Vacuolar ion channels in the liverwort Marchantia polymorpha: influence of ion channel inhibitors

Abstract

Potassium-permeable slow activating vacuolar channels (SV) and chloride-permeable channels in the vacuole of the liverwort Marchantia polymorpha were characterized in respect to calcium dependence, selectivity, and pharmacology. The patch-clamp method was used in the study of ion channel activity in the vacuoles from the liverwort Marchantia polymorpha. The whole-vacuole recordings allowed simultaneous observation of two types of currents-predominant slow activated currents recorded at positive voltages and fast activated currents recorded at negative voltages. Single-channel recordings carried out in the gradient of KCl indicated that slow activated currents were carried by potassium-permeable slowly activating vacuolar channels (SV) and fast activated currents-by chloride-permeable channels. Both types of the channels were dependent in an opposite way on calcium, since elimination of this ion from the cytoplasmic side caused inhibition of SV channels, but the open probability of chloride-permeable channels even increased. The dependence of the activity of both channels on different types of ion channel inhibitors was studied. SV channels exhibited different sensitivity to potassium channel inhibitors. These channels were insensitive to 3 mM Ba²⁺, but were blocked by 3 mM tetraethyl ammonium (TEA). Moreover, the activity of the channels was modified in a different way by calcium channel inhibitors. 200 µM Gd³⁺ was an effective blocker, but 50 µM ruthenium red evoked bursts of the channel activity resulting in an increase in the open probability. Different effectiveness of anion channel inhibitors was observed in chloride-permeable channels. After the application of 100 µM Zn²⁺, a decrease in the open probability was recorded but the channels were still active. 50 µM DIDS was more effective, as it completely blocked the channels.

Keywords: Calcium dependence; Chloride channels; Patch-clamp; Pharmacology; SV channels; Vacuole.

Fig. 1

Activity of ion channels recorded in the whole-vacuole configuration. a Recordings obtained in the symmetrical concentration of 100 mM KCl (the pipette contained 100 mM KCl, 0.1 mM CaCl₂, 2 mM MgCl₂, pH 5.5 buffered by Hepes/TRIS, and the bath—100 mM KCl, 0.1 mM CaCl₂, 2 mM MgCl₂, pH 7.2 buffered by Mes/TRIS). Recordings were obtained by application of 0.5 s holding voltage (0 mV), then 3 s test voltages with 20 mV steps from 100 to −100 mV, and 0.3 s pulse (0 mV) after the test voltage. b Tail currents recorded in the gradient of KCl obtained after reduction of KCl in the pipette to 10 mM (the pipette contained 10 mM KCl, 0.1 mM CaCl₂, 2 mM MgCl₂, pH 5.5 buffered by Hepes/TRIS). Recordings were obtained by application of 0.5 s holding voltage (0 mV), then 2 s voltage which activated SV channels (80 mV), and 1 s test voltages with 10 mV steps from −60 to 60 mV. The reversal potential (dashed line) and the equilibrium potential for K⁺ and Cl⁻ (arrows) are indicated. c J/V curves obtained in the symmetrical concentration of 100 mM KCl (the same conditions as in a, closed circles and solid line, n = 6) and after reduction of the KCl concentration in the pipette to 10 mM (the same conditions as in b, open circles and dashed lines, n = 7)

Fig. 2

Activity of ion channels recorded in the cytoplasm-out configuration. a Recordings obtained in the gradient of KCl (the pipette contained 10 mM KCl, 0.1 mM CaCl₂, 2 mM MgCl₂, pH 5.5 buffered by Hepes/TRIS, and the bath—100 mM KCl, 0.1 mM CaCl₂, 2 mM MgCl₂, pH 7.2 buffered by Mes/TRIS). Solid lines indicate the close state, and dashed lines—open states of the channels. The holding voltages are placed on the left side of the traces. b Recordings obtained at the same patch after replacement of 100 mM KCl in the bath by 100 mM HCl (100 mM HCl, 0.1 mM CaCl₂, 2 mM MgCl₂, pH 7.2 buffered by BIS–TRIS Propane). c Recordings obtained after replacement of 100 mM HCl in the bath by 100 mM K-gluconate (100 mM K-gluconate, 0.1 mM CaCl₂, 2 mM MgCl₂, pH 5.5 buffered by Hepes/TRIS. d I/V curves obtained in the same conditions as in a. Close circles and the solid line denote currents flowing through SV channels (n = 10), and open circles and the dashed line denote currents flowing through chloride channels (n = 11)

Fig. 3

Calcium dependence of SV channels (a) and chloride-permeable channels (b). The cytoplasm-out recordings were obtained at the same patch at +40 mV (a) and −60 mV (b). The traces described as 0.1 Ca²⁺ were obtained in the same conditions as in Fig. 2a. The amplitude histograms indicating the current amplitude (horizontal line) and the number of sample points (vertical line) correspond to the traces obtained in standard conditions (upper histogram) and after elimination of cytoplasmic calcium by application of 2 mM EGTA (lower histogram).The diagonal dashed lines indicate open states and the solid line—close states of the channels. The values of the current amplitude and the open probability (Po) were indicated

Fig. 4

Influence of potassium channel inhibitors, BaCl₂ (a) and TEA (b) on the activity of SV channels. The standard conditions were the same as in Fig. 2a. The cytoplasm-out recordings were obtained at the same patch at +40 mV. The amplitude histograms correspond to the traces obtained in standard conditions (upper histogram) and after application of the inhibitor to the bath solution (lower histogram)

Fig. 5

Influence of chloride channel inhibitors, ZnCl₂ (a) and DIDS (b) on the activity of chloride channels. The standard conditions were the same as in Fig. 2a. The cytoplasm-out recordings were obtained at the same patch at −60 mV

Fig. 6

Influence of calcium channel inhibitors, GdCl₃ (a) and ruthenium red (b) on the activity of SV channels. The standard conditions were the same as in Fig. 2a. The cytoplasm-out recordings were obtained at the same patch at +40 mV. The event detection analysis placed at the bottom of b was based on the presented traces