The exocytotic event in chromaffin cells revealed by patch amperometry

Abstract

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.

Exocytotic catecholamine release is not associated with cation flux through channels in the vesicle membrane but Na+ influx through the fusion pore

“…Fig. 1e shows the ratio of catecholamine flux/fusion pore conductance (flux/conductance ratio) during the foot signal, giving a rather constant value of ~3×10 7 molecules per second per nS of fusion pore conductance (molecules s −1 nS −1 ) for the part indicated by the horizontal line, in agreement with previous estimates 5 . At later times the flux/conductance ratio decreases somewhat, presumably due to depletion of free catecholamine in the vesicle.…”

“…To determine vesicular catecholamine concentrations the number of molecules released from a given vesicle was calculated from the integrated amperometric spike charge assuming a transfer of 2 electrons per molecule and the vesicle volume was estimated from the capacitance step size assuming spherical geometry and a specific membrane capacitance of 9 fF cm −1 as described 5 .…”

“…The initial flux of catecholamines through the early narrow fusion pore can be measured as an amperometric foot signal preceding the amperometric spike 3,5,8 . The amplitude of amperometric foot currents is typically ~5 pA.…”

“…Fusion pores in mast cells and chromaffin cells have typically an initial conductance of ~330 pS 1,3 through which vesicular serotonin and catecholamines are released, respectively 4,5 . The initial fusion pore of a synaptic vesicle is typically >280 pS and release of neurotransmitter should occur with a time constant <500 μs due to the small vesicle volume 6 .…”

Patch amperometry: high-resolution measurements of single-vesicle fusion and release

“…A detailed description of fusion pore analysis for cell-attached capacitance measurements and consideration for choosing the optimal sine wave frequency depending on vesicle size to be studied has been provided elsewhere 3 . Combining patch amperometry with fusion pore analysis has facilitated the analysis of the relation between catecholamine flux during the amperometric foot signal and fusion pore conductance 10 . Specific mutations may be introduced into protein sequences believed to be involved in fusion pore formation to differentially affect fusion pore conductance and catecholamine permeation.…”