THE INITIAL FUSION PORE INDUCED BY BACULOVIRUS GP64 IS LARGE AND FORMS QUICKLY

The formation of the fusion pore is the first detectable event in memb rane fusion (Zimmerberg, J., R. Blumenthal, D.P. Sarkar, M. Curran, an d S.J. Morris. 1994, J. Cell Biol. 127:1885-1894). To date, fusion por es measured in exocytosis and viral fusion have shared features that i nclude reversible closure (flickering), highly fluctuating semistable stages, and a lag time of at least several seconds between the trigger ing and the pore opening. We investigated baculovirus GP64-induced Sf9 cell-cell fusion, triggered by external acid solution, using two diff erent electrophysiological techniques: double whole-cell recording (fo r high time resolution, model-independent measurements), and the more conventional time-resolved admittance recordings. Both methods gave es sentially the same results, thus validating the use of the admittance measurements for fusion pore conductance calculations. Fusion was firs t detected by abrupt pore formation with a wide distribution of initia l conductance, centered around 1 nS. Often the initial fusion pore con ductance was stable for many seconds. Fluctuations in semistable condu ctances were much less than those of other fusion pores, The waiting r ime distribution, measured between pH onset and initial pore appearanc e, fits best to a model with many (similar to 19) independent elements . Thus, unlike previously measured fusion pores, GP64-mediated pores d o not flicker, can have large, stable initial pore conductances lastin g up to a minute, and have typical lag times of < 1 s, These findings are consistent with a barrel-shaped model of an initial fusion pore co nsisting of five to eight GP64 trimers that is lined with lipid.