Red beet (Beta vulgaris L.) vacuoles were mechanically isolated according to the method described previously by Coyaud et al. [36]. Briefly, the vacuoles were directly extruded into the recording chamber (about 1 ml in volume) by cutting a slice of fresh storage tissue and rinsing the surface with bathing solution. The control bath solution was: 100 mM KCl, 2 mM MgCl₂, 0.1 mM CaCl₂, 5 mM MES, 5 mM Tris and 400 mM sorbitol, pH 7.5 (adjusted with 0.1 N NaOH), osmolarity 656 mOsm. The standard pipette solution was: 100 mM KCl, 2 mM MgCl₂, 1 mM CaCl₂, 5 mM MES, 5 mM Tris and 340 mM sorbitol, pH 5.5, osmolarity 587 mOsm. Patch-clamp experiments were performed on the whole-vacuole and excised cytosolic side-out patch configurations using an EPC-7 Plus amplifier (List-Medical-Electronic, Darmstadt, Germany). The current and voltage convention was in accordance with Bertl et al. [37], i.e. the sign of voltage refers to the cytosolic side, and positive (outward) currents represent an efflux of cations into the vacuole. The experimental results were stored using Patch-Master software (HEKA Electronic, Lambrecht, Germany). Signal was filtered by a five-pole Bessel filter and recorded on hard disk with sampling frequency of 1 to 100 kHz. The Bessel filter was an integral part of the EPC-7 Plus amplifier. Patch pipettes were pulled from Kimax-51 (Kimble Products, Toledo, Ohio, USA) using two-stage pipette puller (model L/M-3-PA, List Medical, Germany), fire-polished with a microforge CPZ 101 (List Medical, Germany) and coated with Sylgard (Dow Corning, Midland, MI, USA). The resistance of patch electrodes filled with pipette solution was 2-4 MΩ; seal resistance was in the range 5-21 GΩ. Access to the vacuole interior was gained by breaking the membrane under the pipette with a voltage pulse in of 200 to 800 mV. Effect of Met₃PbCl on SV channels was studied. In the experiments the control bath was changed for a new one of the same salt composition, containing additionally Met₃PbCl at a final concentration of 0.1 to 100 μM. The exchange of bath solution was carried out by continuous perfusion of the measuring chamber using an infusion pump model SP200 (World Precision Instruments, USA). All experiments were carried out at room temperature (22 ± 1 °C).

Data analysis was carried out using the FitMaster (HEKA Electronic, Lambrecht, Germany) and computer software Statistical for Windows (StatSoft 2010; STATISTICA data analysis software system, version 9.1. http://www.statsoft.com, USA).

Patch-clamp experiments performed in the whole-vacuole configuration revealed a typical SV-type channel activity in vacuoles isolated from Beta vulgaris L. taproot. Macroscopic currents recorded in symmetrical 100 mM KCl and Ca²⁺ gradient (0.1 mM in the bath and 1 mM in the pipette) showed slow activation (Fig. 1A, control) and strong outward rectification of the steady-state currents (Fig. 1 C, control). SV channels were closed at negative potentials and activated at voltages more positive than +10 mV (Fig. 1A, C). The addition of Met₃PbCl to the bath solution blocked, in a concentration-dependent manner (Fig. 2), SV currents in red beet vacuoles (see also Fig. 1B, C). When Met₃PbCl at a final concentration of 1.0, 10 and 100 μM was added to the bath solution, SV currents decreased at all potentials between +40 and +100 mV (Fig. 2, insert). For example, in the whole-vacuole configuration addition of 100 μM Met₃PbCl resulted in a 60% decrease of current amplitudes (Fig. 2, insert). Fig. 3 shows the time constant τ of monoexponential function as a function of membrane voltage in control solution and in the presence of trimethyllead chloride (although activation of the channels is biphasic, we chose a monoexponetial function because the fit is satisfactory and includes a smaller number of parameters). As Fig. 3 indicates, the time constant τ increased several times in the presence of 100 μM Met₃PbCl, as compared to the control, at all voltages tested. This means that with organolead the ensemble of channels relax slowly, because the number of the transitions closed-open is smaller compared to control solution; on the macroscopic level, this may reflect a decrease in the open probability of the SV channels in the presence of trimethyllead chloride.

When single channel properties were analyzed (Fig. 4), only little channel activity could be recorded in the presence of 100 μM Met₃PbCl. The open probability of single channels (Fig. 5) decreased significantly in the presence of 100 μM Met₃PbCl. In the control solution, the open probability of single channels increased with increasing membrane voltage from +40 to +100 mV. Below 40 mV the openings, practically, were not observed in the current traces. The addition of 100 μM Met₃PbCl to the bath solution decreased the open probability of single channels by about one order of magnitude as compared to the control (Fig. 5). The single channel recordings obtained in the control solution and in the presence of Met₃PbCl showed that trimethyllead chloride slightly decreased the unitary conductance of single channels; in the presence and absence of 100 μM cytosolic Met₃PbCl the unitary conductance was about 66 ± 4 and 75 ± 6 (n > 7) pS, respectively (Fig. 6). The ratio between the two conductances was 0.88.

Using the FitMaster software, we have performed an analysis of the opening events that included the current amplitude during the open state of the channel, the level of current recording (indicating the number of SV channels in the open state at the same time) and time of opening. Fig. 7 shows the distribution of times of different states of the ensemble of four active channels recorded in control solution as a function of current level. The same dependence determined in the bath solution with 100 μM Met₃PbCl clearly showed (Fig. 8) that trimethyllead chloride decreased significantly the number of openings, as compared to control solution. However, as can be seen in Fig. 7 (insert) and Fig. 8 (insert) trimethyllead chloride did not change the opening times of the channels; the small differences between the opening times found in control solution and in the presence of 100 μM Met₃PbCl are close to statistical errors.

The slow-activating vacuolar (SV) channel is a non-selective cation channel that is activated by cytosol-positive voltages and elevated cytosolic Ca²⁺ concentrations [23,38]. The SV channel shows a characteristically slow activation and its current is predominantly outward (i.e. directed out of cytoplasm), apart from conditions when the K⁺ gradient is directed out of the vacuole into the cytosol [39–41]. Elevation of the cytosolic Ca²⁺ concentration has a dual effect on SV channel activity: an increase in the number of open SV channels at high positive potentials and a shift of the voltage dependence to less positive potentials [21,25,39 for review].

Despite extensive research over the past decade, the physiological function of SV channels is still not clear. Here, we demonstrate that under the experimental conditions of this study the vacuoles isolated from the red beet taproot are characterized by SV channels whose electrical properties, such as slow activation, outward rectification and unitary conductance, are close to those previously described under similar conditions in Beta vulgaris taproots [23,25,42]. The addition of Met₃PbCl to the bath solution blocked, in a concentration-dependent manner, SV currents in red beet vacuoles (Fig. 2). The time constant τ obtained with fitting the macroscopic SV currents by a monoexponential function, showed that addition of Met₃PbCl slowed the activation of SV channels significantly, suggesting (on macroscopic level) a decrease in the open probability of SV channels in the presence of trimethyllead chloride (Fig. 3). However, the open probability of single channels determined in cytosolic side-out configuration clearly showed that in the presence of 100 μM Met₃PbCl its value is by one order of magnitude lower as compared to the control (Fig. 5). This finding suggests that Met₃PbCl stabilizes the closed states of the channels. Taking into account that in the presence of 100 μM Met₃PbCl the unitary conductance of single channels decreased only slightly (Fig. 6) and that the ratio between the current recorded in the presence and in the absence of cytosolic Met₃PbCl did not depend on voltage in the range from +60 to +100 mV (Fig. 2), it can be suggested that trimethyllead chloride binding site is located outside the channel's selectivity filter. As mentioned above, trimethyllead acting on the whole membrane reduced the macroscopic current by about 60% (at 100 μM). However, the results obtained on the single channel level showed that the addition of the same concentration of Met₃PbCl resulted in a reduction of the open probability by approximately one order of magnitude, and a reduction of the single channel current amplitude to 88%. Inconsistency of the whole-vacuole and single channel recordings may be due to two reasons: different affinity of this compound to the membrane (channel) in the whole cell configuration and configuration patches detached (e.g, because of the presence of mechanical stress of lipid phase in the system of patches), and the second reason may be the fact that SV channels operate cooperatively in the system of whole membrane, which does not exist in the patches system.

The experiments carried out on model lipid membranes [33,43] showed that lipophylic properties of organolead compounds are crucial for modification of model membranes. It is noteworthy that experiments on the influence of organolead compounds on the model and biological membrane have shown various effects: erythrocyte hemolysis [32], change in membrane fluidity and its mechanical properties [33], modification of lipids and membrane proteins [34], influence on ion channels and membrane pores [35], and others.

In conclusion, the results presented in this article suggest that the trimethyllead chloride (Met₃PbCl) binding site is located outside the channel selectivity filter and that the inhibitory effect of the compound on SV channel activity probably results from Met₃PbCl-induced disorder in compatibility between membrane lipids and membrane proteins. This suggestion is supported by three lines of evidence:

• • Met₃PbCl only slightly decreased the unitary conductance of single channels;
• • the ratio between the current recorded with and without cytosolic Met₃PbCl did not depend on the voltage in the range +60 ± 100 mV;
• • lipophilicity of organolead compounds.

The authors declare that they have no conflicts of interest concerning this article.