The early steps of vesicular exocytosis involve a so-called fusion pore, the biophysical nature of which is under debate. It has been proposed that the early fusion pore is a proteinaceous structure that may have ion-channel like properties. It has even been proposed that the movement of protons is initially restricted, leading to a suprisingly long delay (>300 ms) between the opening of the pore, measured as a capacitance change and the neutralization of intravesicular pH, measured with pH-sensitive fluorescent probes, which only becomes possible after fusion pore expansion (1). These measurements, however, were done on ensembles of vesicles, greatly reducing the temopral precision of detection.

In order to gain further insight into the funcional properties of the early fusion pore, we have attempted to combine optical and capacitance recordings of single vesicle fusion in rat basophilic leukaemia (RBL-2H3) cells. To monitor exocytosis of single granules at high temporal resolution, we used the piecewise linear technique. Cells were held at -80 mV. The internal solution contained 10mM free Ca²⁺ and 300mM GTPmS, while the bath contained (mM) CaCl₂ 10 and CsCl 10 to stimulate exocytosis and block inward rectifier current, respectively. For fluorescence measurements, the cells were transfected with fusion proteins of highly pH-sensitive (superecliptic, se) GFP and vesicular membrane proteins, so called Phluorins. Two different constructs, synaptopHluorin(2) and sypHy(3) are available. Initially, only SynaptopHluorin was used. Epifluorescence was recorded through a 100 X 1,3 NA oil immersion objective using a CCD Camera (pixel size=6,45 mm).

Using this system, we were able to detect exocytosis of single vesicles both optically and by capacitance recordings. Capacitance steps in RBL-2H3 had mean amplitudes between 5 and10 fF corresponding to a vesicle diameter of 400 - 600 nm. Before perfusion with intrenal solution, punctate fluorescence signals could be detected with excitation at 460 nm, while during perfusion with internal solution and simultaneous excitation at 480 nm, punctate fluorescence signals gradually appeared. The mean diameter of these punctae was 934 +/­ 192 nm. Where such measurements were possible (n=14 cells), appearance of these fluorescent granules broadly coincided with capacitance steps; however, probably due to phototoxic damage of the cell, we have not been able to collect a sufficient number of capacitance steps togetehr with fluorecnece events to submit them to statistical treatment. Phototoxicity appears to be largely due to membrane sorting of synaptoPhluorin; it should, thus be possible to overcome this problem by using sypHy. In summary, however, we can report the first simultaneous electrical and optical measurements of single vesicle exocytosis.

(1) Barg et al.: Neuron, 33, 287–299, 2002, (2) Miesenböck et al.: Nature, 394, 192–195, 1998, (3) Llagnado et al.: Neuron, 57, 773–786, 2006