Effect of Trimethyltin Chloride on Slow Vacuolar (SV) Channels in Vacuoles from Red Beet (Beta vulgaris L.) Taproots

In the present study, patch-clamp techniques have been used to investigate the effect of trimethyltin chloride (Met3SnCl) on the slow vacuolar (SV) channels in vacuoles from red beet (Beta vulgaris L.) taproots. Activity of SV channels has been measured in whole-vacuole and cytosolic side-out patch configurations. It was found that addition of trimethyltin chloride to the bath solution suppressed, in a concentration-dependent manner, SV currents in red beet vacuoles. The time constant, τ, increased significantly in the presence of the organotin. When single channel activity was analyzed, only little channel activity could be recorded at 100 μM Met3SnCl. Trimethyltin chloride added to the bath medium significantly decreased (by ca. threefold at 100 μM Met3SnCl and at 100 mV voltage, as compared to the control medium) the open probability of single channels. Single channel recordings obtained in the presence and absence of trimethyltin chloride showed that the organotin only slightly (by <10%) decreased the unitary conductance of single channels. It was also found that Met3SnCl significantly diminished the number of SV channel openings, whereas it did not change the opening times of the channels. Taking into account the above and the fact that under the here applied experimental conditions (pH = 7.5) Met3SnCl is a non-dissociated (more lipophilic) compound, we suggest that the suppression of SV currents observed in the presence of the organotin results probably from its hydrophobic properties allowing this compound to translocate near the selectivity filter of the channel.


Introduction
In the last several decades, organotin compounds have become an important pollutant due to their widespread use in industry and agriculture as fungicides, biocides, antifouling agents, wood preservatives and plastics stabilizers [1][2][3][4]. Their high toxicity was established e.g. in algae and aquatic plants [5,6]. Although their soil concentrations are low and do not usually exceed the micromolar range [7,8], organotins accumulate in plant tissues and trigger numerous changes in their functioning [9][10][11]. While inorganic tin is rather unavailable to plants and seems to be hardly toxic, its organic derivatives exhibit much stronger toxicity [12][13][14][15]. Despite the large number of papers on organotins, the precise mechanism of their toxic effect on plant cells still remains poorly understood.
Organic compounds of heavy metals, including tin, display significant activity when considering interactions with membranes. According to literature data, the toxic effect of organometallic compounds results from their lipophilic character, facilitating penetration of these radicals through cell membranes and their effect on various cell functions [13,16,17].
In order to avoid metal toxicity, plants have evolved two main mechanisms: (1) tolerance to high metal concentrations in cell cytosol and (2) sequestration of metals in vacuoles [18]. The central vacuole, in various plant cell types covering up to 90% of their volume, may be one of the most effective adaptations for deposition and inactivation of heavy metals, helping to reduce their impact on the cytoplasm, being the site of sensitive biochemical processes. Heavy metals may also change the transport systems across the vacuolar membrane (tonoplast), involving channels, carriers and pumps, which play a key role in the control of cytoplasmic homeostasis and cell osmoregulation. Major ion currents at vacuolar membranes of higher plants are mediated by non-selective cation channels: slow-activating vacuolar channels (SV channels) and fast-activating vacuolar channels (FV channels). SV channels were the first channels to be characterized in a vacuolar membrane [19]. They are ubiquitous in every cell type and plant species investigated so far [20][21][22][23]. FV channels conduct various monovalent cations with poor selectivity, while SV channels conduct K + , Na + , Mg 2+ , Ba 2+ , and Ca 2+ [21]. It has been previously shown that SV channels are regulated by pH [24,25], calmodulin [24], kinases and phosphatases [26], reducing and oxidizing agents [27], flavonoids [28], 14-3-3 proteins [29], polyunsaturated fatty acids [30], ATP [25], polyamines [31], auxin [32] as well as heavy metals, such as zinc [24], nickel [33,34] copper [35], and organolead [36,37].
The main objective of the present study was to determine the effect of trimethyltin chloride on SV channels activity in vacuoles isolated from red beet (Beta vulgaris L.) taproots. To the best of our knowledge, no research has been reported on the effects of this organotin compound on SV channels.

Material and Methods
Vacuoles were isolated from red beet (Beta vulgaris L.) taproots with a mechanical method described previously by Coyaud et al. [38]. Surface of a slice of fresh storage tissue was rinsed with bathing solution, enabling the vacuoles to be directly extruded into the recording chamber (1 ml in volume). The control bath solution used in patch-clamp experiments was: 100 mM KCl, 2 mM MgCl 2 , 0.1 mM CaCl 2 , 5 mM MES, 5 mM Tris and 400 mM sorbitol, pH 7.5 adjusted with 0.1 N NaOH, osmolality 650 mOsm. Pipettes were filled with a solution containing: 100 mM KCl, 2 mM MgCl 2 , 1 mM CaCl 2 , 5 mM MES, 5 mM Tris and 340 mM sorbitol, pH 5.5, osmolality 580 mOsm. Osmolality of all solutions was adjusted under the control of cryoscopic osmometer (Semi-Micro Osmometer K-7400, Knauer, Germany). Under symmetrical 100 mM K + and micromolar cytosolic Ca 2+ concentrations, SV channels conduct outward K + currents (into the vacuole). At Ca 2+ gradient (0.1 mM in the bath and 1 mM in the pipette) the presence of Ca 2+ current into the vacuole at depolarizing voltages is also possible. However, it is very difficult to separate K + and Ca 2+ currents through SV channels because of lack of specific inhibitors of these channels (for review see [22]). All the experiments were performed in two patch-clamp configurations: whole-vacuole and excised cytosolic side-out patch, using the EPC-7 Plus amplifier (List-Medical-Electronic, Darmstadt, Germany), as recently described by Trela et al. [37]. The convention of current and voltage was in accordance with Bertl et al. [39], i.e. the positive (outward) currents indicate an efflux of cations into the vacuole. Experimental data were stored using the Patch-Master software (HEKA Electronic, Lambrecht, Germany). Patch pipettes were pulled from borosilicate glass tubes (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). Patch electrode resistance (for electrodes filled with pipette solution) was 2-4 MO; gigaseal resistance was in the range of 5-20 GO. Access to the vacuole interior was gained by breaking the tonoplast under the pipette with a voltage pulse in the range of 300 to 900 mV.
Effects of 1, 10 and 100 μM trimethyltin (Met 3 SnCl) on SV channel activity were studied. Due to the duration of our experiments (the longest lasting 75 min) we decided to use higher tin concentrations than those present in nature. In the experiments, the control bath was changed for a new one of the same salt composition, containing additionally trimethyltin (Met 3 SnCl). The bath solution was exchanged by continuous perfusion of the measuring chamber using an infusion pump (SP200, World Precision Instruments, USA). All experiments were carried out at room temperature (22 ± 1°C). All the data stored were analyzed with FitMaster (HEKA Electronic, Lambrecht, Germany) and computer software Statistica for Windows (Stat-Soft 2010; STATISTICA data analysis software system, version 9.1., www.statsoft.com, USA). The time course of macroscopic SV currents (whole-vacuole configuration) were fitted with monoexponential function and then two obtained parameters (the steady state current and the time constant of the quasi exponential current) are discussed. In order to compare the results of several experiments, in particular assessing of the effects of trimethyltin, steady state currents were normalized to a value equal to 1 corresponding the steady state current at a membrane voltage of 100 mV in the control bath. In turn, microscopic current traces were used to determine the distribution of the open-close time of the channel and the amplitude of the opening current. Based on current records the opening-closing events characterized by two parameters: duration and amplitude (as average value of the microscopic current during event) were analyzed. Preliminary determination of the levels of the opening-closing currents were carried out by reading the peaks of current histogram (fitted with multimodal gaussian). Individual openclose events are illustrated as a scatterplot. Opening probability was calculated as the total opening time normalized to the total recording time and the number of active channels in patch.

Results
Using the patch-clamp technique, we examined the effect of trimethyltin chloride (Met 3 SnCl) on SV channel activity in red beet (Beta vulgaris L.) taproot vacuoles. Both macroscopic currents (whole-vacuole configuration) and single channel currents (cytosolic side-out configuration) were measured. In the control bath, macroscopic currents showed slow activation typical of steady-state currents (Fig 1A). Addition of Met 3 SnCl to the bath solution, at a final concentration of 100 μM, significantly reduced macroscopic currents (Fig 1B). This effect depended on the time of vacuole incubation in the presence of Met 3 SnCl. As presented in Fig 1C, the greatest changes in macroscopic SV currents, recorded within the first several minutes, were followed by stable and low current values. It is noteworthy that the effect of trimethyltin chloride was irreversible (data not shown). Reduction in macroscopic SV currents progressed with the Met 3 SnCl concentration in bath solution and attained a maximal value at 100 μM (reduction by over 80% at 100 mV, as compared to the control; Fig 2). For example, Met 3 SnCl at 1 μM, applied at the same experimental conditions as 100 μM Met 3 SnCl, diminished SV channel currents by ca. 40% (Fig 2).  fitted to the time courses of macroscopic SV currents. The constants can be interpreted as the rate of SV channel activation after application of voltage pulse. Fig 3 indicates that time constants, τ, increase with organotin concentration, suggesting slower channel activation in the presence of Met 3 SnCl. For example, at 100 μM Met 3 SnCl the time constant, τ, was twofold higher than in the control bath, independently of the applied voltage. When considering microscopic currents in the cytosolic side-out configuration (Fig 4), only little channel activity could be recorded in the presence of 100 μM Met 3 SnCl, compared to the control. This is evident in Fig 5, which shows the distribution of opening-closing events recorded on the same patch of tonoplast both in the control and in the presence of 100 μM Met 3 SnCl. All points in scatter plots (time vs current) indicate events of closing or opening (of one, two, three or four SV channels). Current records have been processed and each closing-opening event in the sequence was assigned to its duration and average current amplitude. In the presence of 100 μM trimethyltin, the number of opening events was significantly reduced, what is evidenced particularly in histograms of opening times (Fig 5C and 5D). Histogram pertaining to the effect of trimethyltin covers a lower number of channel openings (Fig 5D), however it should be noted that its maximum is not significantly shifted compared to the control (Fig 5C). Both histograms, fitted with a lognormal distribution function, displayed similar peak positions (2.32 ms, σ = 0.97 in the control and 2.46 ms, σ = 1.18 in the presence of 100 μM trimethyltin). As presented in Fig 6, activity of single SV channels and the value of macroscopic currents obtained from measurements in the whole-vacuole configuration depended on trimethyltin concentration. The open probability (calculated as the total opening time normalized to the total time of record and the observed number of active channels in the patch) decreased with trimethyltin concentration in the incubation medium (Fig 6). For instance, at a concentration of 100 μM the open probability was reduced several times compared to the control. Interestingly, trimethyltin present in the incubation medium practically did not change the currentvoltage characteristics of microscopic SV currents. Conductivity of single SV channels (slope of the current-voltage function) attained 65.1 ± 9.6 pS in the control and 62.0 ± 11.5 pS in the presence of 100 μM trimethyltin, suggesting that Met 3 SnCl did not affect the unitary conductance of channels.

Discussion
Organotin compounds, as biologically active substances interacting with the cell membrane and organelle membranes, have a direct and indirect effect on ion channel proteins [15,40,41]. They may also influence the lipid phase of membranes and cause changes in their composition and liquidity, resulting in non-specific ion flow [15,42,43]. Trimethyltin chloride (Met 3 SnCl), studied in this work, is well known mainly as a potent neurotoxin. It triggers many neurodegenerative processes, including loss of neurons [44][45][46], as well as other degenerative membrane processes [47]. Trimethyltin has been also shown to decrease intracellular pH, open K + channels and directly inhibit the activity of H + /K + -ATPase in renal intercalated cells [44]. It should be also added that in HeLa cells Met 3 SnCl disrupted cytosolic calcium homeostasis and produced elevated calcium transients [48,49]. While on molecular level the toxic effect of organometals occurs at relatively low concentrations (10 −7 to 10 −6 M), the toxicity on cellular level manifested by changes in mechanical and physicochemical properties of cell walls and membranes appears at much higher concentrations of tin [50,51]. Red beet vacuoles serve as a model for patch-clamp experiments which are crucial for understanding vacuole-related functions of plant cells. An important function of the vacuolar membrane (tonoplast) is the cytoplasm-vacuole transport of metabolites and ions [23,52,53], including the particularly important Ca 2+ ions, involved in intracellular signalling [21,54,55] as well as sequestration of xenobiotics and heavy metals [56]. The last-mentioned are generally harmful substances, inducing free-radical processes and destruction of cell structure [56]. One of the most frequently examined transport systems across the tonoplast is the slow-activating vacuolar (SV) channel. Although SV channels have been well described (reviewed in [22,23]), their physiological function in vivo remains poorly understood. So far, it has been well established that in vitro SV channels are activated by positive voltages and micromolar cytosolic Ca 2+ concentrations [19]. Both SV and FV channels play an important function in the maintenance of Effect of Organotin on Slow Vacuolar (SV) Channels in Red Beet potassium homeostasis, as by operating K + -sensing valves they control K + distribution between vacuole and cytosol (recently reviewed in [57]).
The herein studied trimethyltin chloride irreversibly suppressed the macroscopic outward current in the tonoplast of Beta vulgaris taproots (Figs 1 and 2). Significant loss of macroscopic SV current occurred after several minutes of vacuole incubation in a medium containing trimethyltin. This observation may indicate either that Met 3 SnCl acts directly as a channel blocker or that it indirectly alters the kinetics of transition between the closed and open state of channel. Analysis of the kinetics of relaxation of macroscopic currents may be a source of information on channel gating. For voltage-dependent ion channels, at certain voltage the current reflects the distribution of the number of open channels to the total number of channels in the population. Stepping the membrane voltage (according to the measurement protocol) enforces relaxation of current among the various steady-state currents.
The time constant, τ, calculated by fitting macroscopic SV currents with a monoexponential function, showed that trimethyltin present in medium caused slowing of the macroscopic currents (ca. twofold) as compared to the control (Fig 3). Suppressed macroscopic SV current combined with slowed-down relaxation reflect the lower open probability of channels in the presence of organotin (Fig 4). Scatter plot of opening-closing events in the control and in the presence of trimethyltin chloride (Fig 5) shows a much lower number of openings after incubation in a medium containing the organometal. Trimethyltin changes the kinetics of the channel, however practically does not alter its electrical conductivity (Fig 7), what may suggest an indirect effect of Met 3 SnCl on the channel. The mechanism of SV channel modulation by trimethyltin chloride seems to be explained by its lipophilicity, especially that in the pH of the incubation medium used in the experiments (pH = 7.5) the non-dissociated (more lipophilic) Effect of Organotin on Slow Vacuolar (SV) Channels in Red Beet form of the compound was dominant [58][59][60]. Experiments carried out on erythrocyte membranes, serving as model biological membranes, and on model lipid membranes showed that organic compounds of heavy metals, including tin, indeed affect membranes primarily due to their lipophilic properties [13, 15-17, 51, 61-63]. Such features of organotin compounds cause them to concentrate in the lipid phase of membranes and consequently disturb the interaction between lipids and proteins and therefore indirectly diminish SV channel activity. Similar results were obtained in our previous experiments, performed with the same plant material as here and concerning the effect of an organolead compound (trimethyllead chloride) on SV channels activity [37]. Both trimethyllead chloride and trimethyltin chloride significantly reduced the activity of SV channels, however causing only a small effect on their unitary conductance. This in turn may suggest that the Met 3 SnCl binding site, similarly to Met 3 PbCl [37], is located outside the channel selectivity filter. Interestingly, binding of the organotin compound near the selectivity filter of the ion channel of F-ATP synthase was also previously proposed by von Ballmoos et al. [64]. In agreement with their hypothesis hydrophobic organotin compounds accumulate within the membrane and easily penetrate into the entrance of the channel were they interact with a site near the selectivity filter disabling incoming ions to shed their hydration shell. Such scenario would be also possible in the case of our experiments on SV channels with Met 3 SnCl.
In conclusion, results presented here suggest that the suppression of SV currents by trimethyltin chloride arises probably from its hydrophobic properties allowing this compound to translocate near the selectivity filter of the channel.