Pore formation in lipid bilayer membranes made of phosphatidylinositol and oxidized cholesterol followed by means of alternating current.

The kinetics of porin incorporation into black lipid membranes (BLM) made of phosphatidylinositol (PI) or oxidized cholesterol (Ox Ch) were studied by means of alternating current; the set-up was able to acquire resistance and capacitance simultaneously by means of a mixed double-frequency approach at 1 Hz and 1 KHz, respectively. Conductance was dependent on the interaction between protein-forming pores and lipids. For PI membranes below a porin concentration of 12.54 ng/ml, there was no membrane conductivity, whereas at 200 ng/ml a steady-state value was reached. Different behavior was displayed by Ox Ch membranes, in which a concentration of 12.54 ng/ml was sufficient to reach a steady state. The incorporation kinetics when porin was added after membrane formation were sigmoidal. When porin was present in the medium before membrane formation, the kinetics were sigmoidal for PI membranes but became exponential for Ox Ch membranes. Furthermore, for BLM made of PI, the conductance-versus-porin concentration relationship is sigmoidal, with a Hill coefficient of 5.6 +/- 0.07, which is functional evidence corroborating the six-channel repeating units seen previously. For BLM made of Ox Ch, this relationship followed a binding isotherm curve with a Hill coefficient of 0.934 +/- 0.129.     

Proton transport across transient single-file water pores in a lipid membrane studied by molecular dynamics simulations.

To test the hypothesis that water pores in a lipid membrane mediate the proton transport, molecular dynamic simulations of a phospholipid membrane, in which the formation of a water pore is induced, are reported. The probability density of such a pore in the membrane was obtained from the free energy of formation of the pore, which was computed from the average force needed to constrain the pore in the membrane. It was found that the free energy of a single file of water molecules spanning the bilayer is 108(+/-10) kJ/mol. From unconstrained molecular dynamic simulations it was further deduced that the nature of the pore is very transient, with a mean lifetime of a few picoseconds. The orientations of water molecules within the pore were also studied, and the spontaneous translocation of a turning defect was observed. The combined data allowed a permeability coefficient for proton permeation across the membrane to be computed, assuming that a suitable orientation of the water molecules in the pore allows protons to permeate the membrane relatively fast by means of a wirelike conductance mechanism. The computed value fits the experimental data only if it is assumed that the entry of the proton into the pore is not rate limiting.     

Fluorimetric detection of phospholipid vesicles bound to planar phospholipid membranes.

The first step in the fusion of two phospholipid membranes culminates in the aggregation of the two lipid bilayers. We have used a custom-built fluorimeter to detect multilamellar vesicles (liposomes) containing the fluorescent dye, 6-carboxyfluorescein (6-CF), bound to a planar lipid bilayer (BLM). Liposomes were added to one side of the BLM, and unbound vesicles were perfused out. This left a residual fluorescence from the BLM, but only when the membranes contained anionic lipids, and then only when millimolar levels of calcium were present. This residual fluorescence was consistently detected only when calcium was included in the buffer during the perfusion. This residual fluorescence originated from liposomes bound to the BLM. Breaking the BLM or lysing the adsorbed vesicles with distilled water abolished it. free 6-CF and/or calcium in the absence of liposomes resulted in no residual fluorescence. No residual fluorescence was detected when both the liposomes and the BLM were composed entirely of zwitterionic lipids. This was found to result from the insensitivity of the fluorimeter to a small number of liposomes adsorbed to the BLM. For this system, we conclude that calcium is necessary for both the initiation and maintenance of the state in which the vesicle membrane is bound to the planar bilayer when the membranes contain negatively charged lipids. This attachment is stronger than the interaction between zwitterionic membranes.     

BLM Analyzer: a software tool for experiments on planar lipid bilayers

Planar lipid bilayers represent a versatile platform for studying the functions of various membrane proteins as well as the development of biosensors. Despite the continuing technological progress in the fabrication of low-noise bilayer setups with mechanically and electrically stable planar bilayers, there is still a lack of software utilities for assistance during bilayer formation. We present here a multipurpose software tool, the bilayer lipid membrane (BLM) Analyzer which performs high-resolution measurements of bilayer capacitance and resistance using saw-tooth voltage stimulation. Based on the measured values of capacitance and resistance, the BLM Analyzer detects formation, stabilization, and breakage of lipid bilayer, automatically selects appropriate stimulus protocol, compensates for voltage offsets, and issues sound and voice alerts informing about the state of the measurement cycle. The principle of the BLM Analyzer is based on the integration of current responses within four equivalent time segments. It provides capacitance estimates with standard deviation of several femtofarads at temporal resolution of several tens of milliseconds. The functions of the BLM Analyzer were tested experimentally by monitoring formation and thinning of planar lipid bilayer.     

Increase of (Ca2+ +Mg2+)-ATPase activity of renal basolateral membranes by platelet-derived growth factor through a specific receptor

Studies were made on the direct effect of platelet-derived growth factor (PDGF) on the high-affinity (Ca2+ +Mg2+)-ATPase, a membrane bound Ca2+-extrusion pump enzyme of the basolateral membranes (BLM) of canine kidney (Km for free Ca2+ = 1.0 x 10(-7) M, Vmax = 180 nmol Pi/mg/min). At 1 x 10(-7) M free Ca2+, PDGF (10(-10)-10(-8) M) stimulated the enzyme activity significantly. Addition of 5 - 200 microM suramin, a compound that blocks binding of PDGF to its receptors on cell membranes, inhibited the stimulatory effect of PDGF dose-dependently (IC50 = 40 microM). A high affinity specific receptor for PDGF (Kd = 4.4 x 10(-10) M, Bmax = 460 fmol/mg protein) was detected on BLM preparations by radioreceptor assay with 125I-PDGF and unlabelled PDGF. Suramin (10-1000 microM) also inhibited the binding of PDGF to BLM preparations dose-dependently. From these results, it is proposed that PDGF stimulates (Ca2+ +Mg2+)-ATPase activity of kidney BLM preparations by enhancing its affinity for free Ca2+ through a specific receptor.     

Study of conductance changes of bilayer lipid membrane induced by electric field

Changes in the bilayer lipid membrane (BLM) conductance induced by electric field were studied. BLMs were formed from diphytanoylphosphocholine (DPhPC) solution in squalene. Certain time after a constant voltage (200-500 mV) was applied to the BLM in the voltage-clamp mode, the BLM conductance started to grow up to approximately 10 nS until the BLM ruptured. The conductance often changed abruptly (with the front duration of less than 33 micros) and then stabilized for a relatively long time (up to 10; 300 ms on average) thus resembling the ion channel activity. The mean amplitude of conductance steps was 650 pS. However, in some cases a slow conductance drift was recorded. When N-methyl-D-glucamine/glutamate ions were used instead of KCl, the conductance changes became 5 times smaller. We suggest that formation in the BLM of single pores approximately 1 nm in diameter should result in the observed changes in BLM conductance. The BLM conductance growth was due to consecutive opening of several such pores. When the electric field amplitude was abruptly decreased (down to 50-100 mV), the conductance dropped rapidly to the background value. When we increased the voltage again, the BLM conductance right after the increase depended on the time BLM spent under "weak" electric field. If this time exceeded 500 ms, the conductance was at the background level, but when the time was diminished, the conductance reached the value recorded before the voltage decrease. These data imply that the closure of the pores should lead to the formation in BLM of small defects (prepores) that can be easily transformed into pores when the voltage is increased. The lifetimes of such prepores did not exceed 500 ms.     

The effect of S-layer protein adsorption and crystallization on the collective motion of a planar lipid bilayer studied by dynamic light scattering.

A dedicated dynamic light scattering (DLS) setup was employed to study the undulations of freely suspended planar lipid bilayers, the so-called black lipid membranes (BLM), over a previously inaccessible spread of frequencies (relaxation times ranging from 10(-2) to 10(-6) s) and wavevectors (250 cm(-1) < q < 38,000 cm(-1)). For a BLM consisting of 1,2-dielaidoyl-sn-3-glycero-phosphocholine (DEPC) doped with two different proportions of the cationic lipid analog dioctadecyl-dimethylammonium bromide (DODAB) we observed an increase of the lateral tension of the membrane with the DODAB concentration. The experimentally determined dispersion behavior of the transverse shear mode was in excellent agreement with the theoretical predictions of a first-order hydrodynamic theory. The symmetric adsorption of the crystalline bacterial cell surface layer (S-layer) proteins from Bacillus coagulans E38-66 to a weakly cationic BLM (1.5 mol % DODAB) causes a drastic reduction of the membrane tension well beyond the previous DODAB-induced tension increase. The likely reason for this behavior is an increase of molecular order along the lipid chains by the protein and/or partial protein penetration into the lipid headgroup region. S-layer protein adsorption to a highly cationic BLM (14 mol % DODAB) shows after 7 h incubation time an even stronger decrease of the membrane tension by a factor of five, but additionally a significant increase of the (previously negligible) surface viscosity, again in excellent agreement with the hydrodynamic theory. Further incubation (24 h) shows a drastic increase of the membrane bending energy by three orders of magnitude as a result of a large-scale, two-dimensional recrystallization of the S-layer proteins at both sides of the BLM. The results demonstrate the potential of the method for the assessment of the different stages of protein adsorption and recrystallization at a membrane surface by measurements of the collective membrane modes and their analysis in terms of a hydrodynamic theory.     

Static and dynamic components of renal cortical brush border and basolateral membrane fluidity: Role of cholesterol

Summary Static polarization and differential polarized phase fluorimetry studies on rat renal cortical brush border (BBM) and basolateral membranes (BLM) were undertaken to determine the membrane components responsible for differences in BBM and BLM fluidity, whether these differences were due to the order or dynamic components of membrane fluidity and if a fluidity gradient existed within the bilayer. Surface membrane proteins rigidified both BBM and BLM fluidity. Neutral lipid extraction, on the other hand, caused a larger decrease in BBM than BLM fluorescence polarization (0.104vs. 0.60,P<0.01) using diphenyl hexatriene (DPH). Cholesterol addition to phospholipid fractions restored membrane fluidity to total lipid values in both BBM and BLM phospholipids. The response to cholesterol in the BBM was biphasic, while the BLM response was linear. Lateral mobility, quantitated using dipyrenylpropane, was similar in both BBM and BLM fractions at 35°C. BBM and BLM differed primarily in the order component of membrane fluidity as DPH-limiting anisotropy (r ) (0.212vs. 0.154,P<0.01) differed markedly between the two membrane fractions. The two membrane components also differed with respect to 2 and 12-anthroyloxy stearate (2-AS, 12-AS) probes, indicating a difference in the dynamic component of membrane fluidity may also be present. DPH and 12-As probes were also used to quantitate inner core membrane fluidity and showed the BBM was less fluid than the BLM for intact membranes, total lipid extracts and phospholipids. Results obtained using the surface membrane probes trimethylammonium-DPH (TMA-DPH) and 2-AS suggested a fluidity gradient existed in both BBM and BLM bilayers with the inner core being more fluid in both membranes. These data indicate cholesterol is in large part responsible for fluidity differences between BBM and BLM and that these membranes, while clearly differing in the order component of membrane fluidity, may also difer in the dynamic component as well.     

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.     

The kinetics of DNA damage by bleomycin in mammalian cells.

The kinetics of DNA damage by bleomycin (BLM) was assessed by measuring the amount of DNA breakage induced by BLM at different doses, treatment lengths, and treatment temperatures. DNA degradation was measured with the alkaline unwinding method. Comparison of the curves of DNA cleavage by BLM leads to the conclusion that low doses (1-5 micrograms/ml) and short treatments (5-15 min) produce marked damage in the DNA. High increases in BLM concentration produce relatively small increases in DNA damage above the levels obtained with low doses. Extension of treatment times does not increase the DNA degradation above the rate observed with 15-min treatments. The repair of DNA damage starts at about 15 min after the initiation of treatment. The mending of DNA breaks is very fast and extensive when BLM is no longer present. Repair not only implies the closing of DNA nicks, but very likely the degradation of the BLM molecules intercalated in the DNA interrupting the reactions responsible for the generation of free radicals. Persistence of BLM in the cell environment facilitates the replacement of degraded BLM molecules by new ones. This produces the persistent production of free radicals and the establishment of a balance between the processes of DNA damage and repair.     

Electric capacitance of bilayer lipid membranes from hydrogenated egg lecithin during phase transition from liquid crystalline state to gel

The electrical capacity of planar bilayer lipid membranes (BLM) from natural hydrogenated egg lecithin (HEL) in n-decane at a temperature of phase transition was measured. The temperature of phase transition was determined calorimetrically to be 51 degrees C. The data obtained revealed a phase separation of HEL in BLM into two fractions, one freezing at 42-44 degrees C and one that is converted to a liquid-crystal state at 51-59 degrees C. It was assumed that the first fraction is rich in dipalmitoyl lecithin, and the second fraction is rich in distearoyl lecithin. Freezing and the transition to the liquid-crystal state were accompanied by an increase and decrease in membrane thickness, respectively, in part due to a displacement of the solvent from the torus to the planar part of the bilayer. The displacement of the solvent is explained by changes in the disjoining pressure in BLM, which arises across the lipid bilayer due to van der Waals forces of attraction between water layers on both sides of the BLM.     

Diacylglycerol kinase activity in purified basolateral membranes of kidney tubules. I. Evidence for coupling with phospholipase C

The diacylglycerol kinase (DGK) catalyzes the phosphorylation of diacylglycerol (DAG) yielding phosphatidic acid (PA) signaling molecules which are involved in the modulation of different cell responses. The aim of this work was to characterize the DGK activity associated to the basolateral membranes (BLM) of kidney proximal tubules, in a native preparation that preserves the membrane microenvironment. The Arrhenius plot of DGK activity was non-linear, indicating a complex influence of the lipid environment of the native membrane. The formation of PA was strongly impaired by U73122, an inhibitor of PLC, whereas remained unmodified when exogenous DAG or PLC were added. The Mg.ATP2- complex is the true phosphoryl-donor substrate, and the very narrow peak of activation at pH 7.0 suggests that amino acids that dissociate at this pH, i.e. hystidine residues, play a role by acting in the coordination of the Mg2+ atoms. The renal DGK is almost completely blocked by 0.1 mM sphingosine, but it is insensitive to micromolar free Ca2+ concentrations and to R59499, the most potent inhibitor of the classical DGKs. Taken as a whole, these data suggest that the DGK isoform present in BLM of proximal tubules is different from those included in the type I family, and that membranous PLC could be the main source of DAG for DGK catalysis.     

Measurements of non-electrogenic fluxes through lipid bilayers induced by Men+/nH+-exchangers

In the presence of concentration gradient of metal ions on bilayer lipid membrane (BLM) the addition of non-electrogenic carriers results in a formation of concentration gradient of hydrogen ions in the unstirred layers near membrane. Addition of protonophore under these conditions brings about the formation of diffusion potential of hydrogen ions. This effect underlies the method of measuring non-electrogenic fluxes on BLM initiated in the presence of Men+/nH+ - exchangers. The proposed method was tested on the following Men+/nH+ - exchangers: nigericin, monensin and A23187. The order of cationic selectivity of the given carriers obtained by measuring the potentials on BLM in the presence of protonophores agrees with literature data, which were obtained by direct measurements of ionic fluxes.     

A new method of the measurement of the electrically neutral fluxes of cations through lipid bilayer membranes induced by Men+/nH+-exchangers

Electrically neutral ionophores (nigericin, monencin) incorporated into a planar bilayer lipid membrane (BLM) bring about hydrogen ion gradient formation in the unstirred layers of BLM if a metal ion gradient on the membrane is prepared. Under these conditions a diffusion potential of a hydrogen ion is generated after addition of a protonophore. Cation selectivity of nigericin, monencin and A23187 has been studied by means of electrical potential measurements in the presence of a protonophore and Men+/nH+-exchangers mentioned above. The data on cation selectivity are in a good agreement with the well known results of the direct measurements of metal ion fluxes. This shows that the effect of generation of the potential on BLM in the presence of a protonophore and a Men+/nH+-exchanger can be used for the estimation of electrically neutral ion fluxes through BLM.     

Ca++-induced fusion of fragmented sarcoplasmic reticulum with artificial planar bilayers

Summary Addition of fragmented sarcoplasmic reticulum (SR) vesicles to the aqueous phase of a black lipid membrane (BLM) causes a large increase in BLM conductance within 10 min. The conductance increase is absolutely dependent on three conditions: The presence of at least 0.5mm Ca⁺⁺, an acidic phospholipid such as phosphatidylserine or diphosphatidylglycerol in the BLM phospholipid mixture, and an osmotic gradient across the SR vesicle membrane, with the internal osmolarity greater than the external. These requirements are identical to conditions under which the fusion of phospholipid vesicles occurs.When the early part of the time course of conductance rise is examined at high sensitivity, the conductance is seen to increase in discrete steps. The probability of a step increases with the concentration of Ca⁺⁺ in the medium, with the fraction of acidic phospholipid in the BLM, and with the size of the osmotic gradient across the SR vesicle membrane. On the other hand, the average conductance change per step is independent of the above parameters, but varies with the type and concentration of ions present in the aqueous phase. For a given ion, the mean specific conductance per step is independent of the ion's concentration between 10 and 100mm.The probability distribution of the step-conductances agrees well with the distribution of SR vesicle surface areas, both before and after sonication of the vesicles.The evidence indicates that SR vesicles fuse with the BLM, thereby inserting SR membrane conductance pathways into it. Each discrete conductance jump appears to be the result of the fusion of a single SR vesicle with the BLM. This technique may serve as a general method for inserting membrane vesicles into an electrically accessible system.     

PICH and BLM limit histone association with anaphase centromeric DNA threads and promote their resolution

Centromeres nucleate the formation of kinetochores and are vital for chromosome segregation during mitosis. The SNF2 family helicase PICH (Plk1-interacting checkpoint helicase) and the BLM (the Bloom's syndrome protein) helicase decorate ultrafine histone-negative DNA threads that link the segregating sister centromeres during anaphase. The functions of PICH and BLM at these threads are not understood, however. Here, we show that PICH binds to BLM and enables BLM localization to anaphase centromeric threads. PICH- or BLM-RNAi cells fail to resolve these threads in anaphase. The fragmented threads form centromeric-chromatin-containing micronuclei in daughter cells. Anaphase threads in PICH- and BLM-RNAi cells contain histones and centromere markers. Recombinant purified PICH has nucleosome remodelling activities in vitro. We propose that PICH and BLM unravel centromeric chromatin and keep anaphase DNA threads mostly free of nucleosomes, thus allowing these threads to span long distances between rapidly segregating centromeres without breakage and providing a spatiotemporal window for their resolution.     

Study of F-actin interaction with planar and liposomal bilayer phospholipid membranes

Interaction of the cytoskeletal protein F-actin with planar bilayer lipid membrane (BLM) induced formation of single ionic channels in both NaCl and KCl bathing solutions. We also recorded noiselike high-currentjumps with a mean conductivity of approximately 160 pS, which might represent the simultaneous opening and closing of several channels of lower conductivity. The ratio of cation to anion permeabilities (Pc/Pa) of the BLM with many channels in KCl was 26 +/- 2. Freeze-fracture electron microscopy revealed fibrillar-like structures on the hydrophobic surfaces of liposomal membranes. We also observed some structural features giving evidence for the penetration of F-actin fibers through an artificial phospholipid membrane. We suggest that the F-actin/lipids complexes can transmit electric signals in synaptic and other intercellular contacts.     

Osmotic water permeabilities of brush border and basolateral membrane vesicles from rat renal cortex and small intestine

Summary The osmotic water permeabilityP _f of brush border (BBM) and basolateral (BLM) membrane vesicles from rat small intestine and renal cortex was studied by means of stopped-flow spectrophotometry. Scattered light intensity was used to follow vesicular volume changes upon osmotic perturbation with hypertonic mannitol solutions. A theoretical analysis of the relationship of scattered light intensity and vesicular volume justified a simple exponential approximation of the change in scattered light intensity. The rate constants extracted from fits to an exponential function were proportional to the final medium osmolarity as predicted by theory. For intestinal membranes, computer analysis of optical responses fitted well with a single-exponential treatment. For renal membranes a double-exponential treatment was needed, implying two distinct vesicle populations.P _f values for BBM and BLM preparations of small intestine were equal and amount to 60 m/sec. For renal preparations,P _f values amount to 600 m/sec for the fast component, BBM as well as BLM, and to 50 (BBM) and 99 (BLM) m/sec for the slow component. The apparent activation energy for water permeation in intestinal membranes was 13.3±0.6 and in renal membranes, 1.0±0.3 kCal/mole, between 25 and 35°C. The mercurial sulfhydryl reagentpCMBS inhibited completely and reversibly the highP _f value in renal brush border preparations. These observations suggest that in intestinal membranes water moves through the lipid matrix but that in renal plasma membranes water channels may be involved. From the highP _f values of renal membrane vesicles a transcellular water permeability for proximal tubules can be calculated which amounts to 1 cm/sec. This value allows for an entirely transcellular route for water flow during volume reabsorption.     

多重扫描伏安法在平面双分子脂膜(BLM)研究中的应用

本文阐明了伏安技术对重组膜的各种电性质测量原理和方法,并用多重扫描技术着重研究了膜电导的可逆跃迁变化,以及扫描速度和电压的影响,结果指出,伏安法可能发展成一种研究重组膜离子通道现象的新手段.