scholarly journals Analysis and Minimization of Ligand Concentration Errors at the Internal Face of Excised Patches

2011 ◽  
Vol 100 (3) ◽  
pp. 104a
Author(s):  
Jana Kusch ◽  
Ralf Schmauder ◽  
Christoph Biskup ◽  
Vasilica Nache ◽  
Klaus Benndorf
Keyword(s):  
Ligand Concentration ◽  
Excised Patches

PREDICTIVE SIMULATION AND OPTIMIZATION ORIENTED MODELING FOR THE EXTRACTION OF METAL IONS FROM SOLUTIONS INTO A NOVEL BIS-SCHIFF BASE

2019 ◽  
pp. 1-8
Author(s):  
F. S. Nworie ◽  
S. O. Ngele ◽  
J. C. Onah
Keyword(s):  
Metal Ions ◽  
Metal Concentration ◽  
Acid Concentration ◽  
Aqueous Media ◽  
Industrial Effluents ◽  
Ligand Concentration ◽  
Simulation And Optimization ◽  
Chemometric Technique ◽  
Model Equations ◽  
Level Of Significance

Metal ions present in waste samples, industrial effluents, acid mines and other aqueous media constitute a serious challenge in different human activities. Solvent extraction a technique for preconcentration, separation and identification of trace amount of metal ions coupled with multivariate chemometric technique was used for the determination of Fe(II) and Cr(III) from solutions in the presence of bis(salicylidene)ethylenediamine (SALEN). The influence of main extraction variables affecting the extraction efficiency was simultaneously studied and regression model equations illustrating the relationship between variables predicted. The extraction parameters (time of extraction, acid concentration, ligand concentration, temperature and metal concentration) were optimized using experimental designs with the contributions of the various parameters to extraction of the metal ions bound to the complexone evaluated using SPSS19.0 software. The statistically determined simulated models for the parameters were R2 = 0.946, 0.727, 0.793, 0.53, 0.53, 1.000 and F- values of 70.400, 13. 285, 15.348, 4.646 and 2.569×105 respectively for time of extraction, acid concentration, ligand concentration, temperature and metal concentration for Cr (III). For Fe (II), R2 = 0.243, 0.371, 0.519, 0.446, 1.000 and F-values of 0.964, 2.953, 4.310, 3.216 and 2.516×105 for time of extraction, acid concentration, ligand concentration, temperature and metal concentration respectively. The level of significance of the models as predicted was both lower than 5% making it feasible, efficient, reproducible and accurate. This means that metal ions at the conditions stated could be removed from waste samples, industrial effluents, acid mines and other aqueous media with extension in industrial scale application.

scholarly journals Gating of Na channels in the rat cortical collecting tubule: effects of voltage and membrane stretch.

1996 ◽  
Vol 107 (1) ◽  
pp. 35-45 ◽  
Author(s):  
L G Palmer ◽  
G Frindt
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
Apical Membrane ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Time ◽  
Collecting Tubule ◽  
Excised Patches ◽  
The Mean ◽  
Cortical Collecting Tubule

The gating kinetics of apical membrane Na channels in the rat cortical collecting tubule were assessed in cell-attached and inside-out excised patches from split-open tubules using the patch-clamp technique. In patches containing a single channel the open probability (Po) was variable, ranging from 0.05 to 0.9. The average Po was 0.5. However, the individual values were not distributed normally, but were mainly < or = 0.25 or > or = 0.75. Mean open times and mean closed times were correlated directly and inversely, respectively, with Po. In patches where a sufficient number of events could be recorded, two time constants were required to describe the open-time and closed-time distributions. In most patches in which basal Po was < 0.3 the channels could be activated by hyperpolarization of the apical membrane. In five such patches containing a single channel hyperpolarization by 40 mV increased Po by 10-fold, from 0.055 +/- 0.023 to 0.58 +/- 0.07. This change reflected an increase in the mean open time of the channels from 52 +/- 17 to 494 +/- 175 ms and a decrease in the mean closed time from 1,940 +/- 350 to 336 +/- 100 ms. These responses, however, could not be described by a simple voltage dependence of the opening and closing rates. In many cases significant delays in both the activation by hyperpolarization and deactivation by depolarization were observed. These delays ranged from several seconds to several tens of seconds. Similar effects of voltage were seen in cell-attached and excised patches, arguing against a voltage-dependent chemical modification of the channel, such as a phosphorylation. Rather, the channels appeared to switch between gating modes. These switches could be spontaneous but were strongly influenced by changes in membrane voltage. Voltage dependence of channel gating was also observed under whole-cell clamp conditions. To see if mechanical perturbations could also influence channel kinetics or gating mode, negative pressures of 10-60 mm Hg were applied to the patch pipette. In most cases (15 out of 22), this maneuver had no significant effect on channel behavior. In 6 out of 22 patches, however, there was a rapid and reversible increase in Po when the pressure was applied. In one patch, there was a reversible decrease. While no consistent effects of pressure could be documented, membrane deformation could contribute to the variation in Po under some conditions.

Whole cell and unitary amiloride-sensitive sodium currents in M-1 mouse cortical collecting duct cells

1996 ◽  
Vol 270 (4) ◽  
pp. C998-C1010 ◽  
Author(s):  
M. L. Chalfant ◽  
T. G. O'Brien ◽  
M. M. Civan
Keyword(s):  
Collecting Duct ◽  
Cell Permeability ◽  
Na Channel ◽  
Na Channels ◽  
Cortical Collecting Duct ◽  
Ionic Selectivity ◽  
Whole Cell ◽  
Duct Cells ◽  
Excised Patches ◽  
Collecting Duct Cells

Amiloride-sensitive whole cell currents have been reported in M-1 mouse cortical collecting duct cells (Korbmacher et al., J. Gen. Physiol. 102: 761-793, 1993). We have confirmed that amiloride inhibits the whole cell currents but not necessarily the measured whole cell currents. Anomalous responses were eliminated by removing external Na+ and/or introducing paraepithelial shunts. The amiloride-sensitive whole cell currents displayed Goldman rectification. The ionic selectivity sequence of the amiloride-sensitive conductance was Li+ > Na+ >> K+. Growth of M-1 cells on permeable supports increased the amiloride-sensitive whole cell permeability, compared with cells grown on plastic. Single amiloride-sensitive channels were observed, which conformed to the highly selective low-conductance amiloride-sensitive class [Na(5)] of epithelial Na+ channels. Hypotonic pretreatment markedly slowed run-down of channel activity. The gating of the M-1 Na+ channel in excised patches was complex. Open- and closed-state dwell-time distributions from patches that display one operative channel were best described with two or more exponential terms each. We conclude that 1) study of M-1 whole cell Na+ currents is facilitated by reducing the transepithelial potential to zero, 2) these M-1 currents reflect the operation of Na(5) channels, and 3) the Na+ channels display complex kinetics, involving > or = 2 open and > or = 2 closed states.

Quantitative Study of the Effects of Surface Ligand Concentration on CdSe Nanocrystal Photoluminescence

2007 ◽  
Vol 111 (17) ◽  
pp. 6220-6227 ◽  
Author(s):  
Andrea M. Munro ◽  
Ilan Jen-La Plante ◽  
Marsha S. Ng ◽  
David S. Ginger
Keyword(s):  
Quantitative Study ◽  
Ligand Concentration ◽  
Surface Ligand

scholarly journals The apparent Km is a misleading kinetic indicator

1986 ◽  
Vol 239 (1) ◽  
pp. 175-178 ◽  
Author(s):  
I W Plesner
Keyword(s):  
Substrate Concentration ◽  
Kinetic Data ◽  
Substrate Binding ◽  
Enzymic Activity ◽  
Free Enzyme ◽  
Ligand Concentration ◽  
Double Reciprocal Plot ◽  
Michaelis Constant ◽  
Formal Framework ◽  
Enzyme Species

When information concerning whether or not a ligand interacts with the same enzyme species as do the substrates, the variation of the Michaelis constant Km (for each substrate) with ligand concentration is sometimes used as a diagnostic. It is shown that the Michaelis constant is of no particular value in this respect and may be misleading. Thus, depending on the mechanism, Km may vary with ligand concentration even though the ligand interacts with species far removed in the mechanism from the substrate-binding steps, and it may stay constant in cases where the ligand competes directly for the free enzyme. In contrast, the slope of a double-reciprocal plot of the kinetic data (= Km/Vmax.) (or, equivalently, the ordinate intercept of a Hanes plot A/v versus A, where A is the substrate concentration) independently of the particular mechanism involved uniquely signifies whether or not such interaction occurs. The results clearly indicate that, for purposes other than communicating the substrate concentration yielding control of the enzymic activity, usage of Km and its variation with ligand concentration should be avoided and interest instead focused on the slope, in accordance with the long-established rules of Cleland [Biochim. Biophys. Acta (1963) 67, 188-196], for which the present analysis provides the formal framework.

Actin filaments regulate epithelial Na+ channel activity

1991 ◽  
Vol 261 (5) ◽  
pp. C882-C888 ◽  
Author(s):  
H. F. Cantiello ◽  
J. L. Stow ◽  
A. G. Prat ◽  
D. A. Ausiello
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Functional Role ◽  
Cytochalasin D ◽  
Actin Binding ◽  
Na Channel ◽  
Na Channels ◽  
Channel Activity ◽  
Cortical Actin ◽  
Excised Patches

The functional role of the cytoskeleton in the control of ion channel activity is unknown. In the present study, immunocolocalization of Na+ channels with specific antibodies and fluorescein isothiocyanate-phalloidin to stain the cortical cytoskeleton indicates that actin is always present in close proximity to apical Na+ channels in A6 cells. The patch-clamp technique was used to assess the effect of cortical actin networks on apical Na+ channels in these A6 epithelial cells. The actin filament disrupter, cytochalasin D (5 micrograms/ml), induced Na+ channel activity in cell-attached patches within 5 min of addition. Cytochalasin D also induced and/or increased Na+ channel activity in 90% of excised patches tested within 2 min. Addition of short actin filaments (greater than 5 microM) to excised patches also induced channel activity. This effect was enhanced by addition of ATP and/or cytochalasin D. The effect of actin on Na+ channel activity was reversed by addition of the G actin-binding protein DNase I or completely prevented by treatment of the excised patches with this enzyme. Addition of the actin-binding protein, filamin, reversibly inhibited both spontaneous and actin-induced Na+ channels. Thus actin filament networks, achieved by either depolymerizing endogenous actin filaments by treatment with cytochalasin D, the addition of exogenous short actin filaments plus ATP, or actin plus cytochalasin D, regulate apical Na+ channel activity. This conclusion was supported by the observation that the addition of short actin filaments in the form of actin-gelsolin complexes in molar ratios less than 8:1 was also effective in activating Na+ channels. We have thus demonstrated a functional role for the cortical actin network in the regulation of epithelial Na+ channels that may complement a structural role for membrane protein targetting and assembly.

scholarly journals A Phosphorylation-Based Model for EGFR Activation as a Function of Ligand Concentration

2009 ◽  
Vol 96 (3) ◽  
pp. 329a
Author(s):  
Edwin Li ◽  
Jesse K. Placone ◽  
Kalina Hristova
Keyword(s):  
Ligand Concentration ◽  
Egfr Activation

scholarly journals The structure and dynamics of patch-clamped membranes: a study using differential interference contrast light microscopy.

1990 ◽  
Vol 111 (2) ◽  
pp. 599-606 ◽  
Author(s):  
M Sokabe ◽  
F Sachs
Keyword(s):  
Cell Surface ◽  
Transmural Pressure ◽  
Positive Pressure ◽  
Radius Of Curvature ◽  
Pressure Gradients ◽  
Structure And Motion ◽  
Excised Patches ◽  
Glass Interface ◽  
Pipette Tip ◽  
Flow Through

We have developed techniques for micromanipulation under high power video microscopy. We have used these to study the structure and motion of patch-clamped membranes when driven by pressure steps. Patch-clamped membranes do not consist of just a membrane, but rather a plug of membrane-covered cytoplasm. There are organelles and vesicles within the cytoplasm in the pipette tip of both cell-attached and excised patches. The cytoplasm is capable of active contraction normal to the plane of the membrane. With suction applied before seal formation, vesicles may be swept from the cell surface by shear stress generated from the flow of saline over the cell surface. In this case, patch recordings are made from membrane that was not originally present under the tip. The vesicles may break, or fuse and break, to form the gigasealed patch. Patch membranes adhere strongly to the wall of the pipette so that at zero transmural pressure the membranes tend to be normal to the wall. With transmural pressure gradients, the membranes generally become spherical; the radius of curvature decreasing with increasing pressure. Some patches have nonuniform curvature demonstrating that forces normal to the membrane may be significant. Membranes often do not respond quickly to changes in pipette pressure, probably because viscoelastic cytoplasm reduces the rate of flow through the tip of the pipette. Inside-out patches may be peeled from the walls of the pipette, and even everted (with positive pressure), without losing the seal. This suggests that the gigaseal is a distributed property of the membrane-glass interface.

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