Information is passed between cells by the release of soluble materials via membrane fusion. We are continuing to study the aqueous pore which we discovered initiates fusion of large secretory granules during exocytosis. Over the last year, we have uncovered an effect of granule potential on cell admittance, the time course for the enlargement of the pore from its initial value, and the effect of hyperosmotic conditions on pore growth. The size of the initial pore is heterogeneous and its growth is varied. It would appear that this pore has fundamental differences from membrane channels and gap junctions, and may represent a novel structure composed of both lipids and protein. Our technique reveals pores smaller than the initial pore seen in electron microscopy. We have begun to measure fusion in a system with identified fusion proteins; the influenza virus. We have used digital computer imaging to study the transfer of fluorescent dye from red blood cells to NIH 3T3 cells expressing the viral fusion protein HA. Dye transfer has been documented in lipid/lipid exchange and cyto- phasmic marker exchange.
We aim to establish the temporal sequence of events during membrane fusion. Secretion from exocrine glands is stimulated by acetylcholine released from parasympathetic neurons. We showed that a rundown in activation occurs during intracellular perfusion, that the rundown is due to the loss of a water soluble cytoplasmic compound necessary to the muscarinic response. Thus we can equate rundown and washout. We have succeeded in determining at what point along the chain of events leading from receptor occupancy to channel activation the compound is acting by adding second messengers to depleted cells via a second patch pipette. Our results indicate that a 300 - 500 molecular weight solute is likely to be an unknown cofactor of Gp, the GTP binding protein linked to phospholipase C.
