E.J.A. Lea

The interaction of detergents with bilayer lipid membranes

Detergents are widely used for extracting and purifying membrane proteins. Four such detergents have been studied to find the extent to which they alone can alter black lipid film conductances. The slope of the plot of conductivity versus concentration for Triton X-100 is 4.54 in the range 0.025--0.15 mM; dodecyl sulphate 0.82 in the range 0.01--1 mM; sodium deoxycholate 1.03 in the range 0.01--1 mM and sodium cholate 1.37 in the range 0.1--10 mM. These ranges are below the respective critical micelle concentrations; above these concentrations the membranes break. Bilayer lipid membrane conductivity measured at constant detergent concentration increases with the conductivity of the bathing salt solution with a slope greater than 1, indicating an effect on the putative pore structures induced by detergents.

A new channel-forming antibiotic from Streptomyces coelicolor A3(2) which requires calcium for its activity.

A recently discovered antibiotic (CDA; calcium-dependent antibiotic) of Streptomyces coelicolor A3(2) was found to be effective against a wide range of Gram-positive bacteria only in the presence of calcium ions. Producer and non-producer strains were identified and several media tested for their ability to support antibiotic production. The action of calcium was not simulated by any of the other cations tested. The antibiotic was found to induce discrete conductance fluctuations in planar lipid bilayer consistent with a channel-forming action. The electrical potential difference caused by a concentration difference of various salts across the CDA-containing bilayer, showed the channel to be cation-selective but of a size that discriminated against tetramethyl ammonium and choline ions. The data indicate that the antibiotic activity of CDA is due to its action as a calcium-dependent ionophore.

Modulation of type-1 Ins(1,4,5)P3 receptor channels by the FK506-binding protein, FKBP12.

FK506-binding protein (FKBP12) is highly expressed in neuronal tissue, where it is proposed to localize calcineurin to intracellular calcium-release channels, ryanodine receptors and Ins(1,4,5)P(3) receptors (InsP(3)Rs). The effects of FKBP12 on ryanodine receptors have been well characterized but the nature and function of binding of FKBP12 to InsP(3)R is more controversial, with evidence for and against a tight interaction between these two proteins. To investigate this, we incorporated purified type-1 InsP(3)R from rat cerebellum into planar lipid bilayers to monitor the effects of exogenous recombinant FKBP12 on single-channel activity, using K(+) as the current carrier. Here we report for the first time that FKBP12 causes a substantial change in single-channel properties of the type-1 InsP(3)R, specifically to increase the amount of time the channel spends in a fully open state. In the presence of ATP, FKBP12 can also induce co-ordinated gating with neighbouring receptors. The effects of FKBP12 were reversed by FK506. We also present data showing that rapamycin, at sub-optimal concentrations of Ins(2,4,5)P(3), decreases the rate of calcium release from cerebellar microsomes. These results provide evidence for a direct functional interaction between FKBP12 and the type-1 InsP(3)R.

The cytotoxic domain of colicin E9 is a channel-forming endonuclease.

Bacterial toxins commonly translocate cytotoxic enzymes into cells using channel-forming subunits or domains as conduits. Here we demonstrate that the small cytotoxic endonuclease domain from the bacterial toxin colicin E9 (E9 DNase) shows nonvoltage-gated, channel-forming activity in planar lipid bilayers that is linked to toxin translocation into cells. A disulfide bond engineered into the DNase abolished channel activity and colicin toxicity but left endonuclease activity unaffected; NMR experiments suggest decreased conformational flexibility as the likely reason for these alterations. Concomitant with the reduction of the disulfide bond is the restoration of conformational flexibility, DNase channel activity and colicin toxicity. Our data suggest that endonuclease domains of colicins may mediate their own translocation across the bacterial inner membrane through an intrinsic channel activity that is dependent on structural plasticity in the protein.

Interaction of luminal calcium and cytosolic ATP in the control of type 1 inositol (1,4,5)-trisphosphate receptor channels

Ca2+ within intracellular stores (luminal Ca2+) is believed to play a role in regulating Ca2+ release into the cytosol via the inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3)-gated Ca2+ channel (or Ins(1,4,5)P3 receptor). To investigate this, we incorporated purified Type 1 Ins(1,4,5)P3 receptor from rat cerebellum into planar lipid bilayers and monitored effects at altered luminal [Ca2+] using K+ as the current carrier. At a high luminal [Ca2+] and in the presence of optimal [Ins(1,4,5)P3] and cytosolic [Ca2+], a short burst of Ins(1,4,5)P3 receptor channel activity was followed by complete inactivation. Lowering the luminal [Ca2+] caused the channel to reactivate indefinitely. At luminal [Ca2+], reflecting a partially empty store, channel activity did not inactivate. The addition of cytosolic ATP to a channel inactivated by high luminal [Ca2+] caused reactivation. We provide evidence that luminal Ca2+ is exerting its effects via a direct interaction with the luminal face of the receptor. Activation of the receptor by ATP may act as a device by which cytosolic Ca2+ overload is prevented when the energy state of the cell is compromised.

ompC mutants which allow growth on maltodextrins show increased channel size and greater voltage sensitivity

Misra and Benson [(1988) J. Bacteriol. 170, 3611‐3617] showed that point mutations in theompC gene can allowEscherichia coli to grow on maltotriose in the absence of LamB. This report shows that these mutants produce OmpC porins with increased single channel conductance compared to the wild type. The mutants showed similar voltage dependence to each other and to PhoE by being totally closed at 200 mV. The wild type from various sources was largely insensitive to voltages below 200 mV and thus 6 point mutations at 3 sites appear to increase the voltage dependence of OmpC channels.

Characterisation of channels induced in planar bilayer membranes by detergent solubilised Escherichia coli porins.

Purified OmpF, OmpC, NmpC, PhoE and Lc (Protein 2) porins from the Escherichia coli outer membrane were incorporated into planar phospholipid bilayer membranes and the permeability properties of the pores studied. Triton X-100 solubilised porin samples showed large and reproducible increases in membrane conductivity composed of discreet single-channel events. The magnitude of the cation selectivity found for the porins was in the order OmpC greater than OmpF greater than NmpC = Lc; PhoE was anion selective. For the cation selective porins the cation/anion permeability ratios in a variety of solutes ranged from 6 to 35. Further information on the internal structure of the porins was obtained by examination of the single-channel conductance and this was used to interpret macroscopic observations and to estimate single-channel diameters. The same porins solubilised in SDS exhibited slight conductance increase with no observable single-channel activity. Use of on-line microcomputer techniques confirmed the ohmic current vs. voltage behaviour for all the single porin channels examined.

pH-dependent pore-forming activity of OmpATb from Mycobacterium tuberculosis and characterization of the channel by peptidic dissection

Mycobacteria are characterized by an unusual cell wall that controls nutrient and small hydrophilic compound permeability. Porin‐like proteins are necessary to ensure the transport of molecules into the cell. Here, we investigated the pore‐forming properties of OmpATb, a porin from Mycobacterium tuberculosis, in lipid bilayers. Multi‐channel experiments showed an asymmetric behaviour with channel closures at negative critical voltages (Vc) and a strong decrease in Vc at acidic pH. Single‐channel experiments gave conductance values of about 850 ± 80 pS in 1 M KCl and displayed a weak cationic selectivity in 4–8 pH range. The production and characterization of a series of truncated OmpATb proteins, showed that the central domain (OmpATb73−220) was sufficient to induce the ion channel properties of the native protein in lipid bilayers, i.e. asymmetric insertion, pH‐dependent voltage closure, cationic selectivity and similar conductance values in 1 M KCl. Western blot analysis suggests that the presence of OmpATb is only restricted to certain pathogenic species. Therefore, the propensity of channels of native OmpATb to close at low pH may represent an intrinsic property allowing pathogenic mycobacteria to adapt and survive to mildly acidic conditions, such as those encountered within the macrophage phagosome.

The role of acyl chain character and other determinants on the bilayer activity of A21978C an acidic lipopeptide antibiotic.

An acidic lipopeptide A21978C has previously been shown to have a powerful antibiotic activity against Gram-positive organisms. Due to its ability to increase the K+ permeability of bacterial cells and its specific calcium requirement, which is similar to a previously described ionophore CDA, its effect on planar bilayer membranes has been studied. Although it produces significant increases in the conductivity of lipid bilayers it is shown that this alone cannot account for its in vivo activity. Similarly, unlike the in vivo results, the Ca2+-induced increases in bilayer conductivity can be mimicked by Mg2+ and charged lipids. Results from a series of homologues differing in the length of the acyl moiety show a close similarity between bilayer conductance and LD50 trends from in vivo studies. A complex activity is proposed which depends upon incorporation in, rather than disruption of, the bilayer membrane.

The effects of the membrane-penetrating polypeptide segment of the human erythrocyte MN-glycoprotein on the permeability of model lipid membranes

The insoluble peptide, T(is), prepared by trypsin hydrolysis of the MN-glycoprotein (glycophorin) of the human erythrocyte has been incorporated into phospholipid membranes in the form of liposomes and black lipid membranes. The permeability of liposome membranes to 42K+ and of black lipid membranes to water and ions is increased significantly by the presence of the T(is) peptide. Electrophoresis measurements indicate that these effects are not due to the T(is) peptide carrying a net charge. The results suggest that the peptide causes local disordering of the bilayer membrane structures. This is considered in the light of findings published elsewhere: that the MN-glycoprotein penetrates through the cell membrane via a non-polar segment of its polypeptide chain, which is contained intact within the T(is) peptide; that the T(is) peptide is partially helical when associated with phospholipid and forms multimeric 8.0 nm structures within the hydrophobic plane of phospholipid bilayers.