Showing papers in "The Journal of Membrane Biology in 2009"

Selenium modulates oxidative stress-induced cell apoptosis in human myeloid HL-60 cells through regulation of calcium release and caspase-3 and -9 activities.

Abstract: Selenium is an essential chemopreventive antioxidant element to oxidative stress, although high concentrations of selenium induce toxic and oxidative effects on the human body. However, the mechanisms behind these effects remain elusive. We investigated toxic effects of different selenium concentrations in human promyelocytic leukemia HL-60 cells by evaluating Ca2+ mobilization, cell viability and caspase-3 and -9 activities at different sample times. We found the toxic concentration and toxic time of H2O2 as 100 μm and 10 h on cell viability in the cells using four different concentrations of H2O2 (1 μm–1 mm) and six different incubation times (30 min, 1, 2, 5, 10, 24 h). Then, we found the therapeutic concentration of selenium to be 200 nm by cells incubated in eight different concentrations of selenium (10 nm–1 mm) for 1 h. We measured Ca2+ release, cell viability and caspase-3 and -9 activities in cells incubated with high and low selenium concentrations at 30 min and 1, 2, 5, 10 and 24 h. Selenium (200 nm) elicited mild endoplasmic reticulum stress and mediated cell survival by modulating Ca2+ release, the caspases and cell apoptosis, whereas selenium concentrations as high as 1 mm induced severe endoplasmic reticulum stress and caused cell death by activating modulating Ca2+ release, the caspases and cell apoptosis. In conclusion, these results explained the molecular mechanisms of the chemoprotective effect of different concentrations of selenium on oxidative stress-induced apoptosis.

Membrane porters of ATP-binding cassette transport systems are polyphyletic.

Abstract: The ATP-binding cassette (ABC) superfamily consists of both importers and exporters. These transporters have, by tradition, been classified according to the ATP hydrolyzing constituents, which are monophyletic. The evolutionary origins of the transmembrane porter proteins/domains are not known. Using five distinct computer programs, we here provide convincing statistical data suggesting that the transmembrane domains of ABC exporters are polyphyletic, having arisen at least three times independently. ABC1 porters arose by intragenic triplication of a primordial two-transmembrane segment (TMS)-encoding genetic element, yielding six TMS proteins. ABC2 porters arose by intragenic duplication of a dissimilar primordial three-TMS-encoding genetic element, yielding a distinctive protein family, nonhomologous to the ABC1 proteins. ABC3 porters arose by duplication of a primordial four-TMS-encoding genetic element, yielding either eight- or 10-TMS proteins. We assign each of 48 of the 50 currently recognized families of ABC exporters to one of the three evolutionarily distinct ABC types. Currently available high-resolution structural data for ABC porters are fully consistent with our findings. These results provide guides for future structural and mechanistic studies of these important transport systems.

Slc26A9 - anion exchanger, channel and Na+ transporter

Abstract: The SLC26 gene family encodes anion transporters with diverse functional attributes: (a) anion exchanger, (b) anion sensor, and (c) anion conductance (likely channel). We have cloned and studied Slc26a9, a paralogue expressed mostly in lung and stomach. Immunohistochemistry shows that Slc26a9 is present at apical and intracellular membranes of lung and stomach epithelia. Using expression in Xenopus laevis oocytes and ion-sensitive microelectrodes, we discovered that Slc26a9 has a novel function not found in any other Slc26 proteins: cation coupling. Intracellular pH and voltage measurements show that Slc26a9 is a nCl−-HCO 3 − exchanger, suggesting roles in gastric HCl secretion or pulmonary HCO 3 − secretion; Na+ electrodes and uptakes reveal that Slc26a9 has a cation dependence. Single-channel measurements indicate that Slc26a9 displays discrete open and closed states. These experiments show that Slc26a9 has three discrete physiological modes: nCl−-HCO 3 − exchanger, Cl− channel, and Na+-anion cotransporter. Thus, the Slc26a9 transporter channel is uniquely suited for dynamic and tissue-specific physiology or regulation in epithelial tissues.

Bioinformatic Characterization of P-Type ATPases Encoded Within the Fully Sequenced Genomes of 26 Eukaryotes

Abstract: P-type ATPases play essential roles in numerous processes, which in humans include nerve impulse propagation, relaxation of muscle fibers, secretion and absorption in the kidney, acidification of the stomach and nutrient absorption in the intestine. Published evidence suggests that uncharacterized families of P-type ATPases with novel specificities exist. In this study, the fully sequenced genomes of 26 eukaryotes, including animals, plants, fungi and unicellular eukaryotes, were analyzed for P-type ATPases. We report the organismal distributions, phylogenetic relationships, probable topologies and conserved motifs of nine functionally characterized families and 13 uncharacterized families of these enzyme transporters. We have classified these proteins according to the conventions of the functional and phylogenetic IUBMB-approved transporter classification system ( www.tcdb.org , Saier et al. in Nucleic Acids Res 34:181–186, 2006; Nucleic Acids Res 37:274–278, 2009).

A mathematical model of action potential in cells of vascular plants.

Abstract: A mathematical model of action potential (AP) in vascular plants cells has been worked out. The model takes into account actions of plasmalemma ion transport systems (K(+), Cl(-) and Ca(2+) channels; H(+)- and Ca(2+)-ATPases; 2H(+)/Cl(-) symporter; and H(+)/K(+) antiporter), changes of ion concentrations in the cell and in the extracellular space, cytoplasmic and apoplastic buffer capacities and the temperature dependence of active transport systems. The model of AP simulates a stationary level of the membrane potential and ion concentrations, generation of AP induced by electrical stimulation and gradual cooling and the impact of external Ca(2+) for AP development. The model supports a hypothesis about participation of H(+)-ATPase in AP generation.

Topiramate and vitamin e modulate the electroencephalographic records, brain microsomal and blood antioxidant redox system in pentylentetrazol-induced seizure of rats

Abstract: We investigated the effects of vitamin E and topiramate (TPM) administrations on pentylentetrazol (PTZ)-induced blood and brain toxicity in rats. Forty rats were randomly divided into five equal groups. The first and second groups were used for the control and PTZ groups, respectively. Fifty or 100 mg TPM were administered to rats constituting the third and fourth groups for 7 days, respectively. The TPM and vitamin E combination was given to animals in the fifth group. At the end of 7 days, all groups except the first received a single dose of PTZ. Blood and brain samples were taken at 3 hrs after PTZ administration. Lipid peroxidation levels of plasma, erythrocyte, brain cortex and brain microsomal fraction; nitric oxide levels of serum; and the number of spikes and epileptiform discharges of the EEG were increased by PTZ administration. Plasma and brain vitamin E concentration, erythrocyte glutathione peroxidase (GSH-Px) activity and latency to first spike of the EEG were decreased by PTZ. Plasma lipid peroxidation levels in the third group and plasma and erythrocyte lipid peroxidation levels in the fifth group were decreased compared to the second group, whereas brain vitamin C, vitamin E, erythrocyte GSH-Px and reduced glutathione (GSH) values increased in the fifth group. Brain microsomal GSH levels and EEG records in the third, fourth and fifth groups were restored by the TPM and vitamin E treatment. In conclusion, TPM and vitamin E seems to have protective effects on PTZ-induced blood and brain toxicity by inhibiting free radicals and supporting the antioxidant redox system.

Influences of flavonoids on erythrocyte membrane and metabolic implication through anionic exchange modulation.

Abstract: The antioxidative activity of some natural flavonoids was analyzed against the stable free radical 2,2-diphenyl-1-picryhydrazyl. The results indicate that the scavenging power of the tested flavonols is higher than that of the synthetic antioxidants butylated hydroxyanisole and butylated hydroxytoluene; instead, the flavanones show little activity, as indicated by efficient concentration (EC50) values. Flavonoid autoxidation and interaction with Fe2+ and hydrogen peroxide were tested using erythrocyte membranes as a model. The results show that some compounds, like hesperetin, evidence a pro-oxidant activity higher than the ascorbic acid/iron reference system. The compounds with strong oxidative capability do not only influence cellular redox balance but also activate caspase-3, producing lactate dehydrogenase release and enhancing anionic exchange at the level of band 3 protein.

Regulation of the Kv2.1 potassium channel by MinK and MiRP1

Abstract: Kv2.1 is a voltage-gated potassium (Kv) channel α-subunit expressed in mammalian heart and brain. MinK-related peptides (MiRPs), encoded by KCNE genes, are single–transmembrane domain ancillary subunits that form complexes with Kv channel α-subunits to modify their function. Mutations in human MinK (KCNE1) and MiRP1 (KCNE2) are associated with inherited and acquired forms of long QT syndrome (LQTS). Here, coimmunoprecipitations from rat heart tissue suggested that both MinK and MiRP1 form native cardiac complexes with Kv2.1. In whole-cell voltage-clamp studies of subunits expressed in CHO cells, rat MinK and MiRP1 reduced Kv2.1 current density three- and twofold, respectively; slowed Kv2.1 activation (at +60 mV) two- and threefold, respectively; and slowed Kv2.1 deactivation less than twofold. Human MinK slowed Kv2.1 activation 25%, while human MiRP1 slowed Kv2.1 activation and deactivation twofold. Inherited mutations in human MinK and MiRP1, previously associated with LQTS, were also evaluated. D76N–MinK and S74L–MinK reduced Kv2.1 current density (threefold and 40%, respectively) and slowed deactivation (60% and 80%, respectively). Compared to wild-type human MiRP1–Kv2.1 complexes, channels formed with M54T– or I57T–MiRP1 showed greatly slowed activation (tenfold and fivefold, respectively). The data broaden the potential roles of MinK and MiRP1 in cardiac physiology and support the possibility that inherited mutations in either subunit could contribute to cardiac arrhythmia by multiple mechanisms.

ABC Transporter Pdr10 Regulates the Membrane Microenvironment of Pdr12 in Saccharomyces cerevisiae

Abstract: The eukaryotic plasma membrane exhibits both asymmetric distribution of lipids between the inner and the outer leaflet and lateral segregation of membrane components within the plane of the bilayer. In budding yeast (Saccharomyces cerevisiae), maintenance of leaflet asymmetry requires P-type ATPases, which are proposed to act as inward-directed lipid translocases (Dnf1, Dnf2, and the associated protein Lem3), and ATP-binding cassette (ABC) transporters, which are proposed to act as outward-directed lipid translocases (Pdr5 and Yor1). The S. cerevisiae genome encodes two other Pdr5-related ABC transporters: Pdr10 (67% identity) and Pdr15 (75% identity). We report the first analysis of Pdr10 localization and function. A Pdr10-GFP chimera was located in discrete puncta in the plasma membrane and was found in the detergent-resistant membrane fraction. Compared to control cells, a pdr10∆ mutant was resistant to sorbate but hypersensitive to the chitin-binding agent Calcofluor White. Calcofluor sensitivity was attributable to a partial defect in endocytosis of the chitin synthase Chs3, while sorbate resistance was attributable to accumulation of a higher than normal level of the sorbate exporter Pdr12. Epistasis analysis indicated that Pdr10 function requires Pdr5, Pdr12, Lem3, and mature sphingolipids. Strikingly, Pdr12 was shifted to the detergent-resistant membrane fraction in pdr10∆ cells. Pdr10 therefore acts as a negative regulator for incorporation of Pdr12 into detergent-resistant membranes, a novel role for members of the ABC transporter superfamily.

The Influence of Membrane Lipids in Staphylococcus aureus Gamma-Hemolysins Pore Formation

Abstract: The natural target of Staphylococcus aureus bicomponent γ-hemolysins are leucocyte cell membranes. Because a proteinaceous receptor has not been found yet, we checked for the importance of the different membrane lipid compositions by measuring the activity of the toxin on several pure lipid model membranes. We investigated the effect of membrane thickness, fluidity, and presence of nonbilayer lipids and found that the toxin pore-forming ability increased in the presence of phosphocholines with short saturated acyl chains or with unsaturated chains even though not short. An increase of activity was also evident in the presence of cone-shaped lipids like phosphatidylethanolamine or diphytanoylphosphatidylcholine, whereas cylindrical lipids, like sphingomyelin, did not favor the activity. All these results suggest that γ-hemolysins could bind to the bilayer only if the phosphatidylcholine (PC) head is freely accessible. This condition is satisfied by the concurrent presence of cholesterol and certain lipids, as highlighted by the so-called umbrella model (J. Huang and G. W. Feigenson, Biophys J 76:2142–2157, 1999). According to this model, cholesterol could help to a better exposition of PC head groups only if acyl chains are short or unsaturated. In fact, phosphatidylcholines with more than 13 carbon atoms acyl chains can cover cholesterol molecules; in this way, PC head groups pack tightly, rendering them inaccessible to the toxin, which thus shows a reduced pore-forming ability.

Optical coherence tomography phase measurement of transient changes in squid giant axons during activity

Abstract: Noncontact optical measurements reveal that transient changes in squid giant axons are associated with action potential propagation and altered under different environmental (i.e., temperature) and physiological (i.e., ionic concentrations) conditions. Using a spectral-domain optical coherence tomography system, which produces real-time cross-sectional images of the axon in a nerve chamber, axonal surfaces along a depth profile are monitored. Differential phase analyses show transient changes around the membrane on a millisecond timescale, and the response is coincident with the arrival of the action potential at the optical measurement area. Cooling the axon slows the electrical and optical responses and increases the magnitude of the transient signals. Increasing the NaCl concentration bathing the axon, whose diameter is decreased in the hypertonic solution, results in significantly larger transient signals during action potential propagation. While monophasic and biphasic behaviors are observed, biphasic behavior dominates the results. The initial phase detected was constant for a single location but alternated for different locations; therefore, these transient signals acquired around the membrane appear to have local characteristics.

Cell Migration in BeWo Cells and the Role of Epithelial Sodium Channels

Abstract: Cell migration/proliferation processes associated with wound healing were measured in BeWo cells at 6 h, when mitosis is still scarce. Cells were cultured in medium with 1% fetal bovine serum to minimize proliferation. BeWo cell migration covered 20.6 ± 7.0%, 38.0 ± 5.4%, 16.6 ± 4.8% and 13.7 ± 3.6% of the wound when cultivated under control, aldosterone (100 nM, 12 h), aldosterone plus amiloride (10 μM) and amiloride treatments, respectively. When BeWo cells were treated with aldosterone, there was an increase in wound healing (P < 0.05), which was prevented by adding the ENaC blocker amiloride (P < 0.05, n = 16). Immunocytochemistry studies showed that the three ENaC subunits showed greater expression at the leading edge of the wound 3 h after injury, supporting the notion that these proteins participate in a postinjury signal. Antisense oligonucleotides directed against the α-ENaC subunit decreased the migratory response of the cells compared to the sense treated cells or the cells without oligonucleotides (P < 0.001, n = 16): 30.2 ± 3.7%, 17.6 ± 1.3%, 27.5 ± 1.5% and 20.2 ± 1.5% reinvasion of the wound with aldosterone, aldosterone plus antisense, aldosterone plus sense treatments and control conditions, respectively. Aldosterone and amiloride influence wound healing in BeWo cells, probably by their effects upon ENaCs, transmitting a signal to the cell cytoplasm for the release of several agents that promote cell migration.

Na+-glucose cotransporter SGLT1 protein in salivary glands: potential involvement in the diabetes-induced decrease in salivary flow.

Abstract: Oral health complications in diabetes include decreased salivary secretion. The SLC5A1 gene encodes the Na ? -glucose cotransporter SGLT1 protein, which not only transports glucose, but also acts as a water channel. Since SLC5A1 expression is altered in kidneys of diabetic subjects, we hypothesize that it could also be altered in salivary glands, contributing to diabetic dysfunction. The present study shows a diabetes-induced decrease (p \ 0.001) in salivary secretion, which was accompanied by enhanced (p \ 0.05) SGLT1 mRNA expression in parotid (50%) and submandibular (30%) glands. Immunohistochemical analy- sis of parotid gland of diabetic rats revealed that SGLT1 protein expression increased in the luminal membrane of ductal cells, which can stimulate water reabsorption from primary saliva. Furthermore, SGLT1 protein was reduced in myoepithelial cells of the parotid from diabetic animals, and that, by reducing cellular contractile activity, might also be related to reduced salivary flux. Six-day insulin-treated diabetic rats reversed all alterations. In conclusion, diabetes increases SLC5A1 gene expression in salivary glands, increasing the SGLT1 protein content in the luminal membrane of ductal cells, which, by increasing water

The effects of GPX-1 knockout on membrane transport and intracellular homeostasis in the lens.

Abstract: Glutathione peroxidase-1 (GPX-1) is an enzyme that protects the lens against H2O2-mediated oxidative damage. The purpose of the present study was to determine the effects of GPX-1 knockout (KO) on lens transport and intracellular homeostasis. To investigate these lenses we used (1) whole lens impedance studies to measure membrane conductance, resting voltage and fiber cell gap junction coupling conductance; (2) osmotic swelling of fiber cell membrane vesicles to determine water permeability; and (3) injection of Fura2 and Na+-binding benzofuran isophthalate (SBFI) into fiber cells to measure [Ca2+]i and [Na+]i, respectively, in intact lenses. These approaches were used to compare wild-type (WT) and GPX-1 KO lenses from mice around 2 months of age. There were no significant differences in clarity, size, resting voltage, membrane conductance or fiber cell membrane water permeability between WT and GPX-1 KO lenses. However, in GPX-1 KO lenses, coupling conductance was 72% of normal in the outer shell of differentiating fibers and 45% of normal in the inner core of mature fibers. Quantitative Western blots showed that GPX-1 KO lenses had about 50% as much labeled Cx46 and Cx50 protein as WT, whereas they had equivalent labeled AQP0 protein as WT. Both Ca2+ and Na+ accumulated significantly in the core of GPX-1 KO lenses. In summary, the major effect on lens transport of GPX-1 KO was a reduction in gap junction coupling conductance. This reduction affected the lens normal circulation by causing [Na+]i and [Ca2+]i to increase, which could increase cataract susceptibility in GPX-1 KO lenses.

Alamethicin Aggregation in Lipid Membranes

Abstract: X-ray scattering features induced by aggregates of alamethicin (Alm) were obtained in oriented stacks of model membranes of DOPC(diC18:1PC) and diC22:1PC. The first feature obtained near full hydration was Bragg rod in-plane scattering near 0.11 A−1 in DOPC and near 0.08 A−1 in diC22:1PC at a 1:10 Alm:lipid ratio. This feature is interpreted as bundles consisting of n Alm monomers in a barrel-stave configuration surrounding a water pore. Fitting the scattering data to previously published molecular dynamics simulations indicates that the number of peptides per bundle is n = 6 in DOPC and n ≥ 9 in diC22:1PC. The larger bundle size in diC22:1PC is explained by hydrophobic mismatch of Alm with the thicker bilayer. A second diffuse scattering peak located at q r ≈ 0.7 A−1 is obtained for both DOPC and diC22:1PC at several peptide concentrations. Theoretical calculations indicate that this peak cannot be caused by the Alm bundle structure. Instead, we interpret it as being due to two-dimensional hexagonally packed clusters in equilibrium with Alm bundles. As the relative humidity was reduced, interactions between Alm in neighboring bilayers produced more peaks with three-dimensional crystallographic character that do not index with the conventional hexagonal space groups.

Minimum Membrane Bending Energies of Fusion Pores

Abstract: Membranes fuse by forming highly curved intermediates, culminating in structures described as fusion pores. These hourglass-like figures that join two fusing membranes have high bending energies, which can be estimated using continuum elasticity models. Fusion pore bending energies depend strongly on shape, and the present study developed a method for determining the shape that minimizes bending energy. This was first applied to a fusion pore modeled as a single surface and then extended to a more realistic model treating a bilayer as two monolayers. For the two-monolayer model, fusion pores were found to have metastable states with energy minima at particular values of the pore diameter and bilayer separation. Fusion pore energies were relatively insensitive to membrane thickness but highly sensitive to spontaneous curvature and membrane asymmetry. With symmetrical bilayers and monolayer spontaneous curvatures of −0.1 nm−1 (a typical value) separated by 6 nm (closest distance determined by repulsive hydration forces), fusion pore formation required 43–65 kT. The pore radius of ~2.25 nm fell within the range estimated from conductance measurements. With bilayer separation >6 nm, fusion pore formation required less energy, suggesting that protein scaffolds can promote fusion by bending membranes toward one another. With nonzero spontaneous monolayer curvature, the shape that minimized the energy change during fusion pore formation differed from the shape that minimized its energy after it formed. Thus, a nascent fusion pore will relax spontaneously to a new shape, consistent with the experimentally observed expansion of nascent fusion pores during viral fusion.

Modulation of erythrocyte acetylcholinesterase activity and its association with G protein-band 3 interactions.

Abstract: Circulating acetylcholine, substrate of membrane acetylcholinesterase (AChE), is known to enhance the band 3 protein degree of phosphorylation. The purpose of this study was to verify whether the band 3 phosphorylation status is associated with a G protein and whether it is an influent factor on AChE enzyme activity. From blood samples of healthy donors, erythrocyte suspensions were prepared and incubated with AChE substrate (acetylcholine) and inhibitor (velnacrine), along with protein tyrosine kinase (PTK) and tyrosine phosphatase (PTP) inhibitors. AChE activity was determined by spectrophotometry and extract samples were analyzed by western blotting using primary antibodies to different G protein subunits. Our results with phosphorylated band 3 (PTP inhibitor) show an increase in erythrocyte AChE (p < 0.0001). A dephosphorylated band 3 state (PTK inhibitor) shows a significant decrease. We identified a potential linkage of protein subunits Gαi1/2 and Gβ with band 3 protein. Gαi1/2 and Gβ may be linked to the band 3 C-terminal site. Gαi1/2 is associated with the band 3 N-terminal domain, except for the control and ACh aliquots. Gβ is associated with both phosphorylated and dephosphorylated band 3 in the presence of velnacrine. We conclude that an erythrocyte G protein with subunits Gαi1/2 and Gβ is associated with band 3. AChE depends on the degree of band 3 phosphorylation and its association with Gαi1/2 and Gβ.

Membrane Transporters and Cytoplasmatic pH Regulation on Bovine Sertoli Cells

Abstract: Sertoli cells are responsible for regulating a wide range of processes that lead to the differentiation of male germ cells into spermatozoa. Cytoplasmic pH (pHi) has been shown to be an important parameter in cell physiology, regulating namely cell metabolism and differentiation. However, membrane transport mechanisms involved in pHi regulation mechanisms of Sertoli cells have not yet been elucidated. In this work, pHi was determined using the pH-sensitive fluorescent probe 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Addition of weak acids resulted in rapid acidification of the intracellular milieu. Sertoli cells then recovered pHi by a mechanism that was shown to be sensitive to external Na+. pHi recovery was also greatly reduced in the presence of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and amiloride. These results point toward the action of an Na+-driven HCO3-/Cl- exchanger and/or an Na+/HCO3- cotransporter and the action of the Na+/H+ exchanger on pHi regulation in the experimental conditions used. pHi recovery was only slightly affected by ouabain, suggesting that the inhibition of Na+/K+-ATPase affects recovery indirectly, possibly via the shift on the Na+ gradient. On the other hand, recovery from the acid load was independent of the presence of concanamycin A, a specific inhibitor of the V-type ATPases, suggesting that these pumps do not have a relevant action on pHi regulation in bovine Sertoli cells.

Mechanical Force and Cytoplasmic Ca 2+ Activate Yeast TRPY1 in Parallel

Abstract: The ability to sense mechanical and osmotic stimuli is vital to all organisms from mammals to bacteria. Members of the transient receptor potential (TRP) ion-channel family have attracted intense attention for their involvement in mechanosensation. The yeast homologue TRPY1 can clearly be activated by hypertonic shock in vivo and by stretch force under patch clamp. Like its animal counterparts, TRPY1 is polymodal, being gated by membrane stretch force and by cytoplasmic Ca2+. Here, we investigated how these two gating principles interact. We found that stretch force can induce some channel activation without cytoplasmic Ca2+. Tens of micromolar Ca2+ greatly enhance the observed force-induced activities, with open probabilities following well the Boltzmann distribution, in which the two gating energies are summed as exponents. To map this formalism to structures, we found Ca2+-binding proteins such as calmodulin or calcineurin to be unnecessary. However, removing a dense cluster of negative charges in the C-terminal cytoplasmic domain of TRPY1 greatly diminishes the Ca2+ activation as well as its influence on force activation. We also found a strategic point upstream of this charge cluster, at which insertion of amino acids weakens Ca2+ activation considerably but leaves the mechanosensitivity nearly intact. These results led to a structure–function model in which Ca2+ binding to the cytoplasmic domain and stretching of the membrane-embedded domain both generate gating force, reaching the gate in parallel.

Novel Residues Lining the CFTR Chloride Channel Pore Identified by Functional Modification of Introduced Cysteines

Abstract: Substituted cysteine accessibility mutagenesis (SCAM) has been used widely to identify pore-lining amino acid side chains in ion channel proteins. However, functional effects on permeation and gating can be difficult to separate, leading to uncertainty concerning the location of reactive cysteine side chains. We have combined SCAM with investigation of the charge-dependent effects of methanethiosulfonate (MTS) reagents on the functional permeation properties of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels. We find that cysteines substituted for seven out of 21 continuous amino acids in the eleventh and twelfth transmembrane (TM) regions can be modified by external application of positively charged [2-(trimethylammonium)ethyl] MTS bromide (MTSET) and negatively charged sodium [2-sulfonatoethyl] MTS (MTSES). Modification of these cysteines leads to changes in the open channel current-voltage relationship at both the macroscopic and single-channel current levels that reflect specific, charge-dependent effects on the rate of Cl(-) permeation through the channel from the external solution. This approach therefore identifies amino acid side chains that lie within the permeation pathway. Cysteine mutagenesis of pore-lining residues also affects intrapore anion binding and anion selectivity, giving more information regarding the roles of these residues. Our results demonstrate a straightforward method of screening for pore-lining amino acids in ion channels. We suggest that TM11 contributes to the CFTR pore and that the extracellular loop between TMs 11 and 12 lies close to the outer mouth of the pore.

Regulation of TLR2 Expression and Function in Human Airway Epithelial Cells

Abstract: Toll-like receptor (TLR1–6) mRNAs are expressed in normal human bronchial epithelial cells with higher basal levels of TLR3. TLR2 mRNA and plasma membrane protein expression was enhanced by pretreatment with Poly IC, a synthetic double-stranded RNA (dsRNA) known to activate TLR3. Poly IC also enhanced mRNA expression of adaptor molecules (MyD88 and TIRAP) and coreceptors (Dectin-1 and CD14) involved in TLR2 signaling. Additionally, mRNA expression of TLR3 and dsRNA-sensing proteins MDA5 and RIG-I increased following Poly IC treatment. In contrast, basal mRNA expression of TLR5 and TLR2 coreceptor CD36 was reduced by 77% and 62%, respectively. ELISA of apical and basolateral solutions from Poly IC-stimulated monolayers revealed significantly higher levels of IL-6 and GM-CSF compared with the TLR2 ligand PAM3CSK4. Pretreatment with anti-TLR2 blocking antibody inhibited the PAM3CSK4-induced increase in IL-6 secretion after Poly IC exposure. An increase in IL-6 secretion was also observed in cells stimulated with Alternaria extract after pretreatment with Poly IC. However, IL-6 secretion was not stimulated by zymosan or lipothechoic acid (LTA). These data demonstrated that upregulation of TLR2 following exposure to dsRNA enhances functional responses of the airway epithelium to certain (PAM3CSK4), but not all (zymosan, LTA) TLR2 ligands and that this is likely due to differences in coreceptor expression.

Concentration-Dependent Effects on Intracellular and Surface pH of Exposing Xenopus oocytes to Solutions Containing NH3/NH4+

Abstract: Others have shown that exposing oocytes to high levels of \( {\text{NH}}_{ 3} /{\text{NH}}_{ 4}{}^{ + } \)(10–20 mM) causes a paradoxical fall in intracellular pH (pHi), whereas low levels (e.g., 0.5 mM) cause little pHi change. Here we monitored pHi and extracellular surface pH (pHS) while exposing oocytes to 5 or 0.5 mM NH3/NH4+. We confirm that 5 mM \( {\text{NH}}_{ 3} /{\text{NH}}_{ 4}{}^{ + } \) causes a paradoxical pHi fall (−ΔpHi ≅ 0.2), but also observe an abrupt pHS fall (−ΔpHS ≅ 0.2)—indicative of NH3 influx—followed by a slow decay. Reducing [NH3/NH4+] to 0.5 mM minimizes pHi changes but maintains pHS changes at a reduced magnitude. Expressing AmtB (bacterial Rh homologue) exaggerates −ΔpHS at both \( {\text{NH}}_{ 3} /{\text{NH}}_{ 4}{}^{ + } \) levels. During removal of 0.5 or 5 mM NH3/NH4+, failure of pHS to markedly overshoot bulk extracellular pH implies little NH3 efflux and, thus, little free cytosolic NH3/NH4+. A new analysis of the effects of NH3 vs. NH4+ fluxes on pHS and pHi indicates that (a) NH3 rather than NH4+ fluxes dominate pHi and pHS changes and (b) oocytes dispose of most incoming NH3. NMR studies of oocytes exposed to 15N-labeled \( {\text{NH}}_{ 3} /{\text{NH}}_{ 4}{}^{ + } \) show no significant formation of glutamine but substantial \( {\text{NH}}_{ 3} /{\text{NH}}_{ 4}{}^{ + } \) accumulation in what is likely an acid intracellular compartment. In conclusion, parallel measurements of pHi and pHS demonstrate that NH3 flows across the plasma membrane and provide new insights into how a protein molecule in the plasma membrane—AmtB—enhances the flux of a gas across a biological membrane.

Acetaminophen at different doses protects brain microsomal Ca2+-ATPase and the antioxidant redox system in rats.

Abstract: Acetaminophen, an analgesic and antipyretic drug, rescues neuronal cells from mitochondrial redox impairment and reactive oxygen species (ROS). Excessive administration of acetaminophen above the recommended daily dose range has some negative effects on the brain. We investigated the effects of different doses of acetaminophen on Ca2+-ATPase and the antioxidant redox system in rats. Seventy rats were randomly divided into seven equal groups. The first was used for the control. One dose of 5, 10, 20, 100, 200, and 500 mg/kg acetaminophen was intraperitoneally administered to rats constituting the second, third, fourth, fifth, sixth, and seventh groups, respectively. After 24 h, brain cortical samples were taken and brain microsomal samples were obtained by ultracentrifugation. Brain and microsomal lipid peroxidation (LP) and brain calcium levels in the sixth and seventh groups were increased compared to control. LP levels in the second, third, and forth groups; brain vitamin E levels; brain and microsomal glutathione peroxidase (GSH-Px); and Ca2+-ATPase activity in the sixth and seventh groups were lower than in control, although brain vitamin E concentrations in the second, third, fourth, and fifth groups and microsomal GSH-Px activity in the third and fourth groups were higher than in control. Brain cortical β-carotene and vitamin A concentrations did not differ in the seven groups. In conclusion, 5–100 mg/kg acetaminophen seems to have protective effects on oxidative stress-induced brain toxicity by inhibiting free radicals and supporting the antioxidant redox system.

Distribution of several activating and inhibitory receptors on CD3-CD16+ NK cells and their correlation with NK cell function in healthy individuals.

Abstract: The aim of this study was to estimate the distribution and density of a representative set of activating and inhibitory receptors on gated natural killer (NK) cells, as well as on their bright and dim subsets, and to correlate the receptor expression with NK cell activity for healthy individuals on CD3−CD16+ NK cells. We show that in 43 healthy controls NK cell activity against K562 target cells was 37.34% (E:T, 80:1) by standard chromium release assay. The expression of receptors on NK cells and their subsets was analyzed by flow cytometry. The cytotoxic CD3−CD16bright NK subset constituted 78.97%, while the regulatory CD3−CD16dim NK subset constituted 21.03% of NK cells. We show the distribution of NKG2D, CD161, CD158a, and CD158b receptors on CD3−CD16+ NK cells in peripheral blood lymphocytes (PBLs), on gated NK cells, and on the CD3−CD16bright and CD3−CD16dim subsets. Contrary to CD158a and CD158b killer immunoglobulin-like receptors (KIRs), there is a significant positive correlation of NKG2D and CD161 expression with NK cytotoxicity. We show the kinetics of change in CD3−CD16+NK/K562 conjugate composition, together with the stronger target binding capacity of CD16bright NK cells. Furthermore, we show that after coculture of PBLs with K562 the expression of CD107a, a degranulation marker, on CD3−CD16+NK cells and subsets is time dependent and significantly higher on the cytotoxic CD3−CD16bright NK subset. The novel data obtained regarding expression of NK cell activating and inhibitory receptors for healthy individuals may aid in detecting changes that are associated with various diseases.

Effects of Tryptophan and 5-Hydroxytryptophan on the Hepatic Cell Membrane Rigidity Due to Oxidative Stress

Abstract: The ability of several indoleamines to scavenge free radicals is well documented. Our aim was to evaluate the ability of 0.01–3 mm tryptophan (Trp) and 0.1–5 mm 5-hydroxytryptophan (5-OH-Trp) to protect hepatic cell membranes against 0.1 mm FeCl3 plus 0.1 mm ascorbic acid–induced lipid peroxidation and increases in membrane rigidity. Membrane fluidity was evaluated using fluorescence spectroscopy. Lipid and protein oxidation were estimated by quantifying malondialdehyde (MDA) plus 4-hydroxyalkenals (4-HDA) concentrations and carbonyl group content, respectively. Exposure to FeCl3 plus ascorbic acid increased hepatic cell membrane rigidity, MDA + 4-HDA and carbonyl content. The presence of 5-OH-Trp, but not Trp, attenuated these changes. In the absence of oxidative stress, neither indoleamine modified fluidity, MDA + 4-HDA or carbonylation. These results suggest that C5 hydroxylation determines the ability of Trp to preserve membrane fluidity in the presence of oxidative stress.

The role of H(+)/OH(-) channels in the salt stress response of Chara australis.

Abstract: We investigate the electrophysiological salt stress response of the salt-sensitive charophyte Chara australis as a function of time in saline artificial pond water (saline APW) containing 50 mM NaCl and 0.1 mM CaCl(2). The effects are due to an increase in Na(+) concentration rather than an increase in Cl(-) concentration or medium osmolarity. A previous paper (Shepherd et al. Plant Cell Environ 31:1575-1591, 2008) described the rise in the background conductance and inhibition of proton pumping in saline APW in the first 60 min. Here we investigate the shift of membrane potential difference (PD) to levels above -100 mV and the change of shape of the current-voltage (I/V) profiles to upwardly concave. Arguing from thermodynamics, the I/V characteristics can be modeled by channels that conduct H(+) or OH(-). OH(-) was chosen, as H(+) required an unrealistic increase in the number/permeability of the channels at higher pH levels. Prolonged exposure to saline APW stimulated opening of more OH(-) channels. Recovery was still possible even at a PD near -50 mV, with partial return of proton pumping and a decrease in OH(-) current following APW wash. Upon change of pH from 7 to 9, the response was consistent with previously observed I/V characteristics of OH(-) channels. For a pH change to 6, the response was transient before channel closure but could still be modeled. The consequences of opening of H(+) or OH(-) channels while the cell is under salt stress are discussed.

Derangement of erythrocytic AE1 in beta-thalassemia by caspase 3: pathogenic mechanisms and implications in red blood cell senescence.

Abstract: Considering its complex molecular pathophysiology, beta-thalassemia could be a good in vivo model to study some aspects related to erythrocyte functions with potential therapeutic implications not only within the frame of this particular hemoglobinopathy but also with respect to conditions in which the cellular milieu, altered by a deranged anion exchanger, could display a significant pathogenetic role (i.e., erythrocyte senescence, complications of red cell storage, renal tubular acidosis and some abnormal protein thesaurismosis). This work evaluates the anionic influx across band 3 protein in normal and beta-thalassemic red blood cells (RBCs) and ghosts. Since redox-mediated injury is an important pathway in the destruction of beta-thalassemic RBCs, we studied the anion transport and the activity of caspase 3 in the absence and presence of t-butylhydroperoxide in order to evaluate the effect of an increase of cellular oxidative stress. Interestingly, beta-thalassemic erythrocytes show a faster rate of anion exchange than normal RBCs and absence of any modulation mechanism of anion influx. These findings led us to formulate a hypothesis about the metabolic characteristics of beta-thalassemic erythrocytes, outlining that one of the main targets of caspase 3 in RBCs is the cytoplasmic domain of band 3 protein.

Formation of Two Different Types of Ion Channels by Amphotericin B in Human Erythrocyte Membranes

Abstract: The polyene antibiotic amphotericin B (AmB) is known to form aqueous pores in lipid membranes and biological membranes. Here, membrane potential and ion permeability measurements were used to demonstrate that AmB can form two types of selective ion channels in human erythrocytes, differing in their interaction with cholesterol. We show that AmB induced a cation efflux (negative membrane polarization) across cholesterol-containing liposomes and erythrocytes at low concentrations (≤1.0 × 10−6 M), but a sharp reversal of such polarization was observed at concentrations greater than 1.0 × 10−6 M AmB, an indication that aqueous pores are formed. Cation-selective AmB channels are also formed across sterol-free liposomes, but aqueous pores are only formed at AmB concentrations 10 times greater. The effect of temperature on the AmB-mediated K+ efflux across erythrocytes revealed that the energies of activation for channel formation are negative and positive at AmB concentrations that lead predominantly to the formation of cation-selective channels and aqueous pores, respectively. These findings support the conclusion that the two types of AmB channels formed in human erythrocytes differ in their interactions with cholesterol and other membrane components. In effect, a membrane lipid reorganization, as induced by incubation of erythrocytes with tetrathionate, a cross-linking agent of the lipid raft–associated protein spectrin, led to differential changes in the activation parameters for the formation of both types of channels, reflecting the different lipid environments in which such structures are formed.

Studies of the Mechanism of Activation of the Volume-Regulated Anion Channel in Rat Pancreatic β-Cells

Abstract: There is evidence that depolarization of the pancreatic β cell by glucose involves cell swelling and activation of the volume-regulated anion channel (VRAC). However, it is unclear whether cell swelling per se or accompanying changes in intracellular osmolality and/or ionic strength are responsible for VRAC activation. VRAC activity was measured in rat β cells by conventional or perforated patch whole-cell recording. Cell volume was measured by video imaging. In conventional whole-cell recordings, VRAC activation was achieved by exposure of the cells to a hyposmotic bath solution, by application of positive pressure to the pipette, or by use of a hyperosmotic pipette solution. Increased concentrations of intracellular CsCl also caused channel activation, but with delayed kinetics. In perforated patch recordings, VRAC activation was induced by isosmotic addition of the permeable osmolytes urea, 3-Ο-methyl glucose, arginine, and NH4Cl. These effects were all accompanied by β-cell swelling. It is concluded that increased cell volume, whether accompanied by raised intracellular osmolality or ionic strength, is a major determinant of VRAC activation in the β cell. However, increased intracellular ionic strength markedly reduced the rate of VRAC activation. These findings are consistent with the hypothesis that the accumulation of glucose metabolites in the β cell, and the resultant increase in cell volume, provides a signal coupling glucose metabolism with VRAC activation.

Fibrinogen-Dependent Signaling in Microvascular Erythrocyte Function: Implications on Nitric Oxide Efflux

Abstract: Experimental evidence has shown that plasma fibrinogen plays a key role as a major cardiovascular risk factor, acting directly to trigger erythrocyte aggregation in occlusive vascular disease. However, due to the complex and hitherto unclear interaction between fibrinogen and the erythrocyte membrane, no study has yet evaluated the effects of fibrinogen, under physiological range values, on the erythrocyte nitric oxide (NO) mobilization. Taking into consideration the potential NO-derived molecules, we have raised the hypothesis that fibrinogen, under physiological conditions, may act to influence blood flow via erythrocyte NO modulation. In this in vitro study whole-blood samples were harvested from healthy subjects, erythrocyte suspensions were incubated in the absence (control aliquots) and presence of different fibrinogen concentrations and levels of NO, nitrite, nitrate and S-nitroglutathione (GSNO) were determined. Our results showed, when compared with control aliquots, that the presence of fibrinogen modulates the NO mobilization in erythrocytes by (1) decreasing erythrocyte NO efflux levels (P < 0.001); (2) increasing levels of intraerythrocytic NO oxidative metabolites, namely, nitrite (P < 0.0001) and nitrate (P < 0.0001); and (3) enhancing the formation of GSNO (P < 0.001). In conclusion, this study provides new insights into an unknown mechanism by which fibrinogen modulates the erythrocyte capacity to supply NO, the effects of which on inflammation profiles (generally associated with blood hyperviscosity and hyperaggregation) still need to be elucidated. Also, increased erythrocyte GSNO levels may be associated with platelet NO metabolism, its activation status and hypotension, which may be extremely relevant in the clinical setting as biomarkers.