Voltage-dependent membrane capacitance in rat pituitary nerve terminals due to gating currents

We investigated the voltage dependence of membrane capacitance of pituitary nerve terminals in the whole-terminal patch-clamp configuration using a lock-in amplifier. Under conditions where secretion was abolished and voltage-gated channels were blocked or completely inactivated, changes in membrane potential still produced capacitance changes. In terminals with significant sodium currents, the membrane capacitance showed a bell-shaped dependence on membrane potential with a peak at approximately -40 mV as expected for sodium channel gating currents. The voltage-dependent part of the capacitance showed a strong correlation with the amplitude of voltage-gated Na+ currents and was markedly reduced by dibucaine, which blocks sodium channel current and gating charge movement. The frequency dependence of the voltage-dependent capacitance was consistent with sodium channel kinetics. This is the first demonstration of sodium channel gating currents in single pituitary nerve terminals. The gating currents lead to a voltage- and frequency-dependent capacitance, which can be well resolved by measurements with a lock-in amplifier. The properties of the gating currents are in excellent agreement with the properties of ionic Na+ currents of pituitary nerve terminals.     

Latex phagocytosis by polymorphonuclear leukocytes: Role of sialic acid groups

Polystyrene latex particles are rapidly phagocytized by rabbit polymorphonuclear (PMN) leukocytes. The uptake is influenced by macromolecules which have the effect of altering the surface charge of the latex particle. The influence of polylysines of varying chain length on the surface charge of latex particles and of PMN cells was studied by micro-electrophoresis. Charge reversal at the latex surface was found to occur at concentrations considerably below that at which the surface charge of the PMN cells is reversed. Phagocytosis of latex by PMN cells is enhanced in the presence of low concentrations of long-chain polylysines. The enhancement of phagocytosis is strongly reduced if PMN cells are treated with neuraminidase. This suggests participation of siliac acid groups in a stage of particle-cell interaction which precedes engulfment.     

Inactivation during phagocytosis of leucine aminopeptidase, an ecto-enzyme of polymorphonuclear neutrophils

Changes in enzyme activities of the plasma membrane makers were examined during phagocytosis using guinea-pig polymorphonuclear neutrophils. Incubation of neutrophils with fresh serum-opsonized zymosan particles showed a significant reduction in leucine aminopeptidase activity, whereas 5′-nucleotidase and alkaline phosphodieterase activities remained unchanged. Inactivation of leucine aminopeptidase activity was also observed by exposure of neutrophils to complement-opsonized zymosan particles, but not to non-opsonized zymosan, IgG-coated zymosan or polysterene latex particles. Pretreatment of neutrophils with cytochalasin B, which prevents phagocytosis but not surface binding of particles, provoked inactivation to the same degree as when the cells were allowed to phagocytose the particles. However, the inactivation during phagocytosis was protected by serine protease inhibitors. These findings suggest that loss of leucine aminopeptidase activity from phagocytosing cells may be mediated by certain serine protease inhibitor-sensitive factor(s) which are probably activated by the attachment of an opsonized zymosan particle to a specific membrane receptor, probably the C3b receptor.     

Fibronectin-mediated uptake of gelatin-coated latex particles by peritoneal macrophages

The present study demonstrates the ability of plasma fibronectin or cold-insoluble globulin (Clg) to promote the uptake of 125I-labeled, gelatin-coated latex beads (g-Ltx*) by monolayers of peritoneal macrophages (PM). The uptake of g-Ltx* by PM was enhanced by Clg in a concentration-dependent fashion and required the presence of heparin (10 U/ml) as an obligatory cofactor for maximal particle uptake. Treatment of PM monolayers with trypsin (1 mg/ml) for 15 min at 37 degrees C after particle uptake removed less than 15% of the radioactivity incorporated by the monolayers. However, a similar trypsin treatment of the monolayers before the addition of latex particles depressed Clg-dependent uptake by greater than 75%. Pretreatment of PM monolayers with inhibitors of glycolysis effectively reduced the Clg-dependent uptake of latex. Similarly, pretreatment of monolayers with either inhibitors of protein synthesis or agents that disrupt cytoskeletal elements also significantly depressed Clg- dependent particle uptake. Phagocytosis of g-Ltx* by PM in the presence of Clg and heparin was confirmed by electron microscopy. Finally, g- Ltx* could also be effectively opsonized with Clg at 37 degrees C before their addition to the monolayers. These studies suggest that the recognition of g-Ltx* in the presence of Clg required cell surface protein(s) and that subsequent phagocytosis of these particles by PM was energy dependent and required intact intracellular cytoskeleton elements. Thus, PM monolayers provide a suitable system for further studies on the function of Clg in the recognition and phagocytosis of gelatin-coated particles by phagocytic cells.     

Intramembrane Charge Movement Associated with Endogenous K+ Channel Activity in HEK-293 Cells

1. The use of molecular biology in combination with electrophysiology in the HEK-293 cell line has given fascinating insights into neuronal ion channel function. Nevertheless, to fully understand the properties of channels exogenously expressed in these cells, a detailed evaluation of endogenous channels is indispensable. 2. Previous studies have shown the expression of endogenous voltage-gated K+, Ca2+, and Cl- channels and this predicts that changes in membrane potential will cause intramembrane charge movement, though this gating charge translocation remain undefined. Here, we confirm this prediction by performing patch-clamp experiments to record ionic and gating currents. Our data show that HEK-293 cells express at least two types of K+-selective endogenous channels which sustain the majority of the ionic current, and exclude a significant contribution from Ca2+ and Cl- channels to the whole-cell current. 3. Gating currents were unambiguously resolved after ionic current blockade enabling this first report of intramembrane charge movement in HEK-293 cells arising entirely from endogenous K+ channel activity, and providing valuable information concerning the activation mechanism of voltage-gated K+ channels in these cells.     

MTOC reorientation occurs during FcgammaR-mediated phagocytosis in macrophages

Cell polarization is essential for targeting signaling elements and organelles to active plasma membrane regions. In a few specialized cell types, cell polarity is enhanced by reorientation of the MTOC and associated organelles toward dynamic membrane sites. Phagocytosis is a highly polarized process whereby particles >0.5 microm are internalized at stimulated regions on the cell surface of macrophages. Here we provide detailed evidence that the MTOC reorients toward the site of particle internalization during phagocytosis. We visualized MTOC proximity to IgG-sRBCs in fixed RAW264.7 cells, during live cell imaging using fluorescent chimeras to label the MTOC and using frustrated phagocytosis assays. MTOC reorientation in macrophages is initiated by FcgammaR ligation and is complete within 1 h. Polarization of the MTOC toward the phagosome requires the MT cytoskeleton and dynein motor activity. cdc42, PI3K, and mPAR-6 are all important signaling molecules for MTOC reorientation during phagocytosis. MTOC reorientation was not essential for particle internalization or phagolysosome formation. However Golgi reorientation in concert with MTOC reorientation during phagocytosis implicates MTOC reorientation in antigen processing events in macrophages.     

Modeling of discrete currents of single ion channels of cell membranes using synthetic nanometer pores in polyethylene terephthalate films

Study of the conductivity of single supernarrow pores (1–15 nm in diameter) formed in thin membranes (10–12 μm in thickness) from polyethylene terephthalate (PETP) has revealed discrete changes in the currents passing through such pores when applied from an external source of potential difference of 200–1000 mV. Based on several characteristics, such discrete currents (discrete conductivity changes) appeared to be identical to the so-called currents of single ionic channels in cell membranes. The supernarrow pores whose properties are described in the present work were obtained by alkaline etching of tracks in thin PETP membranes (a variant of the so-called nuclear filters). On the walls of the pores, carboxyl groups, i.e., negative fixed charges, and their compensating counterion (cation) layer are formed. Upon setting the potential difference onto the PETP membrane, this cation layer is able to transfer current, through a process called surface conductance. In the case of nanometer-sized diameters of the pores, such surface conductance can turn out to be dominating. We have shown that these discrete changes of currents passing through the nanometer pores are associated with metastability of their surface conductance. In the highly cation-selective channels in the cell membranes, there should inevitably exist an area with dominating cation surface conductance and, hence, conductance metastability. Therefore, a new explanation is proposed of the characteristic discreteness of the currents of single cation-specific ionic channels in cell membranes. Such an explanation does not rule out the existence of any other traditional explanation of the discreteness of ion channel currents.     

Single channel currents in adrenocortical cells

Single channel currents have been recorded from cell-attached patches of tumoral adrenocortical cells. Our experiments suggest the existence of three sets of potassium channels in the surface membrane of these cells. All channel types can be recorded in a given membrane patch but some patches have only one type of single channel currents. One channel type has a unitary conductance of about 103 pS. The other two channels have smaller conductances and opposite voltage dependence. In one case channels open on depolarization and have a single channel conductance of 31.6 pS. In the other case the probability of being in the open state increases on hyperpolarization and the single channel conductance is of 21 pS. These channels seem to be similar to the delayed and anomalous rectifying potassium channels seen in other preparations. The role of membrane ionic permeability in steroid release induced by ACTH is discussed.     

Altered circadian rhythms of corticosterone, melatonin, and phagocytic activity in response to stress in rats

Corticosterone is thought to be the main glucocorticoid secreted in response to stressful exercise, while melatonin buffers the adverse immunological effects of stress. The present work was aimed to evaluate whether swimming-exercise-induced stress leads to changes in the chronobiology parameters of the circadian rhythms of melatonin and corticosterone, and in the number and phagocytosis of peritoneal macrophages in 3-month-old male Wistar rats. The animals were subjected to a physical activity trial consisting of 2 h of free swimming. Radioimmunoassay was used to determine the plasma levels of melatonin and corticosterone. Phagocytosis was measured by the latex-bead phagocytosis index (PI), i.e., the number of latex beads ingested by 100 macrophages, the phagocytosis percentage (PP), i.e., the percentage of cells that had phagocytosed at least one latex bead, and the phagocytosis efficiency (PE), i.e., the ratio PI: PP which indicates how effectively the phagocytes ingested the particles. Stress significantly decreased the MESOR and amplitude of the melatonin rhythm, and significantly increased the MESOR of the corticosterone rhythm. The control animals' peritoneal macrophage number and PI showed a circadian rhythm with maxima at 02:00 and 03:00, respectively. The stressed group displayed higher values of PI than the controls at most hours of the night, but the number of cells in the peritoneal cavity was practically the same at all hours studied. These data confirm that melatonin and corticosterone act as modulators of the innate immune response, and that the circadian rhythm of the two hormones are altered in situations of stress.     

Effect of phagocytosis on receptor distribution and endocytic activity in macrophages

Phagocytosis requires the internalization of a significant fraction of the plasma membrane and results in the intracellular deposition of large particles. We evaluated the effect of phagocytosis on the cellular distribution of recycling receptors and uptake of ligand to determine whether phagocytosis affects receptor behavior. Phagocytosis of zymosan, latex particles, or IgG-coated red blood cells by rabbit alveolar macrophages did not decrease the number of cell surface receptors for transferrin, alpha 2-macroglobulin X protease complexes, maleylated proteins, or mannosylated proteins. The number of surface receptors for transferrin was also unaltered in J774 cells, a macrophage-like cell line. In both cell types extensive phagocytosis did not affect the rate of receptor-mediated endocytosis or the distribution of receptors between the endosome and the cell surface. However, fluid phase pinocytosis was reduced by phagocytosis. The major reduction appeared to be not in the rate of internalization but rather in the delivery of fluid to the lysosome. These results demonstrate that internalization of a significant amount of the plasma membrane during phagocytosis does not diminish the number of receptors on the cell surface and has no effect on receptor-mediated ligand uptake.     

Phagocytosis and macropinocytosis in Dictyostelium: phosphoinositide-based processes, biochemically distinct

Phagocytosis and macropinocytosis are actin-dependent clathrin-independent processes primarily performed by cells like neutrophils and macrophages that result in the internalization of particles or the formation of fluid-filled macropinosomes, respectively. Phagocytosis consists of a number of stages, including attachment of particles to cell surface receptors, engulfment of the particle dependent on actin polymerization and membrane exocytosis, and formation of phago-lysosomes. In contrast, the molecular steps regulating macropinocytosis are only just now being deciphered. Much remains to be learned concerning the signaling pathways that regulate these processes. Dictyostelium is a genetically and biochemically tractable professional phagocyte that has proven to be a powerful system with which to determine the nature of the molecular steps involved in regulating these internalization processes. This review summarizes what is currently understood concerning the molecular mechanisms governing phagocytosis and macropinocytosis in Dictyostelium and describes recent data concerning the common and distinct pathways that regulate these processes.     

Phagocytic properties of hepatic endothelial cells and splenic macrophages compensating for a decreased phagocytic function of Kupffer cells in the chronically ethanol-fed rats

The balance of phagocytic function among Kupffer cells, hepatic endothelial cells and splenic macrophages in the chronically ethanol-fed rats has been investigated. Clearance of latex particles in the blood was measured to estimate the function of the reticuloendothelial system. Phagocytosis of latex particles by Kupffer cells, hepatic endothelial cells or splenic macrophages in vivo was measured by counting the number of ingested particles in a cell after isolation of hepatic nonparenchymal cells or spleen cells following injection of different amounts of latex particles. Latex particle clearance was suppressed in the ethanol-fed rats, demonstrating a decreased phagocytic capacity of the reticuloendothelial system. Markedly decreased phagocytic function was found in 40% of Kupffer cells of the chronically ethanol-fed rats. In contrast, the number of latex particles in hepatic endothelial cells and in splenic macrophages was increased after injection of a triggering dose of latex particles. From these results it may be concluded that an increased phagocytosis of hepatic endothelial cells and splenic macrophages could compensate for the decreased phagocytic function of Kupffer cells.     

Complement-mediated phagocytosis--the role of Syk

Phagocytosis is a central event in the innate immune responses that are triggered by the association between ligands on the surface of pathogens and receptors on the membrane of phagocytes. Particularly, complement-mediated phagocytosis is accomplished by specific recognition of bound complement components by the corresponding complement receptors on the phagocytes. The protein-tyrosine kinase, Syk, plays a central role in Fcgamma receptor-mediated phagocytosis in the adaptive immune system. From recent studies using a macrophage-like differentiated cell line and serum-treated zymosan, it was found that Syk also plays an essential role in complement-mediated phagocytosis in innate immunity. Serum-treated zymosan particles promptly attached to the cells and were subsequently engulfed via complement receptor3. During this process, Syk became tyrosine-phosphorylated and accumulated around the nascent phagosomes. The transfer of Syk-siRNA or dominant-negative Syk (DN-Syk) into macrophages resulted in impaired engulfment of pathogen. Collectively, Syk is required for the engulfment of pathogen in complement-mediated phagocytosis.     

Membrane capacitance changes associated with particle uptake during phagocytosis in macrophages.

We report the use of capacitance measurements to monitor particle uptake after cellular exposure to phagocytic stimuli. In these studies, human monocyte-derived macrophages (HMDMs) and cells from the murine macrophage-like cell line J774.1 were exposed to immune complexes or sized latex particles (0.8 or 3.2 micron in diameter). An average decrease in cell capacitance of 8 pF was seen after exposure of the cells to immune complexes. Cells in which particle uptake was inhibited by cytochalasin B treatment before exposure to immune complexes showed an average increase of 0.5 pF. The decrease in membrane capacitance after exposure of cells to particulate stimuli was absent with the soluble stimulus, platelet-activating factor, further confirming that decreases in membrane capacitance were due to particle uptake. Exposure of cells to sized latex particles resulted in a graded, stepwise decrease in membrane capacitance. The average step size for 0.8-micron particles was 250 fF, and the average step change for the larger 3.2-micron particles was 480 fF, as calculated from Gaussian fits to the step size amplitude histograms. The predicted step size for the individual particles based upon the minimum amount of membrane required to enclose a particle and a specific capacitance of 10 fF/micron2 was 20 and 320 fF, respectively. The step size for the smaller particles deviates significantly from the predicted size distribution, indicating either a possible lower limit to the size of the phagocytic vacuole or multiple particles taken up within a single phagosome. Dynamic interaction between phagocytosis and exocytosis was observed in a number of cells as a biphasic response consisting of an initial rapid increase in capacitance, consistent with cellular exocytosis, followed by stepwise decreases in capacitance.     

Permeation characteristics of gramicidin conformers.

To investigate the molecular origin of decreased conductance in variant gramicidin channels, we examined the current-voltage (IV) characteristics of single Val1-gramicidin A channels. Unlike standard channels, all variant channels showed pronounced rectification even though bathing solutions were symmetrical. Moreover, channels of lower conductance consistently showed more pronounced rectification. Analysis within the framework of a three-barrier, two-site, single-filing model indicates that the shape of the variant channel IVs could be best explained by an increase in binding affinity near one of the two channel entrances. This conclusion was further tested by characterizing single channel IVs in bi-ionic solutions having different cationic species at each channel entrance. In Cs/Na bi-ionic solutions, reversal potentials of variant channels often differed by a small but significant amount from those of standard channels. When a membrane potential was applied, the ionic currents tended to be reduced more when flowing from the Na+ side than the Cs+ side. These observations support the conclusion that variant channels have increased binding affinity at one end of the channel. Furthermore, H+ currents were increased while Ag+ currents were unaltered for most variant channels exhibiting decreased Na+ or Cs+ currents. The increased H+ conductance argues against long-range coulombic forces as the basis for decreased Na+ or Cs+ conductance while the normal Ag+ conductance suggests that the binding site field strength increases by a change in carbonyl geometry at the channel entrance.     

Modification of potassium channel kinetics by histidine-specific reagents

We have examined the actions of histidine-specific reagents on potassium channels in squid giant axons. External application of 20-500 microM diethylpyrocarbonate (DEP) slowed the opening of potassium channels with little or no effect on closing rates. Sodium channels were not affected by these low external concentrations of DEP. Internal application of up to 2 mM DEP had no effect on potassium channel kinetics. Steady-state potassium channel currents were reduced in an apparently voltage-dependent manner by external treatment with this reagent. The shape of the instantaneous current-voltage relation was not altered. The voltage-dependent probability of channel opening was shifted toward more positive membrane potentials, thus accounting for the apparent voltage-dependent reduction of steady-state current. Histidine-specific photo-oxidation catalyzed by rose bengal produced alterations in potassium channel properties similar to those observed with DEP. The rate of action of DEP was consistent with a single kinetic class of histidine residues. In contrast to the effects on ionic currents, potassium channel gating currents were not modified by treatment with DEP. These results suggest the existence of a histidyl group (or groups) on the external surface of potassium channels important for a weakly voltage-dependent conformational transition. These effects can be reproduced by a simple kinetic model of potassium channels.     

Escherichia coli Membranes Depleted of SecYEG Elicit SecA-Dependent Ion-Channel Activity but Lose Signal Peptide Specificity

We have developed a sensitive method to detect the opening of SecA-dependent, protein-conducting channels in Xenopus oocytes. In this study, we determined the ionic current activities of the SecA-dependent channel from membrane vesicles depleted of SecYEG. We found that these SecYEG-depleted membranes produced SecA-dependent ionic currents in the oocytes, as did membranes containing SecYEG. However, reconstituted membranes depleted of SecYEG required higher concentrations of SecA to elicit ionic currents like those in membranes containing SecYEG. In contrast to membranes containing SecYEG, the proofreading capacity of signal peptides was lost for those membranes lacking SecYEG. These findings are consistent with loss of signal peptide specificity in channel activity from membranes of SecY suppressor or SecY plug domain mutants. The signal peptide specificity of the reconstituted membranes, like SecA-liposomes, can be restored by the addition of SecYEG proteoliposomes. On the other hand, the channel activity efficiency of reconstituted membranes was fully restored, while SecA-liposomes could only be partially enhanced by the addition of SecYEG, indicating that, in addition to SecYEG, other membrane proteins contribute to the efficiency of channel activity. The SecA-dependent channels in membranes that lacked SecYEG also lost ion selectivity to monovalent cations but retained selective permeability to large anions. Thus, the electrophysiological evidence presented here indicates that SecYEG is not obligatory for the channel activity of Escherichia coli membranes, as previously shown for protein translocation, and that SecYEG is important for maintenance of the efficiency and specificity of SecA-dependent channels.     

Cytosolic free Ca(2+) changes and calpain activation are required for beta integrin-accelerated phagocytosis by human neutrophils

Phagocytosis of microbes coated with opsonins such as the complement component C3bi is the key activity of neutrophils. However, the mechanism by which opsonins enhance the rate of phagocytosis by these cells is unknown and has been difficult to study, partly because of the problem of observing and quantifying the events associated with phagocytosis. In this study, C3bi-opsonized particles were presented to neutrophils with a micromanipulator, so that the events of binding, pseudopod cup formation, engulfment, and completion of phagocytosis were clearly defined and distinguished from those involved with chemotaxis. Using this approach in combination with simultaneous phase contrast and Ca(2+) imaging, the temporal relationship between changes in cytosolic free Ca(2+) concentration and phagocytosis were correlated. Here we show that whereas small, localized Ca(2+) changes occur at the site of particle attachment and cup formation as a result of store release, rapid engulfment of the particle required a global change in cytosolic free Ca(2+) which resulted from Ca(2+) influx. This latter rise in cytosolic free Ca(2+) concentration also liberated a fraction of beta2 integrin receptors which were initially immobile on the neutrophil surface, as demonstrable by both fluorescence recovery after laser bleaching and by visualization of localized beta2 integrin labelling. Inhibitors of calpain activation prevented both the Ca(2+)-induced liberation of beta2 integrin and the rapid stage of phagocytosis, despite the persistence of the global Ca(2+) signal. Therefore, we propose that Ca(2+) activation of calpain causes beta2 integrin liberation, and that this signal plays a key role in the acceleration of beta2 integrin-mediated phagocytosis.     

Multiple-state model of ionic channel in reproducing the time course of electrophysiological events.

BACKGROUND: The predictions of the Hodgkin-Huxley model do not accurately fit all the measurements of voltage-clamp currents, gating charge and single-channel currents. There are many quantitative differences between the predicted and measured characteristics of the sodium and potassium channels. For example, the two-state gate model has exponential onset kinetics, whereas the sodium and potassium conductances show S-shaped activation and the sodium conductance shows an exponential inactivation. In this paper we shall examine a more general channel model that can more faithfully represent the measured properties of ionic channels in the membrane of the excitable cell. METHODS: The model is based on the generalisation of the notion of a channel with a discrete set of states. Each state has state attributes such as the state conductance, state ionic current and state gating charge. These variables can have quite different waveforms in time, in contrast with a two-state gate channel model, in which all have the same waveforms. RESULTS: The kinetics of all variables are equivalent: gating and ionic currents give equivalent information about channel kinetics; both the equilibrium values of the current and the time constants are functions of membrane potential. The results are in almost perfect concordance with the experimental data regarding the characteristics of nerve impulse. CONCLUSIONS: The expected values of the gating charge and the ionic conductance are weighted sums of the state occupancy probabilities, but the weights differ: for the expected value of the gating charge the weights are the state gating charges and for the expected value of the ionic conductance the weights are the state conductances. Since these weights are different, the expected values of the gating charge and the ionic conductance will differ.     

Cytoplasmic chloride regulates cation channels in the vacuolar membrane of plant cells

Summary This study is concerned with the characterization of the ionic currents in the vacuolar membrane (tonoplast) of plant cells. Voltage patch-clamp experiments at the whole vacuole and single channel levels were employed to study the effects of cytoplasmic chloride on the tonoplast inward rectifying currents of sugar beet cultured cells. Whole vacuole experiments showed that removal of cytoplasmic chloride induced a decrease in the level of the inward currents, an effect that was reversed upon returning to control levels of cytoplasmic chloride. Substitution of cytoplasmic chloride by any other anion (organic or inorganic) resulted in a reduction in the level of the inward currents. At a given negative tonoplast potential, the inward currents showed a linear relationship with the concentration of cytoplasmic chloride between 10 and 100 mM, with the slope of these relationships increasing as the potential was made more negative. Single channel experiments showed that reduction of cytoplasmic chloride changed the gating mechanism of the channels without affecting the single channel conductance. Reduction of cytoplasmic chloride caused a decrease in the open probability of the tonoplast cation channels by reducing their mean open time and by inducing the appearance of an additional closed state.This work was supported by the National Science and Engineering Research Council of Canada.