The patch amperometry technique: Design of a method to study excytosis of single vesicles.

We developed patch amperometry, a new technique that combines high-resolution cell-attached capacitance measurements with simultaneous amperometric detection by placing the amperometric electrode inside the patch pipette. For this purpose we designed a new electrode holder, which accommodates the connections for two electrodes and has the capability to change the position of the CFE tip versus the pipette tip. The method makes it possible to determine vesicular concentrations, to distinguish transient and full fusion events, and to analyze individual fusion pore openings and the role of the fusion pore in limiting release. While it is now clear how the size of the fusion pore determines release from chromafin granules during the amperometric foot signal, it is not yet well understood what determines the shape of the spike after the fusion pore expansion. After the fusion pore dilates, the molecules are released rapidly, giving rise to the amperometric spike. It is likely that in this phase release is not hindered by the fusion pore restriction any longer. The vesicular contents should be able to diffuse freely to the detector. However, when a CFE is placed very close to the cell surface, spikes are broader than expected from diffusion theory if the release giving rise to the amperometric spike were instantaneous [18–20,36,37]. Using patch amperometry it was shown that in mast cells the upstroke of the amperometric spike is not limited by the fusion pore [35]. It is widely believed that the time course of the amperometric spike reflects dissociation of bound molecules from the matrix followed by diffusion to the CFE. However, recent amperometric measurements from chromaffin cells using four-electrode electrochemical detector arrays have suggested that diffusion near the membrane is very slow, presumably due to reversible binding of catecholamines to the cell surface [22]. Whether a larger fusion pore in chromaffin cells is also participating in limiting release during the amperometric spike is still to be answered.