Transmembrane channel formation by complement: functional analysis of the number of C5b6, C7, C8, and C9 molecules required for a single channel.

Abstract

Earlier studies have shown that sequential treatment of resealed erythrocyte ghosts with C5b6, C7, C8, and C9 leads to insertion ofhydrophobic peptides from these complement proteins into the membrane and assembly of transmembrane channels. The number of molecules of each of the proteins required for assembly of the membrane-associated channel structure was evaluated by measuring the quantitative relationship between the doses ofthe individual proteins and the release of two trapped markers, sucrose and inulin, from ghosts after channel formation. The incubation period was sufficient to attain equilibrium of marker distribution between the ghosts and the extracellular fluid. Two markers of different size (sucrose and inulin, 0.9 and 3 nm molecular diameter, respectively) were used in order to develop information on the molecular composition of small and large channels, respectively. We found that participation of C5b6, C7, and C8 in channel formation displayed one-hit characteristics, regardless of marker size. By contrast, the participation of C9 was one-hit with respect to the sucrose marker, whereas with respect to the inulin marker the C9 reaction was multi-hit. Our results are compatible with the view that these markers are released through a channel structure in the membrane that is a monomer of C5b-9 of the composition C5b61C71C81C9., in which n = 1 for channels permitting passage of sucrose and n = 2 for channels allowing transit of inulin.Membrane attack by complement is initiated when complement protein C5 is cleaved into C5a and C5b by the C5 convertase of either the classical or the alternative activation pathway (1). The subsequent interactions among the terminal complement proteins (C5b-C9) are accompanied by exposure of hydrophobic peptides (2-4). If this occurs in the immediate vicinity of a bilayer membrane, some of the exposed peptides become inserted in the lipid bilayer (5-8) and are assembled into transmembrane channels (9-12).From measurements of the molecular ratios among the individual complement proteins in the membrane-associated complex, Kolb et aL (13) proposed the formula C5b1C6lC7lC8lC9n, with a maximal value of n = 6. Subsequently, Biesecker et al. (14) estimated from hydrodynamic analyses of detergent-extracted complexes that the molecular weight is 1.7 x 106. They concluded from this value that the complex is a dimer of C5b-9 and proposed the formula (C5b1C61C71C81C93)2.By contrast, Bhakdi and Tranum-Jensen proposed that the complex is a monomer of C5b-9. They drew attention to the polydispersity ofthe detergent-solubilized complexes as judged by electron microscopy, ultracentrifugation in density gradients, and gel exclusion chromatography (15). According to their results, the smallest complexes are monomers of C5b-9, which have the structural appearance of hollow cylinders apparently identical to the characteristic complement lesions seen on target membranes. Their heavier ultracentrifugal fractions contained aggregates of these cylinders.An important part of this disa...

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