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

In this work, for the first time the activity of nitrate-permeable channels in the tonoplast of the moss Physcomitrella patens was 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 NO(3) (−). These channels were inwardly rectifying and allowed anions to flow from the cytoplasm to the vacuole. After a decrease in the cytoplasmic NO(3) (−) concentration, the current density recorded in the whole-vacuole configuration and amplitude of the currents flowing through single channels were reduced. Application of the NO(3) (−) gradient caused a shift in the reversal potential (E(rev)) towards E(NO3−), indicating NO(3) (−) permeability. Research of the selectivity of the channels to Cl(−) and NO(3) (−) was also done; it indicated that Cl(−) is less permeable than NO(3) (−) (P(NO3)/P(Cl) = 3.08). Measurements with different concentrations of cytoplasmic Ca(2+) and Mg(2+) revealed that the channel was activated by different concentrations of these ions—100 µM Ca(2+) and 10 mM Mg(2+). 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 Ca(2+). The NO(3) (−) 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00425-015-2250-3) contains supplementary material, which is available to authorized users.