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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 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 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 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 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.

SUBMITTER: Koselski M

PROVIDER: S-EPMC4412613 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Patch-clamp studies carried out on the tonoplast of the moss Physcomitrella patens point to existence of two types of cation-selective ion channels: slowly activated (SV channels), and fast-activated potassium-selective channels. Slowly and instantaneously saturating currents were observed in the whole-vacuole recordings made in the symmetrical KCl concentration and in the presence of Ca(2+) on both sides of the tonoplast. The reversal potential obtained at the KCl gradient (10 mM on the cytoplasmic side and 100 mM in the vacuole lumen) was close to the reversal potential for K(+) (E K), indicating K(+) selectivity. Recordings in cytoplasm-out patches revealed two distinct channel populations differing in conductance: 91.6 ± 0.9 pS (n = 14) at -80 mV and 44.7 ± 0.7 pS (n = 14) at +80 mV. When NaCl was used instead of KCl, clear slow vacuolar SV channel activity was observed both in whole-vacuole and cytoplasm-out membrane patches. There were no instantaneously saturating currents, which points to impermeability of fast-activated potassium channels to Na(+) and K(+) selectivity. In the symmetrical concentration of NaCl on both sides of the tonoplast, currents have been measured exclusively at positive voltages indicating Na(+) influx to the vacuole. Recordings with different concentrations of cytoplasmic and vacuolar Ca(2+) revealed that SV channel activity was regulated by both cytoplasmic and vacuolar calcium. While cytoplasmic Ca(2+) activated SV channels, vacuolar Ca(2+) inhibited their activity. Dependence of fast-activated potassium channels on the cytoplasmic Ca(2+) was also determined. These channels were active even without Ca(2+) (2 mM EGTA in the cytosol and the vacuole lumen), although their open probability significantly increased at 0.1 μM Ca(2+) on the cytoplasmic side. Apart from monovalent cations (K(+) and Na(+)), SV channels were permeable to divalent cations (Ca(2+) and Mg(2+)). Both monovalent and divalent cations passed through the channels in the same direction-from the cytoplasm to the vacuole. The identity of the vacuolar ion channels in Physcomitrella and ion channels already characterised in different plants is discussed.

Project description:BACKGROUND: Hexokinase catalyzes the phosphorylation of glucose and fructose, but it is also involved in sugar sensing in both fungi and plants. We have previously described two types of hexokinases in the moss Physcomitrella. Type A, exemplified by PpHxk1, the major hexokinase in Physcomitrella, is a soluble protein that localizes to the chloroplast stroma. Type B, exemplified by PpHxk2, has an N-terminal membrane anchor. Both types are found also in vascular plants, and localize to the chloroplast stroma and mitochondrial membranes, respectively. RESULTS: We have now characterized all 11 hexokinase encoding genes in Physcomitrella. Based on their N-terminal sequences and intracellular localizations, three of the encoded proteins are type A hexokinases and four are type B hexokinases. One of the type B hexokinases has a splice variant without a membrane anchor, that localizes to the cytosol and the nucleus. However, we also found two new types of hexokinases with no obvious orthologs in vascular plants. Type C, encoded by a single gene, has neither transit peptide nor membrane anchor, and is found in the cytosol and in the nucleus. Type D hexokinases, encoded by three genes, have membrane anchors and localize to mitochondrial membranes, but their sequences differ from those of the type B hexokinases. Interestingly, all moss hexokinases are more similar to each other in overall sequence than to hexokinases from other plants, even though characteristic sequence motifs such as the membrane anchor of the type B hexokinases are highly conserved between moss and vascular plants, indicating a common origin for hexokinases of the same type. CONCLUSIONS: We conclude that the hexokinase gene family is more diverse in Physcomitrella, encoding two additional types of hexokinases that are absent in vascular plants. In particular, the presence of a cytosolic and nuclear hexokinase (type C) sets Physcomitrella apart from vascular plants, and instead resembles yeast, where all hexokinases localize to the cytosol. The fact that all moss hexokinases are more similar to each other than to hexokinases from vascular plants, even though both type A and type B hexokinases are present in all plants, further suggests that the hexokinase gene family in Physcomitrella has undergone concerted evolution.

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

Main conclusion

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