Macroscopic Membrane Research Articles

Preparation of Oriented Mesoporous Carbon Nano-Filaments within the Pores of Anodic Alumina Membranes

Oriented mesoporous carbon nano-filaments were prepared as replicas of Fe-containing silica mesostructures deposited inside the channels of anodic alumina membranes (AAMs). Due to the confinement imposed by the channels of the AAMs, rather unusual mesophase structures showing "circular" or "columnar" mesoscopic channels were formed that were replicated as pure carbon analogues after removal of the templating hosts. These structures are assembled in the forms of macroscopic membranes and are hierarchically ordered first due to the arrangement of the channels of the AAMs and second due to the mesophase counterparts.

The Role of Canonical Transient Receptor Potential 7 in B-cell Receptor-activated Channels

Phospholipase C signaling stimulates Ca2+ entry across the plasma membrane through multiple mechanisms. Ca2+ store depletion stimulates store-operated Ca2+-selective channels, or alternatively, other phospholipase C-dependent events activate Ca2+-permeable non-selective cation channels. Transient receptor potential 7 (TRPC7) is a non-selective cation channel that can be activated by both mechanisms when ectopically expressed, but the regulation of native TRPC7 channels is not known. We knocked out TRPC7 in DT40 B-cells, which expresses both forms of Ca2+ entry. No difference in the store-operated current I(crac) was detected between TRPC7-/- and wild-type cells. Wild-type cells demonstrated nonstore-operated cation entry and currents in response to activation of the B-cell receptor or protease-activated receptor 2, intracellular dialysis with GTPgammaS, or application of the synthetic diacylglycerol oleyl-acetyl-glycerol. These responses were absent in TRPC7-/- cells but could be restored by transfection with human TRPC7. In conclusion, in B-lymphocytes, TRPC7 appeared to participate in the formation of ion channels that could be activated by phospholipase C-linked receptors. This represents the first demonstration of a physiological function for endogenous TRPC7 channels.

On open membranes, cosmic strings and moduli stabilization

We discuss how cosmic strings can be created in heterotic M-theory compactifications with stable moduli. We conclude that the only appropriate candidates seem to be fundamental open membranes with a small length. In four dimensions they will appear as strings with a small tension. We make an observation that, in the presence of the vector bundle moduli, it might be possible to stabilize a five-brane very close to the visible sector so that a macroscopic open membrane connecting this five-brane and the visible brane will have a sufficiently small length. We also discuss how to embed such cosmic strings in heterotic models with stable moduli and whether they can be created after inflation.

Tilt modulus of a lipid monolayer

In addition to the familiar bending and stretching deformations, lipid monolayers and bilayers in their disordered state are often subjected to tilt deformations, occurring for instance in structural rearrangements accompanying membrane fusion, or upon insertion of "oblique" hydrophobic proteins into lipid bilayers. We study the elastic response of a flat lipid monolayer to a tilt deformation, using the spatial and conformational average of the chain end-to-end vector from the membrane normal to define a macroscopic membrane tilt. The physical origin and magnitude of the corresponding tilt modulus kappat is analyzed using two complementary theoretical approaches. The first is a phenomenological model showing that the tilt and bending deformations are decoupled and the effects of inter-chain correlations on the tilt modulus is small. The second is based on a molecular-level mean-field theory of chain packing, enabling numerical evaluation of the tilt modulus for realistic, multi-conformation, chain models. Both approaches reveal that the tilt modulus involves two major contributions. The first is elastic in origin, arising from the stretching of the hydrocarbon chains upon a tilt deformation and reflecting the loss of chain conformational freedom associated with chain stretching. The second, purely entropic, contribution results from the constraints imposed by a tilt deformation on the fluctuations of chain director orientations. Using the chain-packing theory we compute the two contributions numerically as a function of the cross-sectional area per chain. The elastic and entropic terms are shown to dominate the value of kappat for small and large areas per chain, respectively. For typical cross-sectional areas of lipid chains in biological membranes they are of comparable magnitude, yielding kappat approximately 0.2kBT/A2.

The cholinomimetic agent bethanechol activates IK(ACh) in feline atrial myocytes.

The effect of the cholinomimetic agent, bethanechol on macroscopic membrane currents was studied in dispersed cat atrial myocytes, using the whole-cell patch-clamp technique. Bethanechol activated an inward rectifying potassium current similar to I(K(ACh)), and a delayed rectifying-like outward current, similar to I(KM3) activated by pilocarpine, choline, and tetramethylammonium, and I(KM4) activated by 4-aminopyridine. The relatively specific muscarinic receptors subtype antagonists methoctramine (M(2)), and tropicamide (M(4)) inhibited both current components induced by bethanechol, suggesting a lack of specificity of these antagonists on cat atrial myocytes. The specific antagonist of M(3) receptors, para-fluoro-hexahydro-siladifenidol did not significantly inhibit the bethanechol-induced currents. In addition, pretreatment with PTX prevented activation of the bethanechol-induced inward and outward currents, suggesting that M(3) receptors are probably not involved in the bethanechol action. The I(K(ACh)) specific blocker tertiapin inhibited both inward rectifying- and delayed rectifying-like currents. These results suggest that both current components result from activation of a single channel type, likely I(K(ACh)).

Hydration and Lateral Organization in Phospholipid Bilayers Containing Sphingomyelin: A 2H-NMR Study

Interfacial properties of lipid bilayers were studied by 2H nuclear magnetic resonance spectroscopy, with emphasis on a comparison between phosphatidylcholine and sphingomyelin. Spectral resolution and sensitivity was improved by macroscopic membrane alignment. The motionally averaged quadrupolar interaction of interlamellar deuterium oxide was employed to probe the interfacial polarity of the membranes. The D 2O quadrupolar splittings indicated that the sphingomyelin lipid-water interface is less polar above the phase transition temperature T m than below T m. The opposite behavior was found in phosphatidylcholine bilayers. Macroscopically aligned sphingomyelin bilayers also furnished 2H-signals from the amide residue and from the hydroxyl group of the sphingosine moiety. The rate of water-hydroxyl deuteron exchange could be measured, whereas the exchange of the amide deuteron was too slow for the inversion-transfer technique employed, suggesting that the amide residue is involved in intermolecular hydrogen bonding. Order parameter profiles in mixtures of sphingomyelin and chain-perdeuterated phosphatidylcholine revealed an ordering effect as a result of the highly saturated chains of the sphingolipids. The temperature dependence of the 2H quadrupolar splittings was indicative of lateral phase separation in the mixed systems. The results are discussed with regard to interfacial structure and lateral organization in sphingomyelin-containing biomembranes.

Neat macroscopic membranes of aligned carbon nanotubes

Thick macroscopic membranes of magnetically aligned single-wall carbon nanotubes (SWNT) have been produced via high pressure filtration of aqueous surfactant-suspended SWNT in a magnetic field, resulting in membrane thicknesses of 10 μm and surface areas of 125 cm2. Field strengths of 7 and 25 T were used. Polarized Raman spectroscopy shows an anisotropy of (2.5±0.5), indicating good alignment. A similar degree of alignment was seen at both intensities of magnetic field. Furthermore, the degree of alignment is comparable to that achieved in previous work that produced smaller and thinner membranes at 25 T. The membranes also exhibit uniform anisotropy across their surface and throughout their thickness.

Aberrant patterns of cellular communication in diabetes-induced embryopathy I. Membrane signalling

Objective: Our purpose was to investigate the role of membrane signalling in the mechanism of diabetes-induced embryopathy. Methods: Three groups of 70-90-day-old Sprague-Dawley rats were employed in our study: group 1 was normal control rats receiving a normal diet; group 2 represented experimentally induced diabetic rats with malformed offspring (intravenous injection of 65 mg/kg streptozotocin on pregnancy day 6) and group 3 included streptozotocin-induced diabetic rats with normal offspring. Embryos were examined on day 12 under light microscopy, categorized as morphologically normal or defective, and yolk sac cells were harvested from each group. Activities of ERK1 and 2, Raf-1, JNK 1 and 2 in yolk sac cells were analyzed by Western blot with primary antibodies specific to the phosphorylated kinases, respectively. Results: A strong link between hyperglycemia and congenital malformations was confirmed. Key mitogen-activated protein kinases serve as syllabic intermediates: increased activities of Jun-amino-terminal kinase (JNK1 and 2) and decreased activities of extracellular signal-regulated kinase (ERK1 and 2) were observed during hyperglycemia-induced embryopathy. Conclusions: Poorly controlled maternal diabetes results in embryopathy which is mediated via a pattern of aberrant cellular communication manifested by both macroscopic and microscopic membrane injury.

Aberrant patterns of cellular communication in diabetes-induced embryopathy I. Membrane signalling

Objective: Our purpose was to investigate the role of membrane signalling in the mechanism of diabetes-induced embryopathy.Methods: Three groups of 70-90-day-old Sprague-Dawley rats wereemployed in our study: group 1 was normal control rats receiving a normal diet; group 2 represented experimentally induced diabetic rats with malformed offspring (intravenous injection of 65 mg/kg streptozotocinon pregnancy day 6) and group 3 included streptozotocin-induced diabetic rats with normal offspring. Embryos were examined on day 12 under light microscopy, categorized as morphologically normal or defective,and yolk sac cells were harvested from each group. Activities of ERK1 and 2, Raf-1, JNK 1 and 2 in yolk sac cells were analyzed by Western blot with primary antibodies specific to the phosphorylated kinases,respectively.Results: A strong link between hyperglycemia and congenital malformations was confirmed. Key mitogen-activated protein kinases serve as syllabic intermediates: increased activitiesof Jun-amino-terminal kinase (JNK1 and 2) and decreased activities of extracellular signal-regulated kinase (ERK1 and 2) were observed during hyperglycemia-induced embryopathy.Conclusions: Poorlycontrolled maternal diabetes results in embryopathy which is mediated via a pattern of aberrant cellular communication manifested by both macroscopic and microscopic membrane injury.

Mechanisms of Proton Conductance in Polymer Electrolyte Membranes

We provide a phenomenological description of proton conductance in polymer electrolyte membranes, based on contemporary views of proton transfer processes in condensed media and a model for heterogeneous polymer electrolyte membrane structure. The description combines the proton transfer events in a single pore with the total pore-network performance and, thereby, relates structural and kinetic characteristics of the membrane. The theory addresses specific experimentally studied issues such as the effect of the density of proton localization sites (equivalent weight) of the membrane material and the water content of the pores. The effect of the average distance between the sulfonate groups, which changes during membrane swelling, is analyzed in particular, and the factors which determine the temperature dependence of the macroscopic membrane conductance are disclosed. Numerical estimates of the specific membrane conductivity obtained from the theory agree very well with typical experimental data, thereby confirming the appropriateness of the theoretical concepts. Moreover, the versatility of the models offers a useful and transparent frame for combining the analysis of both experimental data and the results of molecular dynamics simulations.

Predicting Single-Component Permeance through Macroscopic Zeolite Membranes from Atomistic Simulations

The atomically ordered crystalline pores of zeolites endow membranes made from zeolites with many desirable properties. We present a theoretical framework that allows predictions of single-component permeance through zeolite membranes directly from atomistic models of the adsorbate and zeolite. In contrast to previous atomistic approaches to zeolite membranes, this framework does not attempt to include an entire membrane in a single nonequilibrium atomistic simulation. Rather, equilibrium molecular simulations are used to derive those data necessary for solving a general macroscopic description of molecular transport through a membrane. This approach allows the behavior of membranes of realistic thicknesses to be assessed over a broad range of temperatures and pressures. To illustrate this new framework, the steady-state permeances of Xe through an AlPO4-31 membrane and CF4 through an AlPO4-5 membrane are predicted directly from atomistic models of these systems.

Cation selective channels formed by a C-terminal fragment of beta-amyloid precursor protein.

The C-terminal 105 amino acid fragment of beta-amyloid precursor protein (CT105) is highly neurotoxic. To obtain insights into its cytotoxic effect, we examined the ionophoric effects of CT105 (10-1000 nM) on artificial lipid membranes. Macroscopic membrane conductance increased with CT105 concentration and its ionophoric effect was comparable to that of amyloid beta protein. The mean unitary conductance of CT105-induced channels was 120 pS and open-state probability was close to 1 at voltages from -80 to +80 mV. CT105induced channels were selective to cations (PK/ P(Cl) = 10.2), being most selective to Ca2+. These findings suggest that CT105 can cause direct neurotoxic effects by forming Ca2+ permeable cation channels on neuronal membranes.

Photofrin II Sensitized Modifications of Ion Transport Across the Plasma Membrane of an Epithelial Cell Line: II. Analysis at the Level of Membrane Patches

In the first part of this study, photofrin II sensitized membrane modifications of OK-cells were investigated at the level of macroscopic membrane currents. In this second part, the inside-out configuration of the patch-clamp technique is applied to analyze the phenomena at the microscopic level. It is shown that the characteristic single channel fluctuations of the electric current disappear after the start of illumination of membrane patches in the presence of photofrin II. This holds for all three types of ion channels investigated: the large-conductance Ca2+-dependent K+ channel (maxi-KCa), a K+ channel of small conductance (sK), and a stretch-activated nonselective cation channel (SA-cat). Part of the experiments show a transient activation of the channels (indicated by an increase of the probability in the open-channel state) before the channels are converted into a closed nonconductive state. Inactivation of all three channel types proceeds by a continuous reduction of their open probability, while the single channel conductance values are not affected. The process of photodynamically induced channel inactivation is followed by a pronounced increase of the leak conductance of the plasma membrane. The latter process - after light-induced initiation - is found to continue in the dark. The ionic pathways underlying the leak conductance also allow permeation of Ca2+ ions. The resulting Ca2+-flux may contribute to the photodynamically induced increase of the intracellular Ca2+ concentration observed in various cell lines.

Noradrenaline modulates the electrical activity of white adipocytes by a cAMP-dependent mechanism.

We studied the effect of the external addition of noradrenaline (NA) on the electrical activity of white adipocytes from rat epididymal tissue. NA modified the active voltage response recorded by the current clamp technique, and decreased the macroscopic membrane potassium currents measured by a whole-cell configuration patch-clamp technique in cells obtained by culturing differentiating preadipocytes. To analyse if cAMP is implicated in the effect of this hormone on potassium conductances, the changes in electrical activity caused by an increase in the intracellular concentration of cAMP were studied using intracellular recording. The addition of forskolin, or NN-dibutyryl cAMP plus 3-isobutyl-1-methylxanthine to the bath reduced active changes in membrane voltage responses to hyperpolarizing and depolarizing pulses. A similar effect was observed when vasoactive intestinal peptide was added to the superfusion chamber. These results suggest that NA could modulate K+ conductances in white adipocytes, mediated by cAMP.

Primary structure and functional expression of a cortical collecting duct Kir channel.

Maintenance of a negative membrane potential in the cortical collecting duct (CCD) principal cell depends on a small-conductance, inward-rectifying basolateral membrane K+ (Kir) channel. In the present study, a candidate cDNA encoding this K+ channel, CCD-IRK3, was isolated from a mouse collecting duct cell line, M1. CCD-IRK3 shares a high degree of homology with a human brain inward-rectifier K+ channel (Kir 2.3). By Northern analysis, CCD-IRK3 transcript (2.9 kb) was readily detected in M1 CCD cells but not in Madin-Darby canine kidney, LLC-PK1, Chinese hamster ovary, or monkey kidney fibroblast cell lines. CCD-IRK3-specific reverse transcription-polymerase chain reaction confirmed bonafide expression in the kidney. Functional expression studies in Xenopus oocytes revealed that CCD-IRK3 operates as strongly inward-rectifying K+ channel. The cation selectivity profile of CCD-IRK3 [ionic permeability values (PK/Pi), Tl > or = Rb > or = K+ >> NH4 > Na; inward-slope conductance (GK/Gi), Tl > or = K+ >> NH4 > Na > Rb] is similar to the macroscopic CCD basolateral membrane K+ conductance (GK/Gi, K+ >> NH4 > Rb; PK/Pi, Rb approximately equal to K+ >> NH4). CCD-IRK3 also exhibits the pharmacological features of the native channel. Patch-clamp analysis reveals that CCD-IRK3 functions as a high open probability, voltage-independent, small-conductance channel (14.5 pS), consistent with the native channel. Based on these independent lines of evidence, CCD-IRK3 is a possible candidate for the small-conductance basolateral Kir channel in the CCD.

The role of L-type Ca2+ current and Na+ current-stimulated Na/Ca exchange in triggering SR calcium release in guinea-pig cardiac ventricular myocytes.

This study examines the relative ability of sodium current (INa)-stimulated reverse mode Na/Ca exchange and the L-type calcium current (ICa) to trigger calcium-induced calcium release (CICR) in guinea-pig ventricular myocytes. Cytosolic Ca2+ transients were recorded from enzymatically dissociated guinea-pig ventricular myocytes using Indo-1. Macroscopic membrane currents were simultaneously recorded using the whole-cell patch-clamp technique. At room temperature (22-25 degrees C) Ca2+ transients were associated with the activation of INa, ICa or INa plus ICa in combination. However, after ICa was blocked by verapamil (10 microM), no Ca2+ transient could be evoked by the activation of INa alone at either -40 or +5 mV. Similar results were obtained with 5 and 8 mM intracellular sodium, and when the temperature of the bathing solution was raised to 35 degrees C and cAMP (10 microM) added to the pipette solution. From consideration of the relative magnitudes of the Ca2+ influx via ICa and Na/Ca exchange and thermodynamic considerations, we suggest that ICa is the major source of 'trigger' calcium for CICR (and cardiac contraction) under normal conditions. Although the Na/Ca exchanger was incapable of triggering CICR under the conditions of these experiments, we suggest that it may become more important when cytosolic Ca2+ is elevated, a condition which will also lead to decrease the amplitude of ICa.

Formation of a macroscopic membrane structure by liposomes containing cationic lipids and exposed to DC electric fields

Application of DC electric fields (from 20–30.4 kV/m) caused a rapid (within 1–5 s) formation of a large membrane structure in an aqueous solution of unilamellar liposomes composed of egg phosphatidylcholine and a cationic amphiphile, either sphingosine or stearylamine (mole fraction X=0.25). The formation of this structure could be readily observed by phase contrast microscopy. Maintenance of the membrane required the DC field and it disappeared rapidly (within ≈5 s) upon the removal of the field. The formation of the assembly was reversible. Such structures were not formed by vesicles composed of phosphatidylcholine only or containing instead of the cationic amphiphile an anionic lipid, phosphatidic acid. Fusion or a hemifusion of the vesicles is likely to take place as fluorescence microscopy of liposomes labeled with a pyrene-containing phospholipid analog revealed domain formation within the field-induced membrane, evident as a depletion of fluorescence in the region subjected to highest field intensity.

4-aminopyridine activates potassium currents by activation of a muscarinic receptor in feline atrial myocytes.

1. The effects of 4-aminopyridine (4-AP) on action potentials, macroscopic membrane currents and single-channel recording from cardiac left atrial myocytes of the adult cat were studied using the whole-cell and cell-attached configurations of the patch-clamp technique. 2. 4-AP (1 mM) produced a hyperpolarization of the resting membrane potential and a shortening of action potential duration. Under voltage-clamp conditions, we have found that 4-AP increased a background current and a delayed rectifier outward current. These effects were antagonized by atropine. In addition, both effects seemed to be mediated through a pertussis toxin-sensitive G protein. 3. The background current induced by 4-AP displayed properties that are highly similar to those of the inwardly rectifying potassium current activated by acetylcholine (IK(ACh)). The time-dependent potassium current activated by 4-AP has kinetic and pharmacological properties different from those of the delayed rectifier potassium current previously identified in cardiac myocytes. 4. The activation of the delayed rectifier-like potassium current could be explained by the activation of a novel muscarinic receptor subtype in which acetylcholine acts as the antagonist. Another possibility is that 4-AP activates IK(ACh) in a time- and voltage-dependent manner.

Voltage-Dependent Potassium Channels in White Adipocytes

Macroscopic membrane currents of white adipocytes were studied using the whole-cell variant of the patch-clamp technique. These cells were obtained by differentiating preadipocytes from rat epididymal tissue. In all differentiated cells outward currents exhibiting sigmoidal activation kinetics were observed with amplitudes ranging from 0.5 to 6 nA. The outward current showed activation thresholds at approximately −25 to −35 mV. The ion channels underlying the macroscopic current were highly selective for K+. Their selectivity was typical for K+channels, with relative permeabilities of K+> NH4+> Cs+, Na+. The potassium current was blocked by tetraethylammonium, 4-aminopyridine, barium and cobalt. These results demonstrate the existence of voltage dependent potassium channels in mature white adipose cells.

Inhibition of prothrombinase at macroscopic lipid membranes: competition between antithrombin and prothrombin.

The kinetics of inhibition of prothrombinase during prothrombin conversion by antithrombin and antithrombin-heparin complexes was studied in a tubular flow reactor. Prothrombinase was assembled at a macroscopic phospholipid membrane, composed of 25 mol % phosphatidylserine and 75 mol % phosphatidylcholine, deposited on the inner wall of a glass capillary, by perfusion with a factor Xa-factor Va mixture. Measurement of thrombin production allowed estimation of the amount of prothrombinase present at the capillary wall. Perfusion with a mixture of prothrombin and antithrombin or antithrombin-heparin complexes caused a progressive decline of the prothrombinase activity. The rate of inactivation steeply decreased with increasing prothrombin concentrations, indicating competitive inhibition. Analysis of competitive inhibition data requires estimation of the time-dependent substrate concentration, Co, near the prothrombin converting surface using earlier developed transport theory [Billy, D., et al. (1995) J. Biol. Chem. 270, 1029-1034]. It appears that the inhibition rate is proportional to the fraction of enzyme, Km/(Km+Co), not occupied by substrate. The value of Km of prothrombinase estimated from the dependence of the inhibition rate on the prothrombin concentration (Km = 2-3 nM) is in excellent agreement with the value estimated from the substrate conversion rate (Km = 3 nM). Therefore inhibition of prothrombinase by antithrombin and antithrombin-heparin complexes is fully competitive with the substrate: prothrombin. Our results show that prothrombinase assembled on macroscopic lipid surfaces by virtue of its low Km value is protected for inhibition due to highly effective competition of prothrombin with antithrombin for the active site of factor Xa.