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Channel fluctuations induced by membrane attack complex C5B-9
1Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, NY 10021.
Insights
The complement (C) system
Area of Science:
- Immunology
- Molecular Biology
- Biophysics
Background:
- The terminal complement pathway, involving C5b-9 complexes, forms membrane attack complexes (MACs) that cause cell lysis.
- The role of C9 polymerization in MAC-mediated cytolysis is debated, as cell lysis can occur without visible tubular formation.
- Previous studies have attributed membrane damage to large tubular structures formed by polymerized C9.
Purpose of the Study:
- To investigate the channel-forming properties of C5b-9 complexes reconstituted in artificial lipid bilayers.
- To characterize the conductance and ion selectivity of C5b-9 channels.
- To elucidate the mechanism of membrane damage by the terminal complement pathway.
Main Methods:
- Reconstitution of C5b-9 complexes into high-impedance planar lipid bilayers.
- Electrophysiological recordings to measure unitary channel conductances.
- Voltage-dependence and ion selectivity measurements using different salt concentrations.
Main Results:
- C5b-9 complexes formed heterogeneous ion channels in lipid bilayers, with the smallest exhibiting 15 pS conductance in 0.1 M NaCl.
- These small channels displayed voltage-dependent gating and cation selectivity, favoring K+ over Na+.
- The observed 15-pS channels resembled those formed by the non-polymerizing C9b fragment, not large polymerized C9 structures.
Conclusions:
- C5b-9 complexes initiate membrane damage by forming small, discrete ion channels.
- These initial small channels may aggregate to form larger, tubular lesions characteristic of MACs.
- C5b-8 increases membrane permeability through lipid perturbation, while C9 addition generates authentic ion channels.
Abstract:
The assembly of complement (C) components C5b-9 in membranes results in the formation of transmembrane lesions. The C9 component has been shown to be mainly responsible for formation of the ultrastructurally visible tubules associated with C5b-9 complexes. Several studies have disputed the role of C9 polymerization in C-mediated cytolysis on the grounds that C5b-9 lyses cells in the absence of tubular formation. Here, C5b-9 complexes were reconstituted into high-impedance planar lipid bilayers and shown to form channels which are heterogenous in size. The smallest channels had unitary conductances of 15 picoSiemens (pS) in 0.1 M NaCl. The closing of these channels showed voltage-dependence at membrane potentials exceeding 40 mV. These channels were more cation-selective, with K+ ions being favored over Na+. The 15-pS channels described here are much smaller than the channels attributed previously to either C5b-9 or polymerized C9 complexes but resemble channels formed by the C9b fragment, which does not polymerize into tubules. These results indicate that C5b-9 complexes are capable of damaging membranes by forming initially small ion channels which then aggregate in the membrane to form tubular lesions with much larger conductances. Like C5b-9, C5b-8 also increased membrane permeability. However, this increase in membrane conductance could not be resolved into single channels, suggesting that C5b-8 may induce membrane leakiness by perturbing the packing of membrane lipids, whereas addition of C9 results in authentic production of ion channels.