Fluorescence combined with excised patch: measuring calcium currents in plant cation channels

Combined application of the patch–clamp technique and fura-2 fluorescence detection enables the study of study calcium fluxes or related increases in cytosolic calcium concentration. Here we used the excised patch configuration, focusing the photomultiplier on the tip of the recording pipette where the fluorescent dye was present (FLEP, fluorescence combined with excised patch). This configuration has several advantages, i.e. a lack of delay in loading the fluorophore, of interference by internal calcium buffers and of photobleaching, due to the quasi-infinite dye reservoir inside the pipette. Upon voltage stimulation of tonoplast patches, sustained and robust fluorescence signals indicated permeation of calcium through the slow vacuolar (SV) channel. Both SV currents and fluorescence signal changes were absent in the presence of SV channel inhibitors and in vacuoles from Arabidopsis tpc1 knockout plants that lack SV channel activity. The fractional calcium currents of this non-selective cation channel were voltage-dependent, and were approximately 10% of the total SV currents at elevated positive potentials. Interestingly, calcium permeation could be recorded as the same time as oppositely directed potassium fluxes. These events would have been impossible to detect using patch–clamp measurements alone. Thus, we propose use of the FLEP technique for the study of divalent ion-selective channels or transporters that may be difficult to access using conventional electrophysiological approaches.