Polarity-dependent voltage-gated porin channels from Escherichia coli in lipid bilayer membranes.

A porin preparation from Escherichia coli 0111:B4 consisting of Omp F and Omp C (with Omp F in excess) was purified by salt extraction procedures and investigated in bilayer lipid membranes formed according to the Montal-Mueller technique. The porin preparation was added to the KCl electrolyte compartment of the Montal-Mueller cell which was connected to the voltage source. As the porin incorporated into the membrane, asymmetric, voltage-gated ion channels were formed. Transmembrane voltages greater than +50 mV (measured with respect to the side of porin addition) caused channel closing, while negative voltages, on the other hand, had no effect on channel behaviour but did increase the rate of porin incorporation at higher voltages. With porin added to both compartments voltage gating no longer occurred. Single-channel conductances corresponded to effective pore diameters of 1.5 nm for opening events and 1.18 nm for channel closing events. The number of charges involved in gating was approximately 2.

Divalent cation-sensitive pores formed by natural and synthetic melittin and by Triton X-100.

Leakage of ions and low-molecular-weight metabolites from Lettre cells is induced by synthetic melittin, as effectively as by melittin isolated from bee venom; in each case leakage is inhibited by Ca2+, Zn2+ or H+. Inhibition of leakage by divalent cations is reversible in that Lettre cells incubated with melittin (or with Triton X-100) in the presence of inhibitory amounts of Zn2+, when freed of Zn2+ by EGTA or by centrifugation, begin to leak (in Zn2(+)-sensitive manner). Electrorotation of Lettre cells is altered by melittin, compatible with membrane permeabilization; melittin plus Zn2+ does not alter electrorotation until Zn2+ (and unbound melittin) are removed. Melittin or Triton X-100 added to calcein-loaded liposomes induces leakage of calcein; divalent cations inhibit. Energy transfer between liposome-associated melittin and 2-, 7- or 12-(9-anthroyloxy)stearate (AS) is maximal with 12-AS; addition of Zn2+ has little effect. Circular dichroism spectra of melittin plus liposomes are unaffected by Zn2+. These results show that the formation of divalent cation-sensitive pores is not dependent on the presence of endogenous membrane proteins and that the action of divalent cations is not by displacement of melittin (or Triton) from the lipid bilayer.

The effect of Ca2+ on virus-cell fusion and permeability changes.

Sendai virus-mediated permeability changes in Lettre cells or red blood cells are affected by extracellular Ca2+ in the following way: the lag period to onset of permeability changes is lengthened and the subsequent extent of leakage is reduced. Ca2+ neither stimulates nor inhibits fusion of the viral envelope to the plasma membrane of Lettre cells or red blood cells. It is concluded that Ca2+ protects cells against virally-induced permeability changes in a manner not involving membrane fusion.

Cell damage by viruses, toxins and complement: common features of pore-formation and its inhibition by Ca2+.

Haemolytic paramyxoviruses interact with cells in the following way: a potentially leaky viral envelope fuses with the plasma membrane, creating a hydrophilic pore of approximately 1 nm in diameter; this allows ions and low molecular weight compounds, but not proteins, to leak into and out of cells. Other viruses act similarly if the pH is reduced to 5. Leakage (measured by collapse of membrane potential, by movement of monovalent cations and by loss of phosphorylated intermediates from cells) is prevented by extracellular Ca2+. Ca2+ does not affect binding or fusion of virus to cells. It inhibits leakage as well as preventing it, and it aids in the recovery (i.e. the restoration of non-leakiness) of cells. Certain 'anti-Ca2+' drugs have an opposite effect. Experiments with the bee venom protein melittin, with the alpha-toxin of Staphylococcus aureus and with activated complement, show that the lesions produced by these agents, too, are sensitive to extracellular Ca2+ and to 'anti-Ca2+' drugs. The mechanisms of these effects are discussed.

A comparative study of treatments for positional sleep apnea.

Sixty male patients all with apnea plus hypopnea indices (A + HI) above 12.5, who met a criterion of positionality by having two or more times the rate of these events during supine sleep in comparison to their lateral sleep rate, were randomly assigned to one of four treatments for 8 weeks. All were restudied for two nights, one with and one without treatment devices. On treatment more than half the patients in each group reduced their A + HI to within normal limits and a third remained WNL without the use of devices. Half of those trained to sleep in the lateral position with the help of an alarm maintained this learning without the alarm as did half of those who were encouraged to learn this sleep posture on their own. There is an additive effect for the positional patient from wearing a tongue retaining device (TRD) if they continue to sleep in the supine position. Factors associated with successful treatment include overall severity, severity in the lateral position, weight, weight change, nasal patency and motivation to help their condition.

Thermal control of drug release by a responsive ion track membrane observed by radio tracer flow dialysis.

The combination of a responsive hydrogel with a rigid porous supporting structure yield a membrane with high mechanical strength and high on-off-permeability ratio. A membrane consisting of an ion track filter with a thermally responsive lining was prepared by penetrating a 19 micron thick foil of poly(ethylene terephthalate) (PET) with swift heavy ions at a fluence of 5 x 10(5) ions/cm2, followed by etching of the ion tracks to generate an ion track filter with 2.9 micron pore diameter, onto which a thin layer of poly(N-isopropylacrylamide) (NIPAAm) hydrogel was grafted. It was revealed that the mass flow of various molecules (water, chloride-, choline+, insulin, and albumin) through the membrane could be thermally controlled. The on-off-permeability ratio ranged between 3 and 10 increasing with molecular weight. Over a storage time of 5 months the permeabilities varied up to a factor of 2.6, while the on-off-permeability ratio and temperature sensitivity remained practically constant.

A novel method for measuring intracellular pH and potassium concentration.

The concentration of Na+ and K+ and the pH in the cytoplasm of Lettré cells was measured by monitoring the net flux of H+, Na+, or K+ across the plasma membrane which had been rendered permeable to these ions by the action of Sendai virus. Ion flux was measured directly by analysis of cell composition, or indirectly by observing the change in membrane potential of cells treated with a specific ionophore. Cytoplasmic concentrations of cations were obtained by establishing the concentration of the cation in the medium at which addition of Sendai virus causes no change in cytoplasmic cation content. The value of Lettré-cell pH was confirmed by direct measurement employing 31P nuclear magnetic resonance, and the values of Na+ and K+ concentration were confirmed by analysis of cell cation and water content. Lettré cells suspended at 32 degrees C in Hepes-buffered saline at pH 7.3 maintain a cytosolic pH of 7.0 and contain 50 mM Na+ and 80 mM K+.

Common action of certain viruses, toxins, and activated complement: pore formation and its prevention by extracellular Ca2+.

Haemolysis by Sendai virus, alpha-toxin, and activated complement is inhibited by high concentrations of divalent cations. In Daudi cells, sublytic amounts of these agents induce the following changes: collapse of surface membrane potential, uptake of Na+ and loss of K+ from cells, and leakage of phosphorylated metabolites from cells. The changes induced by Sendai virus and complement are sensitive to physiological concentrations of extracellular Ca2+. It is concluded that fluctuations in plasma Ca2+ concentration may affect the damaging action of certain pore-forming agents on susceptible cells.

Heat shock proteins induce pores in membranes.

Human heat shock protein (hsp) 70 and bacterial protein groEL promote leakage of calcein from liposomes induced by human serum albumin signal peptide, by S. aureus alpha toxin or by diphtheria toxin. Hsp 70 and groEL, as well as two mycobacterial homologues hsp 71 and hsp 65, induce ion conducting pores across planar lipid bilayers at low or neutral pH. It is concluded that hsp induce pores in membranes and that this may contribute to their action within cells.

Medical record professionals and the performance of concurrent review.

This month we feature two articles which discuss the performance of medical record review activities on the inpatient nursing unit by medical record professionals so that readers may profit by comparing and contrasting the viewpoints of the authors. This article highlights the appropriateness of having medical record professionals perform these activities.

The effect of air resistance on the jumping performance of insects.

A spring gun was constructed to propel objects at known velocities of between 1 and 4.5 m.s-1. This was used to project insects and various models in a vertical trajectory. By comparing the height attained in air by the insects or models with the height theoretically possible in vacuo, the energy lost against air resistance was observed. Small insects have a higher frontal area to mass ratio than larger ones so have relatively more aerodynamic drag and attain lower heights. The observed effect may be expressed in terms of the drag coefficient, CD. Fleas and locusts have CD of about 1. Winged flies have CD of about 1.5 which falls to about 1 when the wings are amputated and to about 0.8 when the legs are amputated. Aptery is advantageous in jumping insects. From experiments with models, it appears that the optimal condition for small jumping insects is that the body should be as compact as possible to reduce the frontal area to mass ratio. Thus dense spherical bodies are favoured. Some species of jumping insect have densities of about 1 mg.mm-3 while some flying beetles and flies have densities between 0.3 and 0.8 mg.mm-3. The Reynolds number at which the experiments were performed was from 65-205 for fleas up to 740-2340 for locusts. The models operated in similar ranges. At a velocity which would propel a larger animal to a height of 1 m, fleas weighing 0.4 mg only reach about 0.4 m. At lower initial velocities, proportionately less energy is wasted against air resistance so the jump efficiency is higher. Most fleas jump to a height of about 0.1 m with an efficiency of 0.8 while locusts jump to a height of 0.35 m with an efficiency of over 0.9. Air resistance is thus an important scale effect in jumping insects and provides its own design constraints.

Action of diphtheria toxin does not depend on the induction of large, stable pores across biological membranes.

Vero cells exposed to diphtheria toxin at pH 4.5 leak monovalent cations but not amino acids or phosphorylated metabolites; affected cells do not take up trypan blue. Monovalent cation leakage is inhibited by 1 mM Cd2+, but not by 1 mM Zn2+ or Ca2+. Cd2+ blocks calcein leakage from liposomes and closes diphtheria toxin-induced channels in lipid bilayers. It is concluded that translocation of the A fragment of diphtheria toxin across biological membranes does not depend on the formation of large stable pores, but that small Cd2(+)-sensitive pores may play a role.

Effect of Ca2+-antagonists on virally-induced cell-permeability changes.

Sendai virus-mediated permeability changes in cells are affected by extracellular Ca2+ or Mn2+ as follows: the lag period to onset of permeability changes is lengthened and the subsequent extent of leakage is reduced. Drugs that block Ca2+ action in excitable cells, such as verapamil and prenylamine, and drugs that inhibit the action of calmodulin, such as trifluoperazine and R24571, have an effect opposite to that of Ca2+: lag is shortened and extent of leakage is increased. The concentration at which either type of drug shows 50% of maximal effect is similar to the concentration at which 50% of binding by drug to calmodulin is achieved. It is concluded that calmodulin may be involved in protecting cells against virally-mediated membrane damage; alternatively the action of calmodulin-binding drugs may not be as specific as currently thought.

Protection against complement-mediated cell damage by Ca2+ and Zn2+.

Ca2+ and Zn2+ prevent antibody-dependent complement-induced permeability changes in tonsil lymphocytes and Lettre cells. Lactate dehydrogenase leaks out from Lettre cells at high complement:cell ratios, under which conditions higher concentrations of Ca2+ and Zn2+ are required for protection. Ca2+ and Zn2+ do not inhibit complement activation or C9 binding to Lettre cells, and prevent leakage through preformed lesions. It is concluded that the extent of complement-induced membrane damage depends on the concentration of extracellular Ca2+, and may be modulated by changes in extracellular Ca2+ or Zn2+.

Ion modulation of membrane permeability: effect of cations on intact cells and on cells and phospholipid bilayers treated with pore-forming agents.

Leakage of ions (Na+, K+) and phosphorylated metabolites (phosphorylcholine, 2-deoxyglucose 6-phosphate) through membrane lesions in intact cells or in cells modified by 'pore-forming' agent has been studied. Leakage from intact cells is induced by protons and by divalent cations such as Cu2+, Cd2+ or Zn2+. Leakage from agent-modified cells--or across phospholipid bilayers modified by agent--is prevented by low concentrations of the same cations and by higher concentrations of Ca2+, Mn2+ or Ba2+; Mg2+, dimethonium, spermine, or spermidine are virtually ineffective. The relative efficacy of a particular cation (e.g. Ca2+) depends more on cell type than on the nature of the pore-forming agent. The predominant effect is on binding of cation to specific sites, not on surface charge. Surface charge, on the other hand, does affect leakage from agent-modified cells in that suspension in nonionic media reduces leakage, which can be restored by increasing the ionic strength: univalent (Na+, K+, Rb+, NH4+) and divalent (Mg2+, dimethonium) cations are equally effective; addition of protons or divalent cations such as Zn2+ to this system inhibits leakage. From this and other evidence here presented it is concluded that leakage across membranes is modulated by the presence of endogenous anionic components: when these are in the ionized state, leakage is favored; when unionized (as a result of protonation) or chelated (by binding to divalent cation), leakage is prevented. It is suggested that such groups are exposed at the extracellular face of the plasma membrane.

Membrane damage by hemolytic viruses, toxins, complement, and other cytotoxic agents. A common mechanism blocked by divalent cations.

Hemolytic viruses, bacterial and animal toxins, the components of activated complement, cationic proteins, and detergents induce a sequence of permeability changes at the plasma membrane that are in every case sensitive to changes in ionic strength and to divalent cations. Individually, each agent exhibits positive cooperativity; when two agents are present together, they show synergy. It is concluded that such cytotoxic agents damage membranes by a common mechanism. Hence permeability changes are unlikely to depend on the formation of specific, protein-lined channels, as previously envisaged in the case of activated complement or certain bacterial toxins.

Conductance studies on trichotoxin_A50E and implications for channel structure.

Trichotoxin_A50E is an 18-residue peptaibol whose crystal structure has recently been determined. In this study, the conductance properties of trichotoxin_A50E have been investigated in neutral planar lipid bilayers. The macroscopic current-voltage curves disclose a moderate voltage-sensitivity and the concentration-dependence suggests the channels are primarily hexameric. Under ion gradients, shifts of the reversal potential indicate that cations are preferentially transported. Trichotoxin displays only one single-channel conductance state in a given experiment, but an ensemble of experiments reveals a distribution of conductance levels. This contrasts with the related peptaibol alamethicin, which produces multiple channel levels in a single experiment, indicative of recruitment of additional monomers into different multimeric-sized channels. Based on these conductance measurements and on the recently available crystal structure of trichotoxin_A50E, which is a shorter and straighter helix than alamethicin, a tightly-packed hexameric model structure has been constructed for the trichotoxin channel. It has molecular dimensions and surface electrostatic potential compatible with the observed conductance properties of the most probable and longer-lived channel.

Membrane damage: common mechanisms of induction and prevention.

Common features in the induction of pores by various agents are as follows: induction is stochastic and progressive; damage by different agents is often synergistic and limited. The prevention of membrane damage is affected by trivalent and divalent cations, by low pH, by low ionic strength and by high osmotic pressure. The inhibitory role of protons and divalent cations is considered in greater detail: pore-forming agents can be classified into two groups: channels across planar lipid bilayers induced by the first group display voltage-sensitive, reversible inhibition by divalent cations; channels of the second group show voltage-insensitive, irreversible inhibition by divalent cations. A search for the ligands to which divalent cations and protons bind has proved elusive. Comparison with the phenomenon of 'surface conductance' through narrow apertures, that is manifest in the absence of any pore-forming agent, may prove fruitful.