MEMBRANE ELECTRIC PROPERTIES BY COMBINED PATCH-CLAMP AND FLUORESCENCERATIO IMAGING IN SINGLE NEURONS

An experimental method has been established to measure the electric pr operties of a cell membrane by combination of patch clamp and dual-wav elength ratio imaging of a fluorescent potentiometric dye, ta[2-(di-n- octylamino)-6-naphthyl]vinyl]pyridinium betaine (di-8-ANEPPS). Pairs o f fluorescence images from the dye-stained membrane of neuroblastoma N 1E-115 cells excited at two wavelengths were initially obtained to cal culate ratio images corresponding to the resting transmembrane potenti al. Subsequently, a whole-cell patch was established and the membrane potential clamped to levels varying from -100 to +60 mV; at each volta ge, a pair of dual-wavelength images were acquired to develop a calibr ation of the fluorescence ratio. Using this method, the resting potent ials could accurately be measured showing that the differentiated cell s were 17 mV more polarized than undifferentiated cells. The combinati on of electrical and optical methods can also follow changes in other membrane electric properties, such as dipole potential, and thus permi t a detailed analysis of the membrane electrical properties underlying the voltage regulation of ion channels.