Plant two-pore channels (TPCs) are non-selective cation channels permeable both to monovalent potassium and divalent calcium. We previously developed a technique that allowed the simultaneous determination of the fluxes of these two ions across the channel by a combined use of patch-clamp and fluorescence. In this paper we studied how potassium and calcium fluxes were influenced by modification of cytosolic concentrations of K+ and Ca2+. A decrease in cytosolic calcium from 2 to 0.5 mM led to a shift of the activation curve of about +60 mV; although at positive potentials currents were very similar, calcium ion permeation was significantly reduced and the ratio between the total and calcium-mediated current increased about two-fold. Upon removal of cytosolic potassium, in the presence of 2 mM cytosolic calcium, the voltage-dependent activation curve was not modified but a dramatic reduction of the currents at positive voltages was apparent. However, calcium permeation did not change significantly in this condition. This work demonstrated that the electrophysiological measurements alone were not capable to predict the extent of the flow of different ions through cation channels. The parallel use of calcium detection by fluorescent dyes proved to be a valuable tool for the correct quantification of the permeation mechanisms in non-selective ion channels.
Keywords: Calcium; Fluorescence; Patch-clamp; Plant vacuole; Potassium.
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