A nitrate-permeable ion channel in the tonoplast of the moss Physcomitrella patens

Abstract

Main conclusion: In this work, for the first time the activity of nitrate-permeable channels in the tonoplast of the mossPhyscomitrella patenswas recorded. The channels allowed nitrate flow in one direction—from the cytoplasm to the vacuole. Selectivity of nitrate over chloride of the channels was proved. The activity of the channels was dependent on cytoplasmic calcium, magnesium, and pH. A patch-clamp study carried out on the vacuolar membrane of the moss Physcomitrella patens has revealed that inhibition of cation-selective channels leads to disclosure of channels permeable to NO3−. These channels were inwardly rectifying and allowed anions to flow from the cytoplasm to the vacuole. After a decrease in the cytoplasmic NO3− concentration, the current density recorded in the whole-vacuole configuration and amplitude of the currents flowing through single channels were reduced. Application of the NO3− gradient caused a shift in the reversal potential (Erev) towards ENO3−, indicating NO3− permeability. Research of the selectivity of the channels to Cl− and NO3− was also done; it indicated that Cl− is less permeable than NO3− (PNO3/PCl = 3.08). Measurements with different concentrations of cytoplasmic Ca2+ and Mg2+ revealed that the channel was activated by different concentrations of these ions—100 µM Ca2+ and 10 mM Mg2+. Calcium dependence of the channels was also modulated by a redox agent—DTT (dithiothreitol), which added on the cytoplasmic side, caused a reduction in the threshold of channel activation with cytoplasmic Ca2+. The NO3− permeable channel was also pH dependent. A decrease in the cytoplasmic pH reduced the open probability of the channel; in turn, an increase in the vacuolar pH did not decrease ion channel activity but lowered its conductance.