Membrane damage by a toxin from the sea anemone Stoichactis helianthus. I. Formation of transmembrane channels in lipid bilayers.

The addition of nanomolar amounts of a toxin preparation derived from the sea anemone Stoichactis helianthus to black lipid membranes increases their electrical conductance by one million-fold. In addition, the membranes become permeable predominantly to monovalent cations. The elevated bilayer conductance is voltage-dependent, and the current-voltage curves of these bilayers display rectification as well as a region of negative resistance. The membrane activity of the toxin is proportional to the third power of its concentration, and at very low concentrations the membrane conductance increases in discrete uniform steps. These observations indicate that the mechanism of toxin action involves the formation of transmembrane channels constructed by the aggregation of protein molecules which are inserted in the bilayer. The voltage-dependent membrane conductance arises from two distinct channel characteristics: (1) the unit conductance of individual channels is dependent on the polarity of applied voltage; (2) the number of ion-conducting channels is influenced by the polarity as well as the magnitude of applied potential. It is believed that these effects are due to the influence of an electric field on the insertion of toxin molecules into the bilayer or on their subsequent association with each other to produce channels. Partial chemical characterization of the toxin material has shown that the membrane active factor is a basic protein with a molecular weight of 17,500.

Membrane damage by a toxin from the sea anemone Stoichactis helianthus. II. Effect of membrane lipid composition in a liposome system.

In the first paper of this series, it was shown that a toxin from the sea anemone Stoichactis helianthus increased the permeability of black lipid membranes due to transmembrane channel formation. In the present study, we have used liposomes to examine the reactivity of the toxin with different phospholipids. Membrane damage was assessed by measuring the release of 86Rb+ and 14C-labeled membrane lipid. For the different lipids, the rank order of marker release was: sphingomyelin greater than C18 : 2 phosphatidylcholine greater than C18 : 1 phosphatidylcholine greater than C18 : 0 phosphatidylcholine greater than C16 : 0 phosphatidylcholine = C14 : 0 phosphatidylcholine. In C14 : 0 and C16 : 0 phosphatidylcholine liposomes there was no 14C-labeled lipid release and only 13 to 16% 86 Rb+ release which corresponds to the 86Rb+ content in the outermost aqueous shell of multilamellar liposomes. This indicates that membrane damage was limited to the outermost bilayer. In liposomes prepared with the other lipids, the extent of release of both markers increased proportionately with the length and the degree of unsaturation of the lipids' acyl side chains. Spingomyelin liposomes were the most susceptible with 47% of the 14C-labeled lipid marker and 90% of the 86Rb+ marker being released. The large extent of 14C-labeled lipid release is attributed to a detergent-like activity of the toxin which presumably is due to the amphipathic nature of the protein. Thus, the toxin can inflict membranrtance of one mechanism or the other apparently varies depending on membrane structure and lipid composition.

Increased ion permeability of planar lipid bilayer membranes after treatment with the C5b-9 cytolytic attack mechanism of complement.

The ion permeability of planar lipid bilayers, as measured electrically, was found to increase modestly upon treatment with purified complement complex C5b,6 and complement components C7 and C8. The subsequent addition C9 greatly amplified this change. No permeability changes occurred when components were added individually to the membrane, or when they were used in paired combinations, or when C5b, C7, C8, and C9 were admixed prior to addition. Thus, there is a significant parallel between the permeability changes induced in the model membrane and damage produced in biological membranes by the C5b-9 complement attack sequence. The efficiency of membrane action by C5b-9 was critically dependent on the order in whcih components were added to the membrane. There were also differences in the electrical properties of membranes treated with C5b-8 and C5b-9, though in both cases the enhanced bilayer permeability is best attributed to the formation of trans-membrane channels. Collectively, the data are consistent with the hypothesis that the mechanism of membrane action by complement involves the production of a stable channel across the lipid bilayer, resulting in cell death by colloid-osmotic lysis.