In mast cells and granulocytes, exocytosis starts with the formation of a fusion pore. It has been suggested that neurotransmitters may be released through such a narrow pore without full fusion. However, owing to the small size of the secretory vesicles containing neurotransmitter, the properties of the fusion pore formed during Ca2+-dependent exocytosis and its role in transmitter release are still unknown. Here we investigate exocytosis of individual chromaffin granules by using cell-attached capacitance measurements combined with electrochemical detection of catecholamines, achieved by inserting a carbon-fibre electrode into the patch pipette. This allows the simultaneous determination of the opening of individual fusion pores and of the kinetics of catecholamine release from the same vesicle. We found that the fusion-pore diameter stays at <3 nm for a variable period, which can last for several seconds, before it expands. Transmitter is released much faster through this pore than in mast cells, generating a 'foot' signals which precedes the amperometric spike. Occasionally, the narrow pore forms only transiently and does not expand, allowing complete transmitter release without full fusion of the vesicle with the plasma membrane.
Exocytosis, the fusion of secretory vesicles with the plasma membrane to allow release of the contents of the vesicles into the extracellular environment, and endocytosis, the internalization of these vesicles to allow another round of secretion, are coupled. It is, however, uncertain whether exocytosis and endocytosis are tightly coupled, such that secretory vesicles fuse only transiently with the plasma membrane before being internalized (the 'kiss-and-run' mechanism), or whether endocytosis occurs by an independent process following complete incorporation of the secretory vesicle into the plasma membrane. Here we investigate the fate of single secretory vesicles after fusion with the plasma membrane by measuring capacitance changes and transmitter release in rat chromaffin cells using the cell-attached patch-amperometry technique. We show that raised concentrations of extracellular calcium ions shift the preferred mode of exocytosis to the kiss-and-run mechanism in a calcium-concentration-dependent manner. We propose that, during secretion of neurotransmitters at synapses, the mode of exocytosis is modulated by calcium to attain optimal conditions for coupled exocytosis and endocytosis according to synaptic activity.
The secretory process requires many different steps and stages. Vesicles must be formed and transported to the target membrane. They must be tethered or docked at the appropriate sites and must be prepared for fusion (priming). As the last step, a fusion pore is formed and the contents are released. Release of neurotransmitter is an extremely rapid event leading to rise times of the postsynaptic response of less than 100 micro s. The release thus occurs during the initial formation of the exocytotic fusion pore. To understand the process of synaptic transmission, it is thus of outstanding importance to understand the molecular structure of the fusion pore, what are the properties of the initial fusion pore, how these properties affect the release process and what other factors may be limiting the kinetics of release. Here we review the techniques currently employed in fusion pore studies and discuss recent data and opinions on exocytotic fusion pore properties.
Patch-Camp experiments have shown that fusion of secretory granules with the plasma membrane does not always occur as an all-or-none event, but can develop slowly in a fluctuating manner or can be transient. These observations suggested that release could be detected during such incomplete fusion events. To test this hypothesis we have combined patch-clamp measurements of the activity of single exocytotic fusion pores in beige mouse mast cells with the electrochemical detection of serotonin released during the exocytotic events. We report here that on fusion pore opening there is a small release of serotonin which is directly proportional to the pore conductance. We also show that a significant release occurs during transient fusion events. These results demonstrate, to our knowledge for the first time, release of a neurotransmitter from a secretory vesicle that did not undergo complete fusion.
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