INFLUENZA HEMAGGLUTININ-MEDIATED FUSION PORES CONNECTING CELLS TO PLANAR MEMBRANES - FLICKERING TO FINAL EXPANSION

We have studied the fusion between voltage-clamped planar lipid bilaye rs and influenza virus infected MDCK cells, adhered to one side of the bilayer, using measurements of electrical admittance and fluorescence . The changes in currents in-phase and 90-degrees out-of-phase with re spect to the applied sinosoidal voltage were used to monitor the addit ion of the cell membrane capacitance to that of the lipid bilayer thro ugh a fusion pore connecting the two membranes. When ethidium bromide was included in the solution of the cell-free side of the bilayer, inc reases in cell fluorescence accompanied the admittance changes, indepe ndently confirming that these changes were due to formation of a fusio n pore. Fusion required acidic pH on the cell-containing side and depe nded on temperature. For fusion to occur, the influenza hemagglutinin (HA) had to be cleaved into HA1 and HA2 subunits. The incorporation of gangliosides into the planar bilayers greatly augmented fusion. Fusio n pores developed in four distinct stages after acidification: (a) a p re-pore, electrically quiescent stage; (b) a flickering stage, with 1- 2 nS pores opening and closing repetitively; (c) an irreversibly opene d stage, in which pore conductances varied between 2 and 100 nS and ex hibited diverse kinetics; (d) a fully opened stage, initiated by an in stantaneous, time-resolution limited, increase in conductance leveling at approximately 500 nS. The expansion of pores by stages has also be en shown to occur during exocytosis in mast cells and fusion of HA-exp ressing cells and erythrocytes. We conclude that essential features of fusion pores are produced with proteins in just one of the two fusing membranes.