability. Resting neuronal [Ca(2+)]i was elevated in FHM1, with loss of compartmentalization between synapses and neuronal shafts. The percentage of calcium-overloaded neurons was increased. Neuronal [Ca(2+)]i surge during CSD was faster and larger, and post-CSD oligemia and hemoglobin desaturation were more severe in FHM1 brains. INTERPRETATION: Our findings provide a mechanism for enhanced CSD susceptibility in hemiplegic migraine. Abnormal synaptic Ca(2+) homeostasis and morphology may contribute to chronic neurodegenerative changes as well as enhanced vulnerability to ischemia in migraineurs.

Biological membranes: the importance of molecular detail.

Are lipid interactions with membrane proteins best described in terms of the physical properties of the lipid bilayer or in terms of direct molecular interactions between particular lipid molecules and particular sites on a protein? A molecular interpretation is more challenging because it requires detailed knowledge of the 3D structure of a membrane protein, but recent studies have suggested that a molecular interpretation is necessary. Here, the idea is explored that lipid molecules modify the ways that transmembrane α-helices pack into bundles, by penetrating between the helices and by binding into clefts between the helices, and that these effects on helix packing will modulate the activity of a membrane protein.

Intracellular Ca(2+) release from endoplasmic reticulum regulates slow wave currents and pacemaker activity of interstitial cells of Cajal.

Interstitial cells of Cajal (ICC) provide pacemaker activity in gastrointestinal muscles that underlies segmental and peristaltic contractions. ICC generate electrical slow waves that are due to large-amplitude inward currents resulting from anoctamin 1 (ANO1) channels, which are Ca(2+)-activated Cl(-) channels. We investigated the hypothesis that the Ca(2+) responsible for the stochastic activation of ANO1 channels during spontaneous transient inward currents (STICs) and synchronized activation of ANO1 channels during slow wave currents comes from intracellular Ca(2+) stores. ICC, obtained from the small intestine of Kit(+/copGFP) mice, were studied under voltage and current clamp to determine the effects of blocking Ca(2+) uptake into stores and release of Ca(2+) via inositol 1,4,5-trisphosphate (IP3)-dependent and ryanodine-sensitive channels. Cyclocpiazonic acid, thapsigargin, 2-APB, and xestospongin C inhibited STICs and slow wave currents. Ryanodine and tetracaine also inhibited STICs and slow wave currents. Store-active compounds had no direct effects on ANO1 channels expressed in human embryonic kidney-293 cells. Under current clamp, store-active drugs caused significant depolarization of ICC and reduced spontaneous transient depolarizations (STDs). After block of ryanodine receptors with ryanodine and tetracaine, repolarization did not restore STDs. ANO1 expressed in ICC has limited access to cytoplasmic Ca(2+) concentration, suggesting that pacemaker activity depends on Ca(2+) dynamics in restricted microdomains. Our data from studies of isolated ICC differ somewhat from studies on intact muscles and suggest that release of Ca(2+) from both IP3 and ryanodine receptors is important in generating pacemaker activity in ICC.

Translational control during endoplasmic reticulum stress beyond phosphorylation of the translation initiation factor eIF2α.

The accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) causes stress to which an unfolded protein response is activated to render cell survival or apoptosis (chronic stress). Transcriptional and translational reprogramming is tightly regulated during the unfolded protein response to ensure specific gene expression. The master regulator of this response is the PERK/eIF2α/ATF4 signaling where eIF2α is phosphorylated (eIF2α-P) by the kinase PERK. This signal leads to global translational shutdown, but it also enables translation of the transcription factor ATF4 mRNA. We showed recently that ATF4 induces an anabolic program through the up-regulation of selected amino acid transporters and aminoacyl-tRNA synthetases. Paradoxically, this anabolic program led cells to apoptosis during chronic ER stress in a manner that involved recovery from stress-induced protein synthesis inhibition. By using eIF2α-P-deficient cells as an experimental system, we identified a communicating network of signaling pathways that contribute to the inhibition of protein synthesis during chronic ER stress. This eIF2α-P-independent network includes (i) inhibition of mammalian target of rapamycin kinase protein complex 1 (mTORC1)-targeted protein phosphorylation, (ii) inhibited translation of a selective group of 5'-terminal oligopyrimidine mRNAs (encoding proteins involved in the translation machinery and translationally controlled by mTORC1 signaling), and (iii) inhibited translation of non-5'-terminal oligopyrimidine ribosomal protein mRNAs and ribosomal RNA biogenesis. We propose that the PERK/eIF2α-P/ATF4 signaling acts as a brake in the decline of protein synthesis during chronic ER stress by positively regulating signaling downstream of the mTORC1 activity. These studies advance our knowledge on the complexity of the communicating signaling pathways in controlling protein synthesis rates during chronic stress.

Regulation of glomerulotubular balance. III. Implication of cytosolic calcium in flow-dependent proximal tubule transport.

In the proximal tubule, axial flow (drag on brush-border microvilli) stimulates Na(+) and HCO3 (-) reabsorption by modulating both Na/H exchanger 3 (NHE3) and H-ATPase activity, a process critical to glomerulotubular balance. We have also demonstrated that blocking the angiotensin II receptor decreases baseline transport, but preserves the flow effect; dopamine leaves baseline fluxes intact, but abrogates the flow effect. In the current work, we provide evidence implicating cytosolic calcium in flow-dependent transport. Mouse proximal tubules were microperfused in vitro at perfusion rates of 5 and 20 nl/min, and reabsorption of fluid (Jv) and HCO3 (-) (JHCO3) were measured. We examined the effect of high luminal Ca(2+) (5 mM), 0 mM Ca(2+), the Ca(2+) chelator BAPTA-AM, the inositol 1,4,5-trisphosphate (IP3) receptor antagonist 2-aminoethoxydiphenyl borate (2-APB), and the Ca-ATPase inhibitor thapsigargin. In control tubules, increasing perfusion rate from 5 to 20 nl/min increased Jv by 62% and JHCO3 by 104%. With respect to Na(+) reabsorption, high luminal Ca(2+) decreased transport at low flow, but preserved the flow-induced increase; low luminal Ca(2+) had little impact; both BAPTA and 2-APB had no effect on baseline flux, but abrogated the flow effect; thapsigargin decreased baseline flow, leaving the flow effect intact. With respect to HCO3 (-) reabsorption, high luminal Ca(2+) decreased transport at low flow and mildly diminished the flow-induced increase; low luminal Ca(2+) had little impact; both BAPTA and 2-APB had no effect on baseline flux, but abrogated the flow effect. These data implicate IP3 receptor-mediated intracellular Ca(2+) signaling as a critical step in transduction of microvillous drag to modulate Na(+) and HCO3 (-) transport.

Entamoeba invadens: Identification of a SERCA protein and effect of SERCA inhibitors on encystation.

Calcium has an important role on signaling of different cellular processes, including growth and differentiation. Signaling by calcium also has an essential function in pathogenesis and differentiation of the protozoan parasites Entamoeba histolytica and Entamoeba invadens. However, the proteins of these parasites that regulate the cytoplasmic concentration of this ion are poorly studied. In eukaryotic cells, the calcium-ATPase of the SERCA type plays an important role in calcium homeostasis by catalyzing the active efflux of calcium from cytoplasm to endoplasmic reticulum. Here, we reported the identification of SERCA of E. invadens (EiSERCA). This protein contains a putative sequence for endoplasmic reticulum retention and all domains involved in calcium transport identified in mammalian SERCA. By immunofluorescence assays, an antibody against SERCA of E. histolytica detected EiSERCA in a vesicular network in the cytoplasm of E. invadens trophozoites, co-localizing with calreticulin. Interestingly, EiSERCA was redistributed close to plasma membrane during encystation, suggesting that this pump could participate in regulate the calcium concentration during this process. In addition, thapsigargin and cyclopiazonic acid, both specific inhibitors of SERCA, affected the number and structure of cysts, supporting the hypothesis that calcium flux mediated by SERCA has an important role in the life cycle of Entamoeba.

Activation of muscarinic acetylcholine receptors elevates intracellular Ca(2+) concentrations in accessory lobe neurons of the chick.

Accessory lobes are protrusions located at the lateral sides of the spinal cord of chicks and it has been proposed that they play a role as a sensory organ for equilibrium during walking. We have reported that functional neurons exist in the accessory lobe. As there is histological evidence that synaptic terminals of cholinergic nerves exist near the somata of accessory lobe neurons, we examined the effects of acetylcholine on changes in intracellular Ca2+ concentrations ([Ca2+]i), as an index of cellular activities. Acetylcholine (0.1-100 µM) caused a transient rise in the [Ca2+]i. Acetylcholine-evoked [Ca2+]i rises were observed in the absence of extracellular Ca2+, and they were abolished in the presence of cyclopiazonic acid, an inhibitor of Ca2+-ATPase of intracellular Ca2+ stores or atropine, a muscarinic receptor antagonist. mRNAs coding M3 and M5 isoforms of the muscarinic receptors were detected in accessory lobes by the RT-PCR. These results indicate that chick accessory lobe neurons express functional muscarinic acetylcholine receptors, and that acetylcholine stimulates Ca2+ mobilization from intracellular Ca2+ stores, which elevates the [Ca2+]i in the somata of accessory lobe neurons, through activation of these receptors. Cholinergic synaptic transmission to the accessory lobe neurons may regulate some cellular functions through muscarinic receptors.