Glenn M. Alder

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

SummaryLeakage 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 isinduced by protons and by divalent cations such as Cu2+, Cd2+ or Zn2+. Leakage from agent-modified cells—or across phospholipid bilayers modified by agent—isprevented 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.

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 α-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 α-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.

Influence of pH on the uptake of 5-fluorouracil into isolated tumour cells.

To investigate the possible dependence of 5-fluorouracil (5FU) uptake in tumours on the intra- (pHi) and extracellular (pHe) pH, a pH gradient (deltapH) was imposed across the plasma membrane of ascites tumour cells in vitro, similar to that known to occur in some solid tumours in vivo, by incubation in media of PHe 5-8. A > or = 2:1 (intracellular/extracellular) accumulation of radiolabelled 5FU occurred after 5 min incubation of the cells with 0.5 mM 5FU at pHe of 5.0, 5.5 or 6.0. 5FU metabolism is slow under these conditions, and 5FU uptake was not affected by longer incubations up to 20 min, nor by the absence of a sodium gradient. pHi was estimated from the distribution of the weak acid, 5.5-dimethyl-2,4-oxazolidione ([14C]DMO) across the cell membrane. There was significant correlation between the intracellular/extracellular 5FU ratio and pHe (from pHe 6-8), deltapH and pHi (P < 0.02). Similar results were obtained with HT29 cells. Incubation with a drug that made plasma membranes permeable to H+ significantly decreased 5FU uptake in Lettre cells. The co-transport of 5FU may occur on a proton symport using the proton motive force of the deltapH.

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, byS. aureus α 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.

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

Haemolysis by Sendal virus, α-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 metabo-tites from cells. The changes induced by Sendal 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.

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

The conductance of pores induced by Staphylococcus aureus α-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 α-toxininduced 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.

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.

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.

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 Sendal 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 3tp 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°C in Hepes-buffered saline at pH 7.3 maintain a cytosolic pH of 7.0 and contain 30 mM Na+ and 80 mM K+.

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

SummaryVero 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 1mmCd2+, but not by 1mmZn2+ 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.