Biomedical subjects

G M Alder

Publications and source records attributed to G M Alder.

At least 19 recordsLinked to original sources

PubMed2004

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.

Alamethicin↗

PubMed1998

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.

Drug Delivery Systems↗

PubMed1998

Structure-based prediction of the conductance properties of ion channels.

The HOLE procedure allows the prediction of the absolute conductance of an ion channel model from its structure. The original prediction method uses an empirically corrected Ohmic method. It is most successful, with predictions being reliable to within a factor of two. A new modification of the procedure is presented in which the self-diffusion coefficients of water molecules from molecular dynamics simulation are used to replace the empirical correction factor. A "prediction" of the conductance for the porin OmpF by the new method is made and shown to be very close to the experimental value. HOLE also allows the prediction of the effect that the addition of non-electrolyte polymers will have on channel conductance. The method has great potential to yield structural information from data provided by single channel recordings but needs further validation by making measurements on channels of known structure. Preliminary results are given of single channel records establishing the effects of non-electrolytes on the conductance of gramicidin D channels. As an example of the potential uses of the procedure application is made to examine the oligomerization of alpha-toxin (alpha-hemolysin) channels. A model for the alpha-toxin hexamer, based on the crystal structure for the heptamer, is generated using molecular mechanics methods. The compatibility of the structures with single channel conductance data is assessed using HOLE.

Computer Simulation↗

PubMed1997

A novel explanation for fluctuations of ion current through narrow pores.

Fluctuation of ion current, between a high conductance and a low conductance state, through biological ion channels and pores is assumed to arise from conformational changes between an "open" and a "closed" configuration. Here we offer an additional mechanism that arises from changes in ionization of fixed charges within, or at the mouth of, a channel or pore. Our hypothesis, which is based on measurements of ion selectivity alongside ion current, applies to pores through some synthetic membranes and through channels-such as those created by certain toxins-that remain (at least partially) open in the low conductance state. It may also explain the phenomena of "open channel noise" and "substate behavior" that characterize several endogenous ion channels and should be considered when modeling the behavior of such channels.

Bacterial Toxins↗

PubMed1996

Pore formation by S. aureus alpha-toxin in liposomes and planar lipid bilayers: effects of nonelectrolytes.

Nonelectrolytes such as polyethylene glycols (PEG) and dextrans (i) promote the association of S. aureus alpha-toxin with liposomes (shown by Coomassie staining) and (ii) enhance the rate and extent of calcein leakage from calcein-loaded liposomes; such leakage is inhibited by H+, Zn2+ and Ca2+ to the same extent as that of nonPEG-treated liposomes. Incubation of liposomes treated with alpha-toxin in the presence of PEG with the hydrophobic photo-affinity probe 3-(trifluoromethyl)-3-m-[125I]iodophenyl) diazirine (125I-TID) labels monomeric and-predominantly-hexameric forms of liposome-associated alpha-toxin; in the absence of PEG little labeling is apparent. At high concentrations of H+ and Zn2+ but not of Ca(2+)-all of which inhibit calcein leakage-the distribution of label between hexamer and monomer is perturbed in favor of the latter. In alpha-toxin-treated planar lipid bilayers from which excess toxin has been washed away, PEGs and dextrans strongly promote the appearance of ion-conducting pores. The properties of such pores are similar in most regards to pores induced in the absence of nonelectrolytes; the differ only in being more sensitive to "closure" by voltage (as are pores induced in cells). In both systems, the stimulation by nonelectrolytes increase with concentration and with molecular mass up to a maximum around 2,000 Da. We conclude (i) that most of the alpha toxin that becomes associated with liposome or planar lipid bilayers does not form active pores and (ii) that the properties of alpha-toxin-induced pores in lipid bilayers can be modulated to resemble those in cells.

Animals↗

PubMed1996

Diffusion through narrow pores: movement of ions, water and nonelectrolytes through track-etched PETP membranes.

The rates at which ions (86Rb+, [3H]-choline, 36Cl), 3H2O and nonelectrolytes ([14C]-urea, [14C]-glycerol, and [14C]-sugars) equilibrate across track-etched polyethyleneterephthalate (PETP) membranes (isotopic diffusion) have been measured by a 'static' and a 'dynamic' technique under conditions where no net flow takes place; the two techniques give essentially the same results. All tracers diffuse faster the longer the membranes are etched, consistent with an increase in pore size. Water and neutral solutes diffuse at rates that are relatively independent of ionic strength, pH or the presence of divalent cations. Diffusion of cations is decreased by high ionic strength, by reducing pH or by addition of divalent cations; diffusion of chloride is increased by these procedures. Treatment of the membrane with diazomethane to reduce the negative fixed charge decreases diffusion of cations and increases that of anions; diffusion of water and neutral solutes is unaffected by methylation except in the membranes with the narrowest pores (i.e., those etched for the shortest time), in which case diffusion is reduced. We conclude (1) that the special features of flow near a charged surface apply to ions but not to water or nonelectrolytes and (2) that calculation of absolute rates of diffusion leads to values for the radii of pores through track-etched PETP membranes that are in remarkably good agreement with measured values.

Anions↗

PubMed1995

Low conductance states of a single ion channel are not 'closed'.

We have used a polymer-exclusion method to estimate the sizes of the high- and low-conductance states of Staphylococcus aureus alpha-toxin channels across planar lipid bilayers. Despite a > 10-fold difference in conductance between high- and low-conductance states, the size differs by < 2-fold. We conclude that factors other than the dimensions have a strong influence on the conductance of alpha-toxin channels. We also show that the high conductance state is destabilized by the presence of high molecular weight polymers outside the channel, compatible with the removal of channel water as the high conductance state "shrinks" to the low conductance state.

Biological Transport, Active↗

PubMed1995

Staphylococcus aureus alpha-toxin-induced pores: channel-like behavior in lipid bilayers and patch clamped cells.

The conductance of pores induced by Staphylococcus aureus alpha-toxin in Lettre cells has been compared to that in bilayers composed of synthetic lipids or Lettre cell membrane constituents. Previously described characteristics of toxin-induced conductance changes in lipid bilayers, namely rectification, voltage-dependent closure, and closure at low pH or in the presence of divalent cations (Menestrina, 1986) are displayed also in bilayers prepared from Lettre cell membranes and in patch clamped Lettre cells. It is concluded that endogenous proteins do not affect the properties of alpha-toxin-induced channels significantly and that the relative lack of ion channels in Lettre cells makes them ideal for studies of pore-forming toxins by the patch clamp technique.

Animals↗

PubMed1992

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.

Animals↗

PubMed1991

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.

Animals↗

PubMed1990

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.

Amino Acid Sequence↗

PubMed1990

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.

Animals↗

PubMed1988

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.

Animals↗

PubMed1988

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+.

Animals↗

PubMed1986

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.

Animals↗

PubMed1985

Oxonol dyes as monitors of membrane potential: the effect of viruses and toxins on the plasma membrane potential of animal cells in monolayer culture and in suspension.

Optical indicators of the cationic, cyanine and anionic oxonol classes were used to evaluate the plasma membrane potential of animal cells in suspension and in monolayer culture. The optical signals were calibrated by using diffusion potentials either of K+ (in the presence of valinomycin) or of H+ (in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone; FCCP); both classes of dye gave similar values of plasma membrane potential, in the range -40 to -90 mV for different cell types. Addition of haemolytic Sendai virus or Staphylococcus aureus alpha-toxin depolarizes cells and causes them to leak monovalent cations; these effects are antagonized by extracellular Ca2+. Cells infected with vesicular stomatitis or Semliki Forest virus become depolarized during an infectious cycle; infection with other viruses was without affect on plasma membrane potential.

Animals↗

PubMed1985

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.

Animals↗

PubMed1984

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.

Calcium↗