Lymphocytes possess an electrogenic H(+)-transporting pathway in their plasma membrane.
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ABSTRACT:
SUBMITTER: Kaldi K
PROVIDER: S-EPMC1137083 | biostudies-other | 1994 Jul
REPOSITORIES: biostudies-other
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Project description:BACKGROUND:The intracellular concentration of heavy-metal cations, such as copper, nickel, and zinc is pivotal for the mycobacterial response to the hostile environment inside macrophages. To date, copper transport mediated by P-type ATPases across the mycobacterial plasma membrane has not been sufficiently explored. RESULTS:In this work, the ATPase activity of the putative Mycobacterium tuberculosis P1B-type ATPase CtpB was associated with copper (I) transport from mycobacterial cells. Although CtpB heterologously expressed in M. smegmatis induced tolerance to toxic concentrations of Cu2+ and a metal preference for Cu+, the disruption of ctpB in M. tuberculosis cells did not promote impaired cell growth or heavy-metal accumulation in whole mutant cells in cultures under high doses of copper. In addition, the Cu+ ATPase activity of CtpB embedded in the plasma membrane showed features of high affinity/slow turnover ATPases, with enzymatic parameters KM 0.19?±?0.04 µM and Vmax 2.29?±?0.10 nmol/mg min. In contrast, the ctpB gene transcription was activated in cells under culture conditions that mimicked the hostile intraphagosomal environment, such as hypoxia, nitrosative and oxidative stress, but not under high doses of copper. CONCLUSIONS:The overall results suggest that M. tuberculosis CtpB is associated with Cu+ transport from mycobacterial cells possibly playing a role different from copper detoxification.
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Project description:Cu-transporting ATPase ATP7B (Wilson disease protein) is essential for the maintenance of intracellular copper concentration. In hepatocytes, ATP7B is required for copper excretion, which is thought to occur via a transient delivery of the ATP7B- and copper-containing vesicles to the apical membrane. The currently available experimental systems do not allow analysis of ATP7B at the cell surface. Using epitope insertion, we identified an extracellular loop into which the HA-epitope can be introduced without inhibiting ATP7B activity. The HA-tagged ATP7B was expressed in Xenopus oocytes and the presence of ATP7B at the plasma membrane was demonstrated by electron microscopy, freeze-fracture experiments, and surface luminescence measurements in intact cells. Neither the deletion of the entire N-terminal copper-binding domain nor the inactivating mutation of catalytic Asp1027 affected delivery to the plasma membrane of oocytes. In contrast, surface targeting was decreased for the ATP7B variants with mutations in the ATP-binding site or the intra-membrane copper-binding site, suggesting that ligand-stabilized conformation(s) are important for ATP7B trafficking. The developed system provides significant advantages for studies that require access to both sides of ATP7B in the membrane.
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Project description:Adrenaline activates transient Cl(-) secretion and sustained K(+) secretion across isolated distal colonic mucosa of guinea-pigs. The Ca(2+)-activated Cl(-) channel inhibitor CaCCinh-A01 (30 μm) significantly reduced electrogenic K(+) secretion, detected as short-circuit current (I(sc)). This inhibition supported the cell model for K(+) secretion in which basolateral membrane Cl(-) channels provide an exit pathway for Cl(-) entering the cell via Na(+)-K(+)-2Cl(-) cotransporters. CaCCinh-A01 inhibited both I(sc) and transepithelial conductance in a concentration-dependent manner (IC(50) = 6.3 μm). Another Cl(-) channel inhibitor, GlyH-101, also reduced sustained adrenaline-activated I(sc) (IC(50) = 9.4 μm). Adrenaline activated whole-cell Cl(-) current in isolated intact colonic crypts, confirmed by ion substitution. This adrenaline-activated whole-cell Cl(-) current was also inhibited by CaCCinh-A01 or GlyH-101. In contrast to K(+) secretion, CaCCinh-A01 augmented the electrogenic Cl(-) secretion activated by adrenaline as well as that activated by prostaglandin E(2). Synergistic Cl(-) secretion activated by cholinergic/prostaglandin E(2) stimulation was insensitive to CaCCinh-A01. Colonic expression of the Ca(2+)-activated Cl(-) channel protein Tmem16A was supported by RT-PCR detection of Tmem16A mRNA, by immunoblot with a Tmem16A antibody, and by detection of immunofluorescence in lateral membranes of epithelial cells. Alternative splices of Tmem16A were detected for exons that are involved in channel activation. Inhibition of K(+) secretion and augmentation of Cl(-) secretion by CaCCinh-A01 support a common colonic cell model for these two ion secretory processes, such that activation of basolateral membrane Cl(-) channels contributes to the production of electrogenic K(+) secretion and limits the rate of Cl(-) secretion. Maximal physiological Cl(-) secretion occurs only for synergistic activation mechanisms that close these basolateral membrane Cl(-) channels.
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Project description:1. Lymphocytes from pig mesenteric lymph node have low permeability to K+ (Rb+), Na+ and Cl-. None of these ions is in Nernst equilibrium with the plasma-membrane potential (delta psi p). 2. delta psi p can be calculated from the transmembrane distribution of the permeant cation methyltriphenylphosphonium (TPMP+) in the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to abolish uptake into intracellular mitochondria. In normal culture medium delta psi p is 56 mV. 3. A similar potential is found in T-enriched pig cells and in mouse thymocytes. 4. The contribution of electrogenic (Na+ + K+)ATPase to delta psi p is about 7 mV. 5. The remainder of the lymphocyte delta psi p is a polyionic potential set up by K+ and Cl- with a permeability coefficient for Cl- of similar magnitude to that for K+.
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Project description:Increases in intracellular Ca2+ concentration ([Ca2+]i) after addition of the carboxylic Ca(2+)-transporting ionophore ionomycin have been reported to be inhibited by depolarization in mouse T- and B-lymphocytes, a result attributed to effects on the plasma-membrane Ca2+ pump. The present experiments were undertaken to test the hypothesis that activation of electrogenic Ca2+ uptake, due to release of Ca2+i from intracellular pools, underlies the membrane-potential (Em)-sensitivity of ionomycin-mediated Ca2+ uptake in T-lymphocytes. To address this proposal we have compared the characteristics of Ca2+ influx induced by ionomycin with those of Ca2+ influx activated by release of Ca2+ from intracellular stores (store-regulated Ca2+ uptake, SRCU). The endosomal Ca(2+)-ATPase inhibitor thapsigargin was used to bring about loss of Ca2+ from intracellular pools. In support of this hypothesis, we report that: (1) ionomycin releases Ca2+ from a thapsigargin-sensitive endosomal pool, a pool previously demonstrated to regulate electrogenic SRCU in rat thymic lymphocytes, (2) Ca2+ and Mn2+ uptake mediated by low doses of ionomycin are inhibited by compounds previously reported to inhibit SRCU, and (3) in the absence of SRCU, ionomycin-mediated Ca2+ uptake displays no sensitivity to Em. We conclude that activation of electrogenic SRCU can adequately account for the Em-sensitivity of Ca2+ flux previously attributed to alterations in Ca(2+)-pump activity. Such a mechanism of action may underlie previous reports of electrogenic Ca2+ transport mediated by ionomycin in other tissues.
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Project description:We have found using imaging techniques that stimulating Jurkat human leukaemic T-cells with ionomycin in the presence of FM1-43, a dye used to monitor exocytosis and endocytosis, causes large (6--10-fold) increases in FM1-43 fluorescence. These responses are too large to be caused by exocytosis. Instead, three lines of evidence suggest that FM1-43 is responding to phospholipid scrambling. First, ionomycin also stimulates increases in the fluorescence of annexin V, a phosphatidylserine-specific probe, while thapsigargin does not stimulate fluorescence increases of either probe. Secondly, cells that exhibit FM1-43 fluorescence increases after ionomycin stimulation stain with annexin V once FM1-43 is washed out. Thirdly, ionomycin stimulates uptake of 7-nitrobenz-2-oxa-1,3-diazole-labelled phosphatidylcholine, a specific assay for scramblase activity, whereas thapsigargin does not. We find that FM1-43 reports phospholipid scrambling with 'better' kinetics than annexin V, and does require extracellular Ca(2+) to report phospholipid scrambling. We suggest that FM1-43 may be a useful probe to study the dynamics of phospholipid scrambling. The results are the first demonstration that FM1-43 can respond significantly to a biological process other than vesicular trafficking.
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Project description:Lymphocyte plasma membrane was isolated from minced pig mesenteric lymph node by differential centrifugation and by centrifuging through a sucrose density gradient. The yield of membrane was approx. 0.1% (dry wt. relative to wet wt. of lymph node). The purified material had a sucrose density of 1.14g/cm(3) and consisted mainly of smooth vesicles. The membrane fraction contained, apart from protein and lipid, 59mug of carbohydrate, 11mug of sialic acid and 28mug of RNA/mg of protein; no DNA was detected. The cholesterol/phospholipid molar ratio was 1.01. Specific activities (mumol of product/h per mg of protein) of 5'-nucleotidase, succinate dehydrogenase, acid phosphatase and glucose 6-phosphatase were 10.1, 0, 0.51 and 0.30 respectively. The membrane vesicles were aggregated by an antiserum against pig lymphocytes and adsorbed the agglutinins to whole lymphocytes present in the antiserum; the membrane fraction was 28 times as effective as whole cells (on a dry wt. basis) in removing the lympho-agglutinins. Antisera against the membrane fraction agglutinated whole lymphocytes. It is concluded that the preparation represents the plasma membrane of small lymphocytes. The plasma membrane of pig thymocytes was isolated by using the same procedure. Its properties were similar to those of the lymphocyte plasma membrane.
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Project description:The mode of activation of an H(+)-conducting pathway present in the membrane of neutrophils was investigated. (1) Resting neutrophils released protons through an electrogenic Cd(2+)-inhibitable (K0.5 approximately 20 microM) route when a pH gradient and appropriate charge compensation was provided. (2) The rate of H+ efflux was stimulated over 2.5-fold by 4 beta-phorbol 12-myristate 13-acetate (PMA; K0.5 approximately 0.7 nM) or by 4 beta-phorbol 12,13-dibutyrate (K0.5 approximately 20 nM) even when the NADPH oxidase was blocked by p-chloromercuribenzoate. (3) Staurosporine inhibited the effect of PMA. (4) The H+ egress was not enhanced by 4 alpha-phorbol 12,13-didecanoate. (5) Low concentrations of Cd2+ (less than 40 microM) inhibited the H+ flux without influencing the oxidase. The results raise the possibility that protein kinase C could be involved in the activation of an electrogenic H(+)-conducting pathway in the membrane of neutrophils. The activation of this route by phorbol esters seems to be independent of the stimulation of NADPH oxidase.
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Project description:Sodium/proton exchangers of the SLC9 family mediate the transport of protons in exchange for sodium to help regulate intracellular pH, sodium levels, and cell volume. In electrogenic Na+/H+ antiporters, it has been assumed that two ion-binding aspartate residues transport the two protons that are later exchanged for one sodium ion. However, here we show that we can switch the antiport activity of the bacterial Na+/H+ antiporter NapA from being electrogenic to electroneutral by the mutation of a single lysine residue (K305). Electroneutral lysine mutants show similar ion affinities when driven by [Formula: see text]pH, but no longer respond to either an electrochemical potential ([Formula: see text]) or could generate one when driven by ion gradients. We further show that the exchange activity of the human Na+/H+ exchanger NHA2 (SLC9B2) is electroneutral, despite harboring the two conserved aspartic acid residues found in NapA and other bacterial homologues. Consistently, the equivalent residue to K305 in human NHA2 has been replaced with arginine, which is a mutation that makes NapA electroneutral. We conclude that a transmembrane embedded lysine residue is essential for electrogenic transport in Na+/H+ antiporters.
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Project description:To understand the transcriptional response of mammalian cells to plasma membrane stress we induced partial loss of plasmid membrane integrity by chemogenetic induction of pore forming proteins (Gasderimin and MLKL) or treatment with the detergent digitonin. We monitored transcription with mRNAseq in the immediate hours after plasma membrane damage and identified conserved transcriptional programs.
2021-10-04 | GSE166538 | GEO