Cation-permeable vacuolar ion channels in the moss Physcomitrella patens: a patch-clamp study

Abstract

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 Ca2+ 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+ (EK), 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 Ca2+ revealed that SV channel activity was regulated by both cytoplasmic and vacuolar calcium. While cytoplasmic Ca2+ activated SV channels, vacuolar Ca2+ inhibited their activity. Dependence of fast-activated potassium channels on the cytoplasmic Ca2+ was also determined. These channels were active even without Ca2+ (2 mM EGTA in the cytosol and the vacuole lumen), although their open probability significantly increased at 0.1 μM Ca2+ on the cytoplasmic side. Apart from monovalent cations (K+ and Na+), SV channels were permeable to divalent cations (Ca2+ and Mg2+). 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.

The role of vacuolar ion channels in salt stress tolerance in the liverwort Conocephalum conicum

“…Since inhibition of SV channel activity by vacuolar calcium is a frequently described phenomenon (Pottosin et al 1997(Pottosin et al , 2004Pottosin and Schӧnknecht 2007;Schönknecht 2013;Beyhl et al 2009;Koselski et al 2013), we decided to study the effect of application of high 1 mM Ca 2+ from the vacuolar side of the membrane during the vacuole-out recordings (Fig. 6).…”

“…They are activated by an unphysiological cytoplasmic Ca 2+ concentration exceeding 10 µM (Schulze et al 2011). A high concentration of luminal calcium (usually present in plant vacuoles) decreases substantially their activity (Pottosin et al 1997(Pottosin et al , 2004Pottosin and Schӧnknecht 2007;Schönknecht 2013;Beyhl et al 2009;Koselski et al 2013). In addition to Ca 2+ , SV/TPC1 channels are regulated by numerous factors playing roles in cell nutrition and signaling, among them: Mg 2+ , Zn 2+ , H + , polyamines, dithiothreitol, glutathione, 14-3-3 proteins, heavy metals, phosphorylation/dephosphorylation, calmodulin, and H 2 O 2 (reviewed by Pottosin and Schӧnknecht 2007;Hedrich and Marten 2011;Peiter 2011).…”

Menthol‐induced action potentials in Conocephalum conicum as a result of unspecific interactions between menthol and the lipid phase of the plasma membrane

“…In our experiments, 2 m M CaCl 2 was applied on both sides of the tonoplast (see section Materials and methods). Interestingly, it is well documented that SV channels are active even at such a ‘high’ calcium concentration and in different plant species (Trebacz and Schönknecht , Dziubinska et al , Dziubinska et al , Koselski et al ). However, according to Pottosin et al () and Ivashikina and Hedrich (), the optimal compositions of calcium ions for activation of maximal SV currents are vestigial amounts in the vacuole lumen and a millimolar concentration on the cytoplasm side.…”

“…They exhibit permeability to Ca 2+ and are outward rectifying. SV channels are present in all investigated plant species (Carpaneto et al , Dziubinska et al , Ivashikina and Hedrich , Beyhl et al , Dziubinska et al , Koselski et al ).…”

Generation of action potential-type changes in response to darkening and illumination as indication of the plasma membrane proton pump status in Marchantia polymorpha

“…However, to date, there has been no report about the characteristics of electrical signals in M. polymorpha gamethophyte cells. Simultaneously, there are reports about intracellular investigations conducted on another liverwort Conocephalum conicum (Dziubińska et al 1983;Favre et al 1999b;Król et al 2007;Zawadzki and Trębacz 1985) and on the moss Physcomitrella patens, a species that is phylogenetically related to liverworts and regarded as a model system in plant functional genomics (Decker et al 2006;Reski 1999) and electrophysiology (Ermolayeva et al 1996(Ermolayeva et al , 1997Koselski et al 2008Koselski et al , 2013Koselski et al , 2015. Conocephalum conicum displays excitability to various stimuli, such as light (Król and Trębacz 1999;Trębacz and Zawadzki 1985;Trębacz et al 1989a), cold (Król et al 2003;Kupisz and Trebacz 2011), wounding (Favre et al 1999a), and direct current (DC) (Dziubińska et al 1983;Król and Trębacz 1999;Paszewski et al 1982).…”