Simultaneous Optical-Electrical Measurement of the Delay between formation of the Fusion Pore and Proton Equilibration in Exocytosis of Single Vesicles

Abstract

Optical detetction of neutralization of pH in granules or vesicles is often used to define exocytotic events. However, combined measurents of ensemble capacitance and pH-dependent vesicular fluorescence changes have suggested that the movement of protons only becomes possible after fusion pore expansion with a mean delay of > 300 ms (1). To enhance the temporal resolution of such measurements, we have combined capacitance recordings of single vesicle fusion in RBL cells transfected with synapthpHluorin as a reporter of vesicular pH. To monitor cell capacitance steps due to exocytosis of single granules in whole cell patch-clamp mode, we used the piecewise linear technique. Internal solution contained 10 μM free Ca2+ and 300 μM GTPγS.View Large Image | View Hi-Res Image | Download PowerPoint Slide Before establishing whole cell recordings, punctate fluorescence signals could be detected with excitation at 460 nm, while during perfusion with internal solution and excitation at 480 nm, punctate fluorescence signals gradually appeared at corresponding sites. Fluorescence increases clearly lagged capacitance steps by several 100 ms- seconds, supporting the idea that pH equilibration through the fusion pore is delayed.(1) Barg et al.: Neuron, 33, 287-299, 2002.