Large cation-selective pores from rat liver peroxisomal membranes incorporated to planar lipid bilayers

Summary Fusion of a highly purified fraction of rat liver peroxisomal membranes to planar lipid bilayers incorporates large, cation-selective voltage-dependent pores. TheP _K/P _Cl ratio of these pores, estimated in KCl gradients, is close to 4. The pores display several conductance states and spend most of the time open at voltages near 0 mV, closing at more positive and negative voltages. At voltages near 0 mV the most frequent open state has a conductance of 2.4 nS in 0.3m KCl. At voltages more positive and more negative than 10 mV the most frequent open state displays a conductance of 1.2 nS in 0.3m KCl. With these results pore diameters of 3 and 1.5 nm, respectively, can be estimated. We suggest that these pores might account for the unusually high permeability of peroxisomes to low molecular weight solutes. Fusion also incorporates a perfectly anion-selective, two-open states channel with conductances of 50 and 100 pS in 0.1m KCl.     

Comparison of transient and successful fusion pores connecting influenza hemagglutinin expressing cells to planar membranes

Time-resolved admittance measurements were used to investigate the evolution of fusion pores formed between cells expressing influenza virus hemagglutinin (HA) and planar bilayer membranes. The majority of fusion pores opened in a stepwise fashion to semistable conductance levels of several nS. About 20% of the pores had measurable rise times to nS conductances; some of these opened to conductances of approximately 500 pS where they briefly lingered before opening further to semistable conductances. The fall times of closing were statistically similar to the rise times of opening. All fusion pores exhibited semistable values of conductance, varying from approximately 2-20 nS; they would then either close or fully open to conductances on the order of 1 microS. The majority of pores closed; approximately 10% fully opened. Once within the semistable stage, all fusion pores, even those that eventually closed, tended to grow. Statistically, however, before closing, transient fusion pores ceased to grow and reversed their conductance pattern: conductances decreased with a measurable time course until a final drop to closure. In contrast, pore enlargement to the fully open state tended to occur from the largest conductance values attained during a pore's semistable stage. This final enlargement was characterized by a stepwise increase in conductance. The density of HA on the cell surface did not strongly affect pore dynamics. But increased proteolytic treatment of cell surfaces did lead to faster growth within the semistable range. Transient pores and pores that fully opened had indistinguishable initial conductances and statistically identical time courses of early growth, suggesting they were the same upon formation. We suggest that transient and fully open pores evolved from common structures with stochastic factors determining their fate.     

Influence of voltage and ATP on ion-channel of (Na,K)ATPase incorporated into solvent-free phospholipid planar bilayers

Purified (Na,K)ATPase was incorporated into solvent free phospholipid bilayers made on patch-clamp pipettes. In the absence of ATP, the incorporated enzyme acted as an ion-channel which underwent opening and closing (switching) upon application of transmembrane potential gradient of more than 40 mV. The minimum conductance was about 40 pS. It was inhibited by ouabain from one side. ATP added to the opposite side shifted the threshold potential for switching of the channel to 80 mV. Furthermore the magnitude of minimum conductance decreased to 6-10 pS in the presence of ATP.     

Conductive properties and gating of channels formed by syringopeptin 25A, a bioactive lipodepsipeptide from Pseudomonas syringae pv. syringae, in planar lipid membranes

Syringopeptin 25A, a pseudomonad lipodepsipeptide, can form ion channels in planar lipid membranes. Pore conductance is around 40 pS in 0.1 M NaCl. Channel opening is strongly voltage dependent and requires a negative potential on the same side of the membrane where the toxin was added. These pores open and close with a lifetime of several seconds. At negative voltages, an additional pore state of around 10 pS and a lifetime of around 30 ms is also present. The voltage dependence of the rates of opening and closing of the stable pores is exponential. This allows estimation of the equivalent charge that is moved across the membrane during the process of opening at about 2.6 elementary charges. When NaCl is present, the pore is roughly 3 times more permeant for anions than for cations. The current voltage characteristic of the pore is nonlinear, i.e., pore conductance is larger at negative than at positive voltages. The maximal conductance of the pore depends on the concentration of the salt present, in a way that varies almost linearly with the conductivity of the solution. From this, an estimate of a minimal pore radius of 0.4 nm was derived.     

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.     

Identification of a hemolysin from Actinobacillus pleuropneumoniae and characterization of its channel properties in planar phospholipid bilayers

A proteinaceous hemolysin secreted by strain 4074 of serotype 1 of Actinobacillus pleuropneumoniae was purified by diafiltration and ion exchange chromatographic techniques. The hemolytic activity is associated with a 107-kDa band as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and confirmed by Western blotting and immunoprecipitation. This hemolysin produces pores in membranes as demonstrated by osmotic protection studies using red blood cells and carbohydrate compounds of various molecular weights. These assays suggest a pore diameter in the order of 2 nm. Phospholipid bilayers composed of 1:1 w/w phosphotidylserine:phosphotidylethanolamine exposed to this toxin display discrete current flow events typical of transmembrane channels and consistent with the interpretation that this toxin acts by forming pores in phospholipid membranes. The linear relationship of current amplitude to holding potential when examined over the -60 to +60 mV range indicates that this pore has a constant mean single channel conductance level of 350-400 pS.     

Proton conduction in gramicidin A and in its dioxolane-linked dimer in different lipid bilayers.

Gramicidin A (gA) molecules were covalently linked with a dioxolane ring. Dioxolane-linked gA dimers formed ion channels, selective for monovalent cations, in planar lipid bilayers. The main goal of this study was to compare the functional single ion channel properties of natural gA and its covalently linked dimer in two different lipid bilayers and HCl concentrations (10-8000 mM). Two ion channels with different gating and conductance properties were identified in bilayers from the product of dimerization reaction. The most commonly observed and most stable gramicidin A dimer is the main object of this study. This gramicidin dimer remained in the open state most of the time, with brief closing flickers (tau(closed) approximately 30 micros). The frequency of closing flickers increased with transmembrane potential, making the mean open time moderately voltage dependent (tau(open) changed approximately 1.43-fold/100 mV). Such gating behavior is markedly different from what is seen in natural gA channels. In PEPC (phosphatidylethanolamine-phosphatidylcholine) bilayers, single-channel current-voltage relationships had an ohmic behavior at low voltages, and a marked sublinearity at relatively higher voltages. This behavior contrasts with what was previously described in GMO (glycerylmonooleate) bilayers. In PEPC bilayers, the linear conductance of single-channel proton currents at different proton concentrations was essentially the same for both natural and gA dimers. g(max) and K(D), obtained from fitting experimental points to a Langmuir adsorption isotherm, were approximately 1500 pS and 300 mM, respectively, for both the natural gA and its dimer. In GMO bilayers, however, proton affinities of gA and the dioxolane-dimer were significantly lower (K(D) of approximately 1 and 1.5 M, respectively), and the g(max) higher (approximately 1750 and 2150 pS, respectively) than in PEPC bilayers. Furthermore, the relationship between single-channel conductance and proton concentration was linear at low bulk concentrations of H+ (0.01-2 M) and saturated at concentrations of more than 3 M. It is concluded that 1) The mobility of protons in gramicidin A channels in different lipid bilayers is remarkably similar to proton mobilities in aqueous solutions. In particular, at high concentrations of HCl, proton mobilities in gramicidin A channel and in solution differ by only 25%. 2) Differences between proton conductances in gramicidin A channels in GMO and PEPC cannot be explained by surface charge effects on PEPC membranes. It is proposed that protonated phospholipids adjacent to the mouth of the pore act as an additional source of protons for conduction through gA channels in relation to GMO bilayers. 3) Some experimental results cannot be reconciled with simple alterations in access resistance to proton flow in gA channels. Said differences could be explained if the structure and/or dynamics of water molecules inside gramicidin A channels is modulated by the lipid environment and by modifications in the structure of gA channels. 4) The dioxolane ring is probably responsible for the closing flickers seen in the dimer channel. However, other factors can also influence closing flickers.     

Reconstitution of expressed KCa channels from Xenopus oocytes to lipid bilayers.

Reconstitution of large conductance calcium-activated potassium (KCa) channels from native cell membranes into planar lipid bilayers provides a powerful method to study single channel properties, including ion conduction, pharmacology, and gating. Recently, KCa channels derived from the Drosophila Slowpoke (Slo) gene have been cloned and heterologously expressed in Xenopus oocytes. In this report, we describe the reconstitution of cloned and expressed Slo KCa channels from Xenopus oocyte membranes into lipid bilayers. The reconstituted channels demonstrate functional properties characteristic of native KCa channels. They possess a mean unitary conductance of approximately 260 pS in symmetrical potassium (250 mM), and they are voltage- and calcium-sensitive. At 50 microM Ca2+, their half-activation potential was near -20 mV; and their affinity for calcium is in the micromolar range. Reconstituted Slo KCa channels were insensitive to external charybdotoxin (40-500 nM) and sensitive to micromolar concentrations of external tetraethylammonium (KD = 158 microM, at 0 mV) and internal Ba2+ (KD = 76 microM, at 40 mV). In addition, they were blocked by internally applied "ball" inactivating peptide (KD = 480 microM, at 40 mV). These results demonstrate that cloned KCa channels expressed in Xenopus oocytes can be readily incorporated into lipid bilayers where detailed mechanistic studies can be performed under controlled internal and external experimental conditions.     

Influenza hemagglutinin-mediated fusion pores connecting cells to planar membranes: flickering to final expansion

We have studied the fusion between voltage-clamped planar lipid bilayers and influenza virus infected MDCK cells, adhered to one side of the bilayer, using measurements of electrical admittance and fluorescence. The changes in currents in-phase and 90 degrees out-of- phase with respect to the applied sinusoidal voltage were used to monitor the addition of the cell membrane capacitance to that of the lipid bilayer through a fusion pore connecting the two membranes. When ethidium bromide was included in the solution of the cell-free side of the bilayer, increases in cell fluorescence accompanied tee admittance changes, independently confirming that these changes were due to formation of a fusion pore. Fusion required acidic pH on the cell- containing side and depended on temperature. For fusion to occur, the influenza hemagglutinin (HA) had to be cleaved into HA1 and HA2 subunits. The incorporation of gangliosides into the planar bilayers greatly augmented fusion. Fusion pores developed in four distinct stages after acidification: (a) a pre-pore, electrically quiescent stage; (b) a flickering stage, with 1-2 nS pores opening and closing repetitively; (c) an irreversibly opened stage, in which pore conductances varied between 2 and 100 nS and exhibited diverse kinetics; (d) a fully opened stage, initiated by an instantaneous, time- resolution limited, increase in conductance leveling at approximately 500 nS. The expansion of pores by stages has also been shown to occur during exocytosis in mast cells and fusion of HA-expressing cells and erythrocytes. We conclude that essential features of fusion pores are produced with proteins in just one of the two fusing membranes.     

Patch clamp studies of single intact secretory granules.

The membrane of secretory granules is involved in the molecular events that cause exocytotic fusion. Several of the proteins that have been purified from the membrane of secretory granules form ion channels when they are reconstituted in lipid bilayers and, therefore, have been thought to form part of the molecular structure of the exocytotic fusion pore. We have used the patch clamp technique to study ion conductances in single isolated secretory granules from beige mouse mast cells. We found that the membrane of the intact granule had a conductance of < 50 pS. No abrupt changes in current corresponding to the opening and closing of ion channels were observed, even under conditions where exocytotic fusion occurred. However, mechanical tension or a large voltage pulse caused the breakdown of the granule membrane resulting in the abrupt opening of a pore with an ion conductance of about 1 nS that fluctuated rapidly and could expand to an immeasurably large conductance or close completely. Surprisingly, the behavior of these pores resembled the pattern of conductance changes of exocytotic fusion pores observed in degranulating beige mast cells. This similarity supports the view that the earliest fusion pore is formed upon the breakdown of a bilayer such as that formed during hemifusion.     

A large, voltage-dependent channel, isolated from mitochondria by water-free chloroform extraction

We examined ion channels derived from a chloroform extract of isolated, dehydrated rat liver mitochondria. The extraction method was previously used to isolate a channel-forming complex containing poly-3-hydroxybutyrate and calcium polyphosphate from Escherichia coli. This complex is also present in eukaryotic membranes, and is located primarily in mitochondria. Reconstituted channels showed multiple subconductance levels and were voltage-dependent, showing an increased probability of higher conductance states at voltages near zero. In symmetric 150 mM KCl, the maximal conductance of the channel ranged from 350 pS to 750 pS. For voltages >+/-60 mV, conductance fluctuated in the range of approximately 50- approximately 200 pS. In the presence of a 1:3 gradient of KCl, at pH = 7.4, selectivity periodically switched between different states ranging from weakly anion-selective (V(rev) approximately -15 mV) to ideally cation-selective (V(rev) approximately +29 mV), without a significant change in its conductance. Overall, the diverse, but highly reproducible, channel activity most closely resembled the behavior of the permeability transition pore channel seen in patch-clamp experiments on native mitoplasts. We suggest that the isolated complex may represent the ion-conducting module from the permeability transition pore.     

A high-conductance mode of a poly-3-hydroxybutyrate/calcium/polyphosphate channel isolated from competent Escherichia coli cells

Reconstitution into planar lipid bilayers of a poly-3-hydroxybutyrate/calcium/polyphosphate (PHB/Ca(2+)/polyP) complex from Escherichia coli membranes yields cationic-selective, 100 pS channels (Das, S., Lengweiler, U.D., Seebach, D. and Reusch, R.N. (1997) Proof for a non-proteinaceous calcium-selective channel in Escherichia coli by total synthesis from (R)-3-hydroxybutanoic acid and inorganic polyphosphate. Proc. Natl. Acad. Sci. USA 94, 9075-9079). Here, we report that this complex can also form larger, weakly selective pores, with a maximal conductance ranging from 250pS to 1nS in different experiments (symmetric 150mM KCl). Single channels were inhibited by lanthanum (IC(50)=42+/-4microM, means+/-S.E.M.) with an unusually high Hill coefficient (8.4+/-1.2). Transition to low-conductance states (<250pS) was favored by increased membrane polarization (/V/ >or=50mV). High conductance states (>250pS) may reflect conformations important for genetic transformability, or "competence", of the bacterial cells, which requires the presence of the PHB/Ca(2+)/polyP complex in the membrane.     

Batrachotoxin-modified sodium channels from squid optic nerve in planar bilayers. Ion conduction and gating properties

Squid optic nerve sodium channels were characterized in planar bilayers in the presence of batrachotoxin (BTX). The channel exhibits a conductance of 20 pS in symmetrical 200 mM NaCl and behaves as a sodium electrode. The single-channel conductance saturates with increasing the concentration of sodium and the channel conductance vs. sodium concentration relation is well described by a simple rectangular hyperbola. The apparent dissociation constant of the channel for sodium is 11 mM and the maximal conductance is 23 pS. The selectivity determined from reversal potentials obtained in mixed ionic conditions is Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+. Calcium blocks the channel in a voltage-dependent manner. Analysis of single-channel membranes showed that the probability of being open (Po) vs. voltage relation is sigmoidal with a value of 0.5 between -90 and -100 mV. The fitting of Po requires at least two closed and one open state. The apparent gating charge required to move through the whole transmembrane voltage during the closed-open transition is four to five electronic charges per channel. Distribution of open and closed times are well described by single exponentials in most of the voltage range tested and mean open and mean closed times are voltage dependent. The number of charges associated with channel closing is 1.6 electronic charges per channel. Tetrodotoxin blocked the BTX-modified channel being the blockade favored by negative voltages. The apparent dissociation constant at zero potential is 16 nM. We concluded that sodium channels from the squid optic nerve are similar to other BTX-modified channels reconstituted in bilayers and to the BTX-modified sodium channel detected in the squid giant axon.     

Depolarization increases the single-channel conductance and the open probability of crayfish glutamate channels.

We have studied the voltage sensitivity of glutamate receptors in outside-out patches taken from crayfish muscles. We found that single-channel conductance, measured directly at the single-channel level, increases as depolarization rises. At holding potentials from -90 mV to approximately 20 mV, the conductance is 109 pS. At holding potentials positive to 20 mV, the conductance is 213 pS. This increase in single-channel conductance was also observed in cell-attached patches. In addition, desensitization, rise time, and the dose-response curve were all affected by depolarization. To further clarify these multifaceted effects, we evaluated the kinetic properties of single-channel activity recorded from cell-attached patches in hyperpolarization (membrane potential around -75 mV) and depolarization (membrane potential approximately 105 mV). We found that the glutamate dissociation rate constant (k_) was affected most significantly by membrane potential; it declined 6.5-fold under depolarization. The rate constant of channel closing (k(c)) was also significantly affected; it declined 1.8-fold. The rate constant of channel opening (k(o)) declined only 1.2-fold. The possible physiological significance of the depolarization-mediated changes in the above rate constants is discussed.     

Single-channel properties of a rat brain endoplasmic reticulum anion channel.

Many intracellular membranes contain ion channels, although their physiological roles are often poorly understood. In this study we incorporated single anion channels colocalized with rat brain endoplasmic reticulum (ER) ryanodine-sensitive Ca(2+)-release channels into planar lipid bilayers. The channels opened in bursts, with more activity at negative (cytoplasm-ER lumen) membrane potentials, and they occupied four open conductance levels with frequencies well described by the binomial equation. The probability of a protomer being open decreased from approximately 0.7 at -40 mV to approximately 0.2 at +40 mV, and the channels selected between different anions in the order PSCN > PNO3 > PBr > PCl > PF. They were also permeant to cations, including the large cation Tris+ (PTris/PCl = 0.16). Their conductance saturated at 170 pS in choline Cl. The channels were inactivated by 15 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and blocked with low affinity (KD of 1-100 microM) by anthracene-9-carboxylic acid, ethacrynic acid, frusemide (furosemide), HEPES, the indanyloxyacetic acid derivative IAA-94, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), and Zn2+. Unlike protein translocation pores, the channels were unaffected by high salt concentrations or puromycin. They may regulate ER Ca2+ release, or be channel components en route to their final cellular destinations. Alternatively, they may contribute to the fusion machinery involved in intracellular membrane trafficking.     

Biophysics of gating phenomena in voltage-dependent OmpC mutant porin channels (R74C and R37C) of <Emphasis Type="Italic">Escherichia</Emphasis><Emphasis Type="Italic">coli</Emphasis> outer membranes

The mechanism by which the membrane potential closes and opens voltage-dependent beta-barrel membrane channels is not fully understood. OmpC porins form trimeric water-filled channels when incorporated into artificial bilayers, each monomer having a conductance of approximately 510 pS in 1 M KCl. These channels are relatively insensitive to membrane potential difference (pd) and close only when the pd exceeds +/-250 mV. Another well-known trimer, OmpF, has a monomer conductance of approximately 780 pS in 1 M NaCl, is more sensitive to pd, and can be closed reversibly when a pd of more than +/-150 mV is applied to the channel-containing membranes. With the aid of the 3D atomic structure of these channels determined by X-ray crystallography, and using site-directed mutagenesis, specific amino acids can be substituted in desired locations in the channel lumen. In this study we have used mutants 37C and 74C and attached fluorescence probes to them to monitor polarity changes in the channel lumen during gating. From the observed changes in polarity, we conclude that conformational changes occur in the channel which interrupt the electrolyte conducting pathway.     

Channel reconstitution in liposomes and planar bilayers with HPLC-purified MIP26 of bovine lens

Summary The major intrinsic protein (MIP26) of bovine lens membranes, purified by HPLC, was incorporated into liposomes and planar bilayers. Permeability of MIP26 channels was studied in liposomes by a spectrophotometric osmotic-swelling assay, and channel electrical properties were monitored in planar bilayers following liposome fusion. Particle formation in liposomes was determined by freeze fracture. MIP26 channels were permeable to KCl and sucrose. In planar bilayers, channel-conductance transitions were observed only after addition of liposomes to both chambers and with voltages greater than ±20 mV. Channel open probability decreased progressively as voltage increased, and an open probability of 50% was at 60–80 mV, indicating that the channels are voltage dependent. Histograms of single-channel current amplitudes at 80 mV showed a Gaussian distribution that peaked at 10 pA (120 pS), after subtraction of 1 pA baseline current. Frequency distributions of open and closed times at 80 mV were single exponential functions with time constants of 0.13 and 1.9 sec, respectively. Open time constants ranged from 0.1 to 0.3 sec, and closed time constants ranged from 1 to 7 sec. Cs⁺ did not decrease conductance, but reduced mean open time from 0.2 to 0.038 sec and mean closed time from 1.5 to 0.38 sec. The increase in channel flickering with Cs⁺ occurred in bursts. TEA affected neither conductance nor kinetics. Channel events were also observed in Na⁺ solutions (zero K⁺). These data indicate that MIP26 channels are not K⁺-selective channels. Channel characteristics such as: permeability to molecules larger than small ions, conductance greater than 100 pS, long open and closed time constants, etc., are similar to those of gap junction channels.     

Mode of action of the staphylococcinlike peptide Pep 5: voltage-dependent depolarization of bacterial and artificial membranes.

The cationic staphylococcinlike peptide Pep 5 is shown to depolarize bacterial and planar lipid membranes in a voltage-dependent manner. An artificial valinomycin-induced potassium diffusion potential across the cytoplasmic membrane of Staphylococcus cohnii 22 was sufficient to promote Pep 5 action. Thus, evidence is provided that a membrane potential of sufficient magnitude is the only prerequisite for Pep 5 activity. The voltage dependence was elucidated by macroscopic conductance measurements with black lipid membranes. A threshold potential of about -90 to -100 mV, which was deduced from experiments with bacterial cells, could be confirmed. Single pores were resolved which often occur as short-lived bursts and fluctuate among different conductance levels. Pore diameters were calculated ranging from 0.1 to 1 nm. Succinylation of the lysine residues of Pep 5 resulted in prolonged pore lifetimes and maintenance of distinct conductance levels. However, the succinylated peptide required a higher threshold potential, approximately -150 mV, than the native peptide, which is probably the reason for the reduced activity of the modified peptide against intact gram-positive bacteria.     

Kinetics of the Opening and Closing of Individual Excitability-Inducing Material Channels in a Lipid Bilayer

The kinetics of the opening and closing of individual ion-conducting channels in lipid bilayers doped with small amounts of excitability-inducing material (EIM) are determined from discrete fluctuations in ionic current. The kinetics for the approach to steady-state conductance during voltage clamp are determined for lipid bilayers containing many EIM channels. The two sets of measurements are found to be consistent, verifying that the voltage-dependent conductance of the many-channel EIM system arises from the opening and closing of individual EIM channels. The opening and closing of the channels are Poisson processes. Transition rates for these processes vary exponentially with applied potential, implying that the energy difference between the open and closed states of an EIM channel is linearly proportional to the transmembrane electric field. A model incorporating the above properties of the EIM channels predicts the observed voltage dependence of ionic conductance and conductance relaxation time, which are also characteristic of natural electrically excitable membranes.     

Microfabricated teflon membranes for low-noise recordings of ion channels in planar lipid bilayers

We present a straightforward, accessible method for the fabrication of micropores with diameters from 2 to 800 micro m in films of amorphous Teflon (Teflon AF). Pores with diameters 相似文献