Certain calcium channel inhibitors exhibit a number of secondary effects on the physiological properties in Nitellopsis obtusa: a voltage clamp approach.

An unsolved problem of contemporary plant electrophysiology is the identity of Ca2+ channels responsible for the initiation of the action potential. We took a pharmacological approach and applied several Ca2+ channel blockers (verapamil, tetrandrine, and NED-19) on a Characean (Nitellopsis obtusa ) algae model system. The impact of the selected pharmaceuticals on the parameters of excitation transients of a single cell was analysed employing the two-electrode voltage clamp technique. It was revealed that tetrandrine exerted no effect, while both verapamil and NED-19 prolonged activation and inactivation durations of the excitatory Cl- current. NED-19 also significantly depolarised the excitation threshold membrane potential and shifted Ca2+ current reversal potential. Thus, NED-19 most specifically targeted Ca2+ channels. A viability assay paired with observations of cytoplasmic streaming revealed that verapamil affected not only Ca2+ channels but also exhibited non-specific effects, which eventually lead to cell death. Since many potential Ca2+ channel blockers exert additional undesirable non-specific effects, our study underlines the necessity to search for new more specific modulators of plant Ca2+ transport systems.

Evolution of long-distance signalling upon plant terrestrialization: comparison of action potentials in Characean algae and liverworts.

BACKGROUND: In this review, we summarize data concerning action potentials (APs) - long-distance electrical signals in Characean algae and liverworts. These lineages are key in understanding the mechanisms of plant terrestrialization. Liverworts are postulated to be pioneer land plants, whereas aquatic charophytes are considered the closest relatives to land plants. The drastic change of the habitat was coupled with the adaptation of signalling systems to the new environment. SCOPE: APs fulfil the 'all-or-nothing' law, exhibit refractory periods and propagate with a uniform velocity. Their ion mechanism in the algae and liverworts consists of a Ca2+ influx (from external and internal stores) followed by/coincident with a Cl- efflux, which both evoke the membrane potential depolarization, and a K+ efflux leading to repolarization. The molecular identity of ion channels responsible for these fluxes remains unknown. Publication of the Chara braunii and Marchantia polymorpha genomes opened up new possibilities for studying the molecular basis of APs. Here we present the list of genes which can participate in AP electrogenesis. We also point out the differences between these plant species, e.g. the absence of Ca2+-permeable glutamate receptors (GLRs) and Cl--permeable SLAC1 channel homologues in the Chara genome. Both these channels play a vital role in long-distance signalling in liverworts and vascular plants. Among the common properties of APs in liverworts and higher plants is their duration (dozens of seconds) and the speed of propagation (mm s-1), which are much slower than in the algae (seconds, and dozens of mm s-1, respectively). CONCLUSIONS: Future studies with combined application of electrophysiological and molecular techniques should unravel the ion channel proteins responsible for AP generation, their regulation and transduction of those signals to physiological responses. This should also help to understand the adaptation of the signalling systems to the land environment and further evolution of APs in vascular plants.

Impact of Mammalian Two-Pore Channel Inhibitors on Long-Distance Electrical Signals in the Characean Macroalga Nitellopsis obtusa and the Early Terrestrial Liverwort Marchantia polymorpha.

Inhibitors of human two-pore channels (TPC1 and TPC2), i.e., verapamil, tetrandrine, and NED-19, are promising medicines used in treatment of serious diseases. In the present study, the impact of these substances on action potentials (APs) and vacuolar channel activity was examined in the aquatic characean algae Nitellopsis obtusa and in the terrestrial liverwort Marchantia polymorpha. In both plant species, verapamil (20-300 µM) caused reduction of AP amplitudes, indicating impaired Ca2+ transport. In N. obtusa, it depolarized the AP excitation threshold and resting potential and prolonged AP duration. In isolated vacuoles of M. polymorpha, verapamil caused a reduction of the open probability of slow vacuolar SV/TPC channels but had almost no effect on K+ channels in the tonoplast of N. obtusa. In both species, tetrandrine (20-100 µM) evoked a pleiotropic effect: reduction of resting potential and AP amplitudes and prolongation of AP repolarization phases, especially in M. polymorpha, but it did not alter vacuolar SV/TPC activity. NED-19 (75 µM) caused both specific and unspecific effects on N. obtusa APs. In M. polymorpha, NED-19 increased the duration of repolarization. However, no inhibition of SV/TPC channels was observed in Marchantia vacuoles, but an increase in open probability and channel flickering. The results indicate an effect on Ca2+ -permeable channels governing plant excitation.

Glutamate and NMDA affect cell excitability and action potential dynamics of single cell of macrophyte Nitellopsis obtusa.

The effect of glutamate and N-methyl-d-aspartate (NMDA) on electrical signalling - action potentials (AP) and excitation current transients - was studied in intact macrophyte Nitellopsis obtusa (Characeaen) internodal cell. Intracellular glass electrode recordings of single cell in current clamp and two-electrode voltage clamp modes indicate that glutamate (Glu, 0.1-1.0 mM) and NMDA (0.01-1.0 mM) increase electrically induced AP amplitude by hyperpolarising excitation threshold potential (Eth) and prolong AP fast repolarisation phase. Amplitude of Cl- current transient, as well as its activation and inactivation durations were also increased. Both Glu and NMDA act in a dose-dependent manner. The effect of NMDA exceeds that of Glu. Ionotropic glutamate receptor inhibitors AP-5 (NMDA-type receptors) and DNQX (AMPA/Kainate-type) have no effect on Nitellopsis cell electrical signalling per se, yet robustly inhibit excitatory effect of NMDA. This study reinforces NMDA as an active component in glutamatergic signalling at least in some plants and stresses the elaborate fine-tuning of electrical signalling.

Modeling the Action Potential in Characeae Nitellopsis obtusa: Effect of Saline Stress.

Action potentials (AP) of characean cells were the first electrical transients identified in plants. APs provide information about plethora of environmental cues. Salinity stress is critical for plants and impacts on excitability. The AP of brackish Characeae Nitellopsis obtusa, obtained in artificial pond water (APW) and under osmotic stress of 90 or 180 mM sorbitol APW or saline stress of 50 or 100 mM NaCl APW, were simulated by the Thiel-Beilby model (Beilby and Al Khazaaly, 2016). The model is based on a paradigm from animal systems, featuring the second messenger inositol 1,4,5-triphosphate (IP3) mediating the opening of Ca2+ channels on internal stores. In plants the IP3 receptors have not been identified, so other second messengers might translate the threshold plasma membrane depolarization to Ca2+ release. The increased Ca2+ concentration in the cytoplasm activates Cl- channels, which lead to the depolarizing phase of the AP. The repolarization to normal resting potential difference (PD) results from the Ca2+ being re-sequestered by the Ca2+ pumps, the closure of the Cl- channels, efflux of K+ through the depolarization-activated outward rectifier channels and the continuing activity of the proton pump. The Nitellopsis AP form is longer in APW compared to that of Chara, with more gradual repolarization. The tonoplast component of the AP is larger than that in Chara australis. The plasma membrane AP is prolonged by the exposure to saline to a "rectangular" shape, similar to that in Chara. However, the changes are more gradual, allowing more insight into the mechanism of the process. It is possible that the cells recover the original AP form after prolonged exposure to brackish conditions. Some cells experience tonoplast APs only. As in Chara, the proton pump is transiently inhibited by the high cytoplasmic Ca2+ and gradually declines in saline media. However, if the cells are very hyperpolarized at the start of the experiment, the pump inhibition both by the AP and by the saline medium is mitigated. The model parameters and their changes with salinity are comparable to those in Chara.

Asparagine alters action potential parameters in single plant cell.

Effect of amino acid L-asparagine on electrical signalling of single Nitellopsis obtusa (Characeaen) cell was investigated using glass-microelectrode technique in current-clamp and voltage-clamp modes. Cell exposure for 30 min to 0.1 mM and 1 mM of asparagine resulted in changes of electrically stimulated action potential (AP) parameters in comparison to standard conditions. Results indicate that asparagine acts in dose-dependent manner: increases AP amplitude by hyperpolarizing AP threshold potential (Eth), prolongs action potential repolarization, increases maximum Cl- efflux amplitude along with the increase of activation and inactivation durations. Presented findings provide new aspects of exogenous amino acids' effect on plants' electrical signalling with emphasis on separate single plant cell excitability and AP characteristics.

Electrical signalling in Nitellopsis obtusa: potential biomarkers of biologically active compounds.

The Nitellopsis obtusa (N.A.Desvaux) J.Groves cell provides a model system for complex investigation of instantaneous effects of various biologically active compounds (BC) on the generation of plant bioelectrical signals in vivo. Experimental evidence using multiple electrical signals as biomarkers of the effects of BC (acetylcholine, asparagine, glutamate, nicotine, aluminium, nickel and cadmium ions) is provided. The effect of BC on membrane transport systems involved in the cell excitability were tested by current clamp, voltage clamp and patch clamp methods. Membrane potential (MP) alterations and action potential (AP) patterns in response to BC were shown to represent the cell state. High discretisation frequency allows precise, high time resolution analysis of real-time processes measuring changes in excitation threshold, AP amplitude and velocity of repolarisation values after application of BC indicating the effect on ion channels involved in AP generation. Application of voltage clamp revealed that changes in AP peak value were caused not only by increment in averaged maximum amplitude of the Cl- current, but in prolonged Cl- channels' opening time also. The cytoplasmic droplet can serve as a model system in which the effects of BC on single tonoplast ion channel can be studied by patch clamping. Investigation of electrical cell-to-cell communication revealed evidence on the electrical signal transduction through plasmodesmata.

The effects of Ni(2+) on electrical signaling of Nitellopsis obtusa cells.

The effect of nickel (Ni) on the generation of plant bioelectrical signals was evaluated in Nitellopsis obtusa, a Characean model organism. Conventional glass-microelectrode technique and K(+)-anaesthesia method in current-clamp and voltage-clamp modes were used for the measurement and analysis of electrical parameters. Ni(2+) treatment rapidly influenced the action potential (AP) parameters namely, excitation threshold, AP peak and duration, membrane potential at various voltages and dynamics of ion currents. We conclude that altered electrical signaling pathway in the test organism constituted the early target for Ni toxicity imposition. The observed Ni interference could be ascribed to disturbed [Ca(2+)]cyt content, impaired Cl(-) and K(+) channels activity resulting in decreased excitability and repolarization rate in generated AP.

Charophyte electrogenesis as a biomarker for assessing the risk from low-dose ionizing radiation to a single plant cell.

The impact of low-dose ionizing radiation on the electrical signalling pattern and membrane properties of the characea Nitellopsis obtusa was examined using conventional glass-microelectrode and voltage-clamp techniques. The giant cell was exposed to a ubiquitous radionuclide of high biological importance - tritium - for low-dose irradiation. Tritium was applied as tritiated water with an activity concentration of 15 kBq L(-1) (an external dose rate that is approximately 0.05 µGy h(-1) above the background radiation level); experiments indicated that this was the lowest effective concentration. Investigating the dynamics of electrical excitation of the plasma membrane (action potential) showed that exposing Characeae to tritium for half an hour prolonged the repolarization phase of the action potential by approximately 35%: the repolarization rate decreased from 39.2 ± 3.1 mV s(-1) to 25.5 ± 1,8 mV s(-1) due to tritium. Voltage-clamp measurements showed that the tritium exposure decreased the Cl(-) efflux and Ca(2+) influx involved in generating an action potential by approximately 27% (Δ = 12.4 ± 1.1 µA cm(-2)) and 64% (Δ = -5.3 ± 0.4 µA cm(-2)), respectively. The measured alterations in the action potential dynamics and in the chloride and calcium ion transport due to the exogenous low-dose tritium exposure provide the basis for predicting possible further impairments of plasma membrane regulatory functions, which subsequently disturb essential physiological processes of the plant cell.