Angiotensin II-induced formation of ionic channels in bilayer lipid membranes.

The interaction of angiotensin II (ANG II) with membrane was studied by measuring conductance and current-voltage characteristics (IVC) of bilayer lipid membranes (BLM) prepared of a mixture of egg lecithin with cholesterol, and of gramicidin D-modified membranes of the same composition. Addition of physiological concentrations of ANG II (approx. 15 mumol/l) into the electrolyte (1 mol/l KCl, pH = 7) in contact with one side of BLM resulted in the appearance of discrete membrane conductance (symbol; see text) = (39.5 +/- 1.07) pS with a duration of the conductivity state tau = (52.15 +/- 6.44) s. Raising ANG II concentration to 75 mumol/l resulted in an additional conductance level of approx. 130 pS with a lifetime of approx. 1s. The electrolyte pH markedly influenced ANG II modified BLM conductance. A decrease of the electrolyte pH to 2.8 resulted in a reduction of the discrete conductance level to approx. 14 pS, whereas ANG did not induce any conductivity at pH = 11.5. The results obtained suggest that ion channels are formed consisting at least of two ANG II molecules. IVC of ANG II-modified BLM are superlinear within the range of electrolyte concentrations studied (between 0.01 and 3 mol/l KCl), i.e, the limiting stage of ion transport is the internal area of the conducting pore. ANG II affects in a cooperative manner the gramicidin D (GRD)-mediated transport, most likely by forming ANG II aggregates in the area of local inhomogeneities in the BLM structure of GRD channels.     

Interaction between filamentous actin and lipid bilayer causes the increase of syringomycin E channel-forming activity

The effect of filamentous (F) actin on the channel-forming activity of syringomycin E (SRE) in negatively charged and uncharged bilayer lipid membranes (BLM) was studied. F-actin did not affect the membrane conductance in the absence of SRE. No changes in SRE-induced membrane conductance were observed when the above agents were added to the same side of BLM. However, the opposite side addition of F-actin and SRE provokes a multiple increase in membrane conductance. The similar voltage dependence of membrane conductance, equal values of single channel conductance and the effective gating charge of the channels upon F-actin action suggests that the actin-dependent increase in BLM conductance may result from an increase in the number of opened SRE-channels. BLM conductance kinetics depends on the sequence of SRE and F-actin addition, suggesting that actin-dependent rise of conductance may be induced by BLM structural changes that follow F-actin adsorption. F-actin exerted similar effect on membrane conductance of both negatively charged and uncharged bilayers, as well as on conductance of BLM with high ionic strength bathing solution, suggesting the major role for hydrophobic interactions in F-actin adsorption on lipid bilayer.     

Capacitive and ionic currents in BLM from phosphatidic acid in Ca2+-induced phase transition

The development of electric current with time in a bilayer lipid membrane (BLM) formed from dipalmitoylphosphatidic acid on introducing Ca2+ ions into the medium was studied at constant temperature and pH. The phase transition in the Ca2+-induced BLM is accompanied by the initial capacitive current followed by the occurrence of single ionic channels. The amount of transported charges in the capacitive current is 5 C/ microF. The conductivity of the single ionic channels ranges from 50 to 100 pSm.     

Reconstitution of the Mitochondrial ATP-Dependent Potassium Channel into Bilayer Lipid Membrane1

Electrical properties and regulation of the mitochondrialATP-dependent potassium channel were studied. The channel protein wassolubilized from the mitochondrial membrane using an ethanol/water mixture.Reconstituted into a bilayer lipid membrane BLM), the protein formed aslightly voltage-dependent channel with a conductance of 10 pS in 100 mM KCl.Often, several channels worked simultaneously (clusters) when many channelswere incorporated into the BLM. The elementary channel and the clusters wereboth highly potassium selective. At concentrations of 1 to 10 M, ATPfavors channel opening, while channels become closed at 1–3 mM ATP. GDP(0.5 mM) reactivated the ATP-closed channels without affecting the untreatedchannels. The sulfhydryl-reducing agent ditiothreitol increased the openprobability at concentrations of 1 to 3 mM, but damaged the selectivity ofthe channel.     

Protein from beef heart mitochondria inducing the potassium channel conductivity of bilayer lipid membrane

Protein (M. m. 60 000) inducing selective potassium conductance of bilayer lipid membranes (BLM) was isolated from mitochondria and homogenate of the beef heart. This protein was obtained by means of alcohol (ethanol) extraction and was purified by gel-filtration on Sephadex G-15 and G-50 followed by electrophoresis in the 10% polyacrylamide gel. 6-10 g/ml of the protein produced the conductivity channels on BLM with amplitude divisible of 24 +/- 4 pmho. The channels of 175 +/- 7 pmho were the most typical ones. The modification of BLM by K+-transport in protein under the conditions of potassium gradient resulted in the appearance of the membrane potential close to the theoretical Nernst potential.     

Bongkrekic acid and atractyloside inhibits chloride channels from mitochondrial membranes of rat heart

The aim of this work was to characterize the effect of bongkrekic acid (BKA), atractyloside (ATR) and carboxyatractyloside (CAT) on single channel properties of chloride channels from mitochondria. Mitochondrial membranes isolated from a rat heart muscle were incorporated into a bilayer lipid membrane (BLM) and single chloride channel currents were measured in 250/50 mM KCl cis/trans solutions. BKA (1-100 microM), ATR and CAT (5-100 microM) inhibited the chloride channels in dose-dependent manner. The inhibitory effect of the BKA, ATR and CAT was pronounced from the trans side of a BLM and it increased with time and at negative voltages (trans-cis). These compounds did not influence the single channel amplitude, but decreased open dwell time of channels. The inhibitory effect of BKA, ATR and CAT on the mitochondrial chloride channel may help to explain some of their cellular and/or subcellular effects.     

Coupled gating between individual cardiac ryanodine calcium release channels

In order to study interactions between ryanodine receptor calcium release (RyR2) channels during excitation-contraction coupling in cardiac muscle, we used bilayer lipid membrane (BLM) and improved the method of cardiac sarcoplasmic vesicle fusion into BLM. We increased fusion gradient for the vesicles, used chloride ions for fusion up to concentration of 1.2 mol/l and fused the vesicles by adding them directly to the forming BLM. Under these conditions, increased probability of fusion of vesicles containing 2-7 ryanodine channels into BLM was observed. Interestingly about 10% of the channels did not gate into BLM independently, but their gating was coupled. At 53 mmol/l calcium solution, two coupled gating channels had double conductance (191 +/- 15 pS) in comparison with the noncoupled channels (93 +/- 10 pS). Activities of the coupled channels were decreased by 5 micromol/l ryanodine and inhibited by 10 micromol/l ruthenium red similarly as single RyR2 channels. We suppose that cardiac sarcoplasmic vesicles contain single as well as coupled RyR2 channels.     

The thiazole derivative reduces transmembrane currents via ionic channels formed by alpha-latrotoxin and sea anemone toxin in the bilayer lipid membranes

It was shown that the thiazole derivative 3-decyloxycarbonylmethyl-4-methyl- 5-(2-hydroxyethyl)thiazole chloride (DMHT) (0.1 mM) reversibly reduced the transmembrane current in solutions of 10 mM CaCl2 and 100 mM KCl via ionic channels produced by alpha-latrotoxin from black widow spider (alpha-LT) and sea anemone toxin (RTX) in the bilayer lipid membranes (BLM). Introduction of DMHT from the cis-side of BLM inhibited transmembrane current by 31.6 +/- 3% and by 61.8 +/- 3% from the trans-side of BLM for alpha-LT channels. Application of DMHT to the cis-side BLM decreased the inward current through the RTX channels by 50 +/- 5%. Addition of Cd(2+) (0.1 mM) to the cis- or trans-side of a membrane after the DMHT induced depression of transmembrane current across the alpha-LT channels caused its further decrease by 85 +/- 5% that coincides completely with the intensity of Cd(2+)-inhibition in the control experiments without DMHT. These data suggest that DMHT may exert its inhibitory action on alpha-LT channels without considerable influence on the ionogenic groups inside the channel cavity. The comparative analysis of effective radii measured for alpha-LT and RTX channels on the cis- (0.9 nm and 0.55 nm, respectively) and the trans-side of BLM (< 0.467 nm for alpha-LT) allowed to propose the blocking action of DMHT for alpha-LT and RTX channels to result from direct penetration into the channel, achieved due to similar hydrodynamic size of blocking molecules and the size of toxin pores.     

Bongkrekic acid and atractyloside inhibits chloride channels from mitochondrial membranes of rat heart

The aim of this work was to characterize the effect of bongkrekic acid (BKA), atractyloside (ATR) and carboxyatractyloside (CAT) on single channel properties of chloride channels from mitochondria. Mitochondrial membranes isolated from a rat heart muscle were incorporated into a bilayer lipid membrane (BLM) and single chloride channel currents were measured in 250/50 mM KCl cis/trans solutions. BKA (1-100 μM), ATR and CAT (5-100 μM) inhibited the chloride channels in dose-dependent manner. The inhibitory effect of the BKA, ATR and CAT was pronounced from the trans side of a BLM and it increased with time and at negative voltages (trans-cis). These compounds did not influence the single channel amplitude, but decreased open dwell time of channels. The inhibitory effect of BKA, ATR and CAT on the mitochondrial chloride channel may help to explain some of their cellular and/or subcellular effects.     

Potassium-chloride promiscuous channels in mitochondrial membranes

Mitochondrial membranes isolated from a rat heart muscle were incorporated into a bilayer lipid membrane (BLM) and channel currents were measured in 250/50 mmol/l KCl cis/trans solutions. The channel currents measured from -40 to +40 mV had various linear voltage-current relationships and K(+)/Cl(-) permeability ratios at distinct voltage ranges. The channels possessed K(+)-Cl(-) promiscuous property. Depending on voltage, membrane permeability suddenly switched from K(+) over Cl(-) to Cl(-) over K(+) and back. The channels had Cl(-)/K(+) > 1 permeability at potentials around 0 mV and the permeability was switched to K(+)/Cl(-) > 1 at more negative and positive potentials. The chloride channel blocker, 5-nitro-2-(phenylpropylamino)-benzoate (NPPB, 5 x 10(-5) mol/l), influenced properties of the promiscuous channels - it activated potassium conductance of the channels.     

Low molecular channel-former protein from the mitochondrial membrane

Individual low molecular weight protein component was isolated by gel-filtration method from the inner mitochondrial membrane. This membrane component increases the conductivity of bilayer lipid membranes (BLM) in the presence of K+ and Ca2+-ions. This phenomenon may be explained by the formation of single conductivity channels. The voltage - current characteristics of this channel is nonlinear, which may be the result of asymmetrical operation of the channel - former in the polarized membrane, in spite of equal concentration of protein on both sides of the membrane.     

Gravity dependency of the gramicidin A channel conductivity

Theoretical investigations involving the membrane-solution interface have revealed that the density of the solution varies appreciably within interfacial layers adjacent to charged membrane surfaces. The hypothesis that gravity interacts with this configuration and modifies transport rates across horizontal and vertical membranes differently was supported by initial experiments with gramicidin A channels in phosphatidylserine (PS) membranes in 0.1 M KCl. Channel conductivity was found to be about 1.6 times higher in horizontal membranes than in vertical membranes. Here we present the results of further experiments with gramicidin A channels (incorporated into charged PS- and uncharged phosphatidylcholine (PC) membranes in KCl- and CsCl-solutions) to demonstrate that the hypothesis is more generally applicable. Again, channel conductivity was found to be higher in horizontal PS membranes by a factor of between 1.20 and 1.75 in 0.1 M CsCl. No difference in channel conductivity was found for uncharged PC membranes in 0.1 M KCl and in 0.1 M CsCl. However, for PC membranes in 0.05 M KCl the channel conductivity was significantly higher in horizontal membranes by a factor of between 1.07 and 1.14. These results are consistent with the results of our model calculations of layer density and extension, which showed that the layer formation is enhanced by increasing membrane surface charge and decreasing electrolyte ion concentration. The mechanism of gravity interaction with membrane transport processes via interface reactions might be utilized by biological systems for orientational behaviour in the gravity field, which has been observed even for cellular systems. Received: 16 October 1995 / Accepted: 23 April 1996     

Chemical composition of calcium channels of characean algal cells

The present study was performed to determine the chemical composition and molecular weight of channel-forming complex by fractioning the cell homogenate supernatant by different means (chromatography, gel-electrophoresis, using dissociating and denaturating agents). Availability of the channel-forming complexes in individual fractions is judged from reconstruction of channels in BLM. The Ca-channels reconstructed in BLM are formed by molecules of a protein nature (presumably, peptides). Their molecular weight is no more than 20000 (presumably, 5000). A high stability of spacial organization of channel forming molecules was observed. The channels--subunits aggregate without any change in properties of the selective filter. The thermodynamically preferred agagregates have conductivity of 200 pmho in 0.1 M KCl and seem to consist of 80 channels--subunits which are switched off and on all together.     

Conductivity and diameter of latrotoxin channels in lipid bilayers

It is shown that approximately 9 A Ca-selective ion channels were induced in bilayer lipid membrane (BLM) from phosphatidylserine by nonpurified spider venom (Latrodectus tredicimguttatus) and by alpha-latrotoxin obtained from it. It is established that channels greatly different in conductivity have the same diameter and nearly the same charge constitution, that evidences for their claster organization. The purification process as well as freezing-thawing and long time keeping influence the channel conductivity without changing its diameter and selectivity.     

Vitamin B1 thiazole derivative reduces transmembrane current through ionic channels formed by toxins from black widow spider venom and sea anemone in planar phospholipid membranes

The vitamin B1 (thiamine) structural analogue 3-decyloxycarbonylmethyl-4-methyl-5-(beta-hydroxyethyl) thiazole chloride (DMHT) (0.1 mM) reversibly reduced transmembrane currents in CaCl2 and KCl solutions via ionic channels produced by latrotoxins (alpha-latrotoxin (alpha-LT) and alpha-latroinsectotoxin (alpha-LIT)) from black widow spider venom and sea anemone toxin (RTX) in the bilayer lipid membranes (BLMs). Introduction of DMHT from the cis-side of BLM bathed in 10 mM CaCl2 inhibited transmembrane current by 31.6+/-3% and by 61.8+/-3% from the trans-side of BLM for alpha-LT channels. Application of DMHT in the solution of 10 mM CaCl2 to the cis-side of BLM decreased the current through the alpha-LIT and RTX channels by 52+/-4% and 50+/-5%, respectively. Addition of Cd2+ (1 mM) to the cis- or trans-side of the membrane after the DMHT-induced depression of Ca2+-current across the alpha-LT channels caused its further decrease by 85+/-5% that coincides favorably with the intensity of Cd2+ blocking in control experiments without DMHT. These data suggest that DMHT inhibiting is not specific for latrotoxin channels only and DMHT may exert its action on alpha-LT channels without considerable influence on the ionogenic groups of Ca2+-selective site inside the channel cavity. The binding kinetics of DMHT with the alpha-LT channel shows no cooperativity and allows to expect that the DMHT binding site of the toxin is formed by one ionogenic group as the slopes of inhibition rate determined in log-log coordinates are 1.25 on the trans-side and 0.68 on the cis-side. Similar pK of binding (5.4 on the trans-side and 5.7 on the cis-side) also suggest that DMHT may interact with the same high affinity site of alpha-LT channel on either side of the BLM. The comparative analysis of effective radii measured for alpha-LT, alpha-LIT and RTX channels on the cis-side (0.9 nm, 0.53 nm and 0.55 nm, correspondingly) and for alpha-LT channel on the trans-side (0.28+/-0.18 nm) with the intensity of DMHT inhibitory action obtained on these channels allowed to conclude that the potency of DMHT inhibition increased on toxin pores of smaller lumen.     

Interaction of two heads of myosin with F-actin: binding of H-meromyosin with F-actin in the absence of nucleotide

The bindings of S-1 and the two heads of HMM with pyrene-labeled F-actin were studied using the change in light-scattering intensity or that in the fluorescence intensity of the pyrenyl group. At low ionic strength (50 mM KCl), both S-1 and HMM became bound tightly with F-actin (Kd less than 0.1 microM) and both heads of HMM became bound to F-actin. The affinities of S-1 and HMM for F-actin decreased with increasing KCl concentration. In 1 M KCl, the Kd values of S-1 and HMM for F-actin were 11 and 0.58 microM, respectively. Thus, HMM was bound to F-actin 19 times more tightly than S-1. We compared the extent of binding of HMM to F-actin measured by a centrifugation method with that measured by the fluorescence change of pyrenyl-group, and found that even in 1 M KCl, HMM became bound to F-actin with a two-headed attachment. We measured the kinetics of binding and dissociation of acto-S-1 and acto-HMM from the time course of the change in light-scattering intensity after mixing S-1 or HMM with F-actin at 1 M KCl and that after mixing 1 M KCl with acto-S-1 or acto-HMM formed at low ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)     

hERG ion channel pharmacology: cell membrane liposomes in porous-supported lipid bilayers compared with whole-cell patch-clamping

The purpose of this study was to obtain functional hERG ion channel protein for use in a non-cell-based ion channel assay. hERG was expressed in Sf9 insect cells. Attempts to solubilise the hERG protein from Sf9 insect cell membranes using non-ionic detergents (NP40 and DDM) were not successful. We therefore generated liposomes from the unpurified membrane fraction and incorporated these into porous Teflon-supported bilayer lipid membranes. Macroscopic potassium currents (1 nA) were recorded that approximated those in whole-cell patch-clamping, but the channels were bidirectional in the bilayer lipid membrane (BLM). Currents were partially inhibited by the hERG blockers E4031, verapamil, and clofilium, indicating that the protein of interest is present at high levels in the BLM relative to endogenous channels. Cell liposomes produced from Sf9 insect cell membranes expressing voltage-gated sodium channels also gave current responses that were activated by veratridine and inhibited by saxitoxin. These results demonstrate that purification of the ion channel of interest is not always necessary for liposomes used in macro-current BLM systems.     

Inward-rectifier potassium channels in basolateral membranes of frog skin epithelium

Inward-rectifier K channel: using macroscopic voltage clamp and single- channel patch clamp techniques we have identified the K+ channel responsible for potassium recycling across basolateral membranes (BLM) of principal cells in intact epithelia isolated from frog skin. The spontaneously active K+ channel is an inward rectifier (Kir) and is the major component of macroscopic conductance of intact cells. The current- voltage relationship of BLM in intact cells of isolated epithelia, mounted in miniature Ussing chambers (bathed on apical and basolateral sides in normal amphibian Ringer solution), showed pronounced inward rectification which was K(+)-dependent and inhibited by Ba2+, H+, and quinidine. A 15-pS Kir channel was the only type of K(+)-selective channel found in BLM in cell-attached membrane patches bathed in physiological solutions. Although the channel behaves as an inward rectifier, it conducts outward current (K+ exit from the cell) with a very high open probability (Po = 0.74-1.0) at membrane potentials less negative than the Nernst potential for K+. The Kir channel was transformed to a pure inward rectifier (no outward current) in cell- attached membranes when the patch pipette contained 120 mM KCl Ringer solution (normal NaCl Ringer in bath). Inward rectification is caused by Mg2+ block of outward current and the single-channel current-voltage relation was linear when Mg2+ was removed from the cytosolic side. Whole-cell current-voltage relations of isolated principal cells were also inwardly rectified. Power density spectra of ensemble current noise could be fit by a single Lorentzian function, which displayed a K dependence indicative of spontaneously fluctuating Kir channels. Conclusions: under physiological ionic gradients, a 15-pS inward- rectifier K+ channel generates the resting BLM conductance in principal cells and recycles potassium in parallel with the Na+/K+ ATPase pump.     

Effect of insulin on lipid bilayer viscoelasticity

Changes in the Young elasticity modulus in perpendicular direction to the membrane surface E perpendicular, in the coefficient of dynamic viscosity eta, in the electric capacitance C, in the surface charge U1, in the conductivity g and in the coefficient of non-linearity beta of current-voltage characteristic caused by insulin were studied in bilayer lipid membranes (BLM) prepared from a mixture of egg lecithin and cholesterol (4:1, w/w) in n-heptane. Even relatively small concentrations of insulin in electrolyte (ci approximately 4.8 x 10(-11) mol/l) caused a diminution in parameters E perpendicular and eta. Negative surface charge emerged on the membrane due to the insulin absorption, and U1 gradually increased depending on the concentration of the hormone in the electrolyte. Addition of insulin was also followed by an increase in membrane conductivity and affected the value of the coefficient of non-linearity beta of current-voltage characteristic. The effect of insulin on the BLM structure was discussed on the basis of the results obtained.     

Bilayer lipid membranes supported on Teflon filters: a functional environment for ion channels

Many ion channel proteins have binding sites for toxins and pharmaceutical drugs and therefore have much promise as the sensing entity in high throughput technologies and biosensor devices. Measurement of ionic conductance changes through ion channels requires a robust biological membrane with sufficient longevity for practical applications. The conventional planar BLM is 100-300 μm in diameter and typically contains fewer than a dozen channels whereas pharmaceutical screening methods in cells use current recordings for many ion channels. We present a new, simple method for the fabrication of a disposable porous-supported bilayer lipid membrane (BLM) ion channel biosensor using hydrated Teflon (polytetrafluoroethylene, PTFE) filter material (pore size 5 μm, filter diameter=1 mm). The lipid layer was monitored for its thickness and mechanical stability by electrical impedance spectroscopy. The results showed membrane capacitances of 1.8±0.2 nF and membrane resistances of 25.9±4.1 GΩ, indicating the formation of lipid bilayers. The current level increased upon addition of the pore-forming peptide gramicidin. Following addition of liposomes containing voltage-gated sodium channels, small macroscopic sodium currents (1-80 pA) could be recorded. By preloading the porous Teflon with sodium channel proteoliposomes, prior to BLM formation, currents of 1-10 nA could be recorded in the presence of the activator veratridine that increased with time, and were inhibited by tetrodotoxin. A lack of rectification suggests that the channels incorporated in both orientations. This work demonstrates that PTFE filters can support BLMs that provide an environment in which ion channels can maintain their functional activity relevant for applications in drug discovery, toxin detection, and odour sensing.