Resolution of fusion pore formation in a cell-attached patch

Exocytosis of neurotransmitter is initiated by formation of a fusion pore, a narrow channel connecting the vesicle lumen to the extracellular space. Opening of the fusion pore can produce a flow of capacitative current, which was used to resolve the exocytosis of mast cell giant secretory granules by Breckenridge and Almers [Breckenridge LJ, Almers W. Currents through the fusion pore that forms during exocytosis of a secretory vesicle. Nature 1987;328:814-7]. We present an extension of this method to resolve fusion pore formation initiating exocytosis of single vesicles in bovine chromaffin cells. Cell-attached patch recordings revealed a capacitative current that was evoked by an increase of intracellular calcium (Ca2+) from application of a Ca2+ ionophore or by the opening of a co-localized Ca2+ channel. Calculated values for fusion pore conductance and vesicular membrane potential were in accord with previous estimates. Finally, we show that a single opening of a co-localized calcium channel evoked fusion pore formation, with delay times between channel opening and exocytosis agreeing with other methods. This method can be applied to resolve exocytosis regardless of what the vesicle contains or where exocytosis occurs, giving the possibility of using this method to resolve the release of synaptic vesicles.