Conformational changes in the HA2 subunit of influenza hemagglutinin (HA) are coupled to membrane fusion. We investigated the fusogenic activity of the polypeptide FHA2 representing 127 amino terminal residues of the ectodomain of HA2. While the conformation of FHA2 both at neutral and at low pH is nearly identical to the final low pH conformation of HA2, we found that FHA2 induces lipid mixing between bound cells in a low pH-dependent manner, indicating that the """"""""spring-loaded"""""""" energy is not required for FHA2-mediated membrane merger. Although, unlike HA, FHA2 did not form an expanding fusion pore, both acidic pH and membrane concentrations of FHA2, required for lipid mixing, have been close to those required for HA-mediated fusion. The same genetic modification of the fusion peptide inhibits both HA- and FHA2- fusogenic activities. The ability of FHA2 to mediate lipid mixing very similar to HA-mediated lipid mixing is consistent with the hypothesis that hemifusion requires just a portion of the energy released in the conformational change of HA at acidic pH. In contrast opening of an expanding fusion pore connecting two membrane compartments requires low pH-triggered refolding of multiple full-sized HA molecules. To better understand energetics of a pore in membrane we studied voltage-induced pores in protein-free lipid bilayers. Electric fields promote pore formation in both biological and model membranes. We clamped unmodified planar bilayers at 150-550 mV to monitor transient single pores for a long period of time. We observed fast transitions between different conductance levels reflecting opening and closing of metastable lipid pores. The mean amplitude of conductance fluctuations was independent of voltage and close (~500 pS) for bilayers of different area indicating the local nature of the conductive defects. Based on the conductance value and its dependence of the ion size, the radius of the average pore was estimated as ~ 1 nm. Short bursts of conductance spikes (opening and closing of pores) were often separated by periods of background conductance. Within the same burst the conductance between spikes was indistinguishable from the background. The mean time interval between spikes in the burst was much smaller than that between adjacent bursts. These data indicate that opening and closing of lipidic pores proceed through some electrically invisible (""""""""silent"""""""") pre-pores. Similar pre-pore defects and metastable conductive pores might be involved in remodeling of cell membranes in different biologically relevant processes.
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