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TOXICOLOGY

Inorganic Mercury and Methylmercury Inhibit the Ca_v3.1 Channel Expressed in Human Embryonic Kidney 293 Cells by Different Mechanisms

Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovakia

Received September 9, 2005; accepted November 30, 2005.

	Abstract

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Part of the neurotoxic effects of inorganic mercury (Hg²⁺) and methylmercury (MeHg) was attributed to their interaction with voltage-activated calcium channels. Effects of mercury on T-type calcium channels are controversial. Therefore, we investigated effects of Hg²⁺ and MeHg on neuronal Ca_v3.1 (T-type) calcium channel stably expressed in the human embryonic kidney (HEK) 293 cell line. Hg²⁺ acutely inhibited current through the Ca_v3.1 calcium channel in concentrations 10 nM and higher with an IC₅₀ of 0.63 ± 0.11 µM and a Hill coefficient of 0.73 ± 0.08. Inhibition was accompanied by strong deceleration of current activation, inactivation, and deactivation. The current-voltage relation was broadened, and its peak was shifted to a more depolarized membrane potentials by 1 µM Hg²⁺. MeHg in concentrations between 10 nM and 100 µM inhibited the current through the Ca_v3.1 calcium channel with an IC₅₀ of 13.0 ± 5.0 µM and a Hill coefficient of 0.47 ± 0.09. Low concentration of MeHg (10 pM to 1 nM) had both positive and negative effects on the current amplitude. Micromolar concentrations of MeHg reduced the speed of current activation and accelerated current inactivation and deactivation. The current-voltage relation was not affected. Up to 72 h of exposure to 10 nM MeHg had no significant effect on current amplitude, whereas 72-h-long exposure to 1 nM MeHg increased significantly current density. Acute treatment with Hg²⁺ or MeHg did not affect HEK 293 cell viability. In conclusion, interaction with the Ca_v3.1 calcium channel may significantly contribute to neuronal symptoms of mercury poisoning during both acute poisoning and long-term environmental exposure.

Direct interaction with voltage- and ligand-gated ion channels may contribute to toxic effects of both methylmercury and inorganic mercury. Inhibition of neuronal sodium, potassium, or calcium channels alters action potential shape and may result in the observed neuronal symptoms. Indeed, it was shown that an acute exposure to 1 µM or more of MeHg reduced potassium currents of cultured dorsal root ganglion cells (Leonhardt et al., 1996a). At least 10 µM Hg²⁺ was necessary for inhibition of potassium channels in outer hair cells (Liang et al., 2003). Sodium channels of cultured dorsal root ganglion cells were blocked by MeHg in concentrations above 10 µM (Leonhardt et al., 1996a). Among neuronal voltage-dependent ion channels, voltage-dependent calcium channels are the most sensitive to mercury being acutely affected by its nanomolar concentrations (Shafer et al., 2002).

Effects of inorganic and/or organic mercury on voltage-dependent calcium channels were investigated in both native cell preparations and in recombinant expression systems. Results reported so far are partly controversial. Nanomolar concentrations of inorganic mercury increased the amplitude of high-voltage-activated (HVA) calcium current, presumably an L-type, in rat PC12 cells (Rossi et al., 1993). Micromolar concentrations of Hg²⁺ transiently increased low-voltage-activated (LVA) calcium current in rat hippocampal pyramidal cells (Szücs et al., 1997). In dorsal root ganglion (DRG) neurons and in Aplysia neurons, micromolar Hg²⁺ inhibited L-, N-, and T-type calcium currents (Pekel et al., 1993).

MeHg was shown to inhibit both HVA and LVA calcium channels in native preparations. In rat hippocampal neurons, HVA channels were less sensitive than LVA channels (Szücs et al., 1997). Although some authors reported shift of current-voltage (I-V) relation by MeHg in the depolarizing direction in DRG neurons (Leonhardt et al., 1996a,b), other authors did not observe any voltage dependence of current inhibition in granule cells (Sirois and Atchison, 2000). In addition to its effects on voltage-gated ion channels, inorganic mercury induced a background current in DRG neurons (Pekel et al., 1993), and organic mercury did so in rat Purkinje cells (Yuan and Atchison, 2005).

It is difficult to separate individual calcium channel types in native preparations. Therefore, some authors investigated subtype-specific action of mercury on recombinant channels in an expression system. Until now, only the effects of mercury on recombinant HVA calcium channels were investigated. Micromolar concentrations of MeHg inhibited the current through the Ca_v1.2 (L-type) calcium channel transiently expressed in human embryonic kidney (HEK) 293 cells (Peng et al., 2002). Currents through the Ca_v2.2 (N-type) and Ca_v2.3 (R-type) calcium channels were inhibited by comparable concentrations of both Hg²⁺ and MeHg in the same expression system (Hajela et al., 2003).

Interaction of mercury with recombinant T-type calcium channels was not yet investigated. These channels are highly expressed in various neuronal tissues (for review, see Lacinova, 2004), where they contribute to neuronal excitability; e.g., they generate low-threshold calcium spikes or initiate burst firing. Therefore, their modulation by mercury may significantly add to neuronal symptoms of mercury poisoning. In this study, we have compared the effects of Hg²⁺ and MeHg on Ca_v3.1 (T-type) calcium channel stably expressed in HEK 293 cells. Hg²⁺ was slightly more effective than MeHg in current inhibition. In addition, it affected the shape of I-V relation and decelerated kinetics of current gating. Furthermore, micromolar concentrations of inorganic mercury induced unspecific background current in HEK 293 cells. Effects of MeHg on current amplitude were more complex. Nanomolar concentrations caused both activation and inhibition of current amplitude. Organic mercury decelerated kinetics of channel activation, whereas inactivation and deactivation were accelerated. The shape of I-V relation was not altered. Chronic application of 10 nM MeHg caused minor increase in average current density. However, both forms of mercury did not have cytotoxic effects on HEK 293 cells.

	Materials and Methods

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Cell Culture. Experiments were carried out on HEK 293 cells stably expressing the Ca_v3.1 subunit of T-type Ca²⁺ channel (CACNA1G; accession no. AJ012569 [GenBank] , European Molecular Biology Laboratory database). Construction of the expression vector was described previously (Klugbauer et al., 1999). HEK 293 line was purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH (Braunschweig, Germany). The cells were grown in minimal essential medium (MEM) with Earle's salts, containing 10% (v/v) fetal calf serum, 100 U/ml penicillin-streptomycin, and 0.04% (w/v) G418 (Geneticin; Duchefa Biochemie, Haarlem, The Netherlands) at 37°C in a humidified atmosphere of air/CO₂ 95:5. The cells were harvested from their culture flasks by trypsinization and plated out 24 to 48 h before use in electrophysiological experiments. Background current activated by Hg²⁺ was analyzed in nontransfected HEK 293 cells. Nontransfected cells were cultured as described above, except that G418 was absent in culture media.

Whole-Cell Ca²⁺ Current Recording. The extracellular solution contained 160 mM NaCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, and 5 mM glucose, pH 7.4 (NaOH). The intracellular solution contained 130 mM CsCl, 1 mM EGTA, 1 mM MgCl₂, 10 mM tetra-ethyl ammonium chloride, 10 mM HEPES, and 5 mM Na-ATP, pH 7.4 (CsOH). All chemicals were obtained from Sigma-Aldrich (St. Louis, MO). Osmolarity of the internal solution was approximately 300 mOsm. Osmolarity of the external solution was adjusted by adding glucose so that its final value was 2 to 3 mOsm below that of the internal solution. One molar stock solutions of both HgCl₂ and MeHg were prepared in deionized water every week, stored at 4°C, and diluted in a bath solution prior to the experiment. Deionized water was made by Millipore Elix Water Purification Systems (Millipore Corporation, Billerica, MA). Extracellular solutions were exchanged by a gravity-driven flow system with manually controlled valves.

Ionic currents were recorded in the whole-cell configuration of the patch-clamp method using the HEKA-10 patch-clamp amplifier (HEKA Electronic, Lambrecht, Germany). Patch-clamp pipettes were manufactured from borosilicate glass with the input resistance ranging from 1.8 to 2.1 M. The capacitance of individual cells ranged between 10 and 22 pF. The series resistance ranged from 2.5 to 5 M. Both capacitance and series resistance were compensated by built-in circuits of the HEKA-10 amplifier. The bath was grounded using an AgCl pellet connected to the experimental chamber through an agar bridge.

The holding potential (HP) in all experiments was –100 mV. The effect of mercurial salts was investigated using series of 40-ms-long depolarizing pulses applied from the HP to the membrane potential of –30 mV with a frequency of 0.2 Hz. Current-voltage relations were measured by pulse protocol or by ramp protocol. Pulse protocol represented a series of 40-ms-long depolarizing pulses applied every 3 s from the HP to membrane potentials between –70 and +70 mV. Ramp protocol represented series of 100-ms-long linear voltage ramps from –80 to +20 mV repeated every 3 s.

Cell Survival Rate Tested with 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl Tetrazolium Bromide Assay. Nontransfected HEK 293 cells were plated onto 96-well plates (1 x 10⁴ cells/well) and cultured overnight to allow for cell attachment. Cells were then incubated with control MEM, MEM containing 10 µM MeHg, or MEM containing 1 µM Hg²⁺ for 10 min or for 4 h. After incubation, cells were centrifuged, and 200 µl of fresh MEM containing 10 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (5 mg/ml) was added and incubated for further 3 h and centrifuged again. The supernatant was discarded, and the pellet was dissolved in 150 µl of dimethyl sulfoxide. The optical density at 540 nm was recorded on a MicroQuant Microplate Spectrophotometer (Biotek, Winooski, VT). Cell viability was determined relative to untreated controls.

Detection of Cell Viability and Apoptotic Cells. Cell viability and apoptosis were measured by annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining using the Annexin V-FITC Apoptosis Detection Kit (Sigma-Aldrich). Approximately 1 x 10⁶ nontransfected HEK 293 cells/ml were incubated in the control medium, in a medium with 10 µM MeHg, or in medium with 1 µM Hg²⁺ for 10 min or for 4 h. After the indicated time, cells were harvested, washed twice with phosphate-buffered saline, and stained with annexin V-FITC and PI according to manufacturer's instructions. The cells were then analyzed using a Coulter Epics Altra flow cytometer (Beckman Coulter, Fullerton, CA).

Data Analysis. Data were recorded using a HEKA Pulse 8.5 program and analyzed with HEKA Pulsefit 8.5 and Origin 7.5 software. Capacity transients and series resistance were compensated on-line by procedures built in the EPC 10 amplifier. The currents measured during Hg²⁺ application were corrected for linear time-independent component of the leak current, which was calculated individually for each current trace according to eq. 1:

where I_sub is leak-subtracted current, I(V_membr) is nonsubtracted current measured at the membrane potential V_membr, I(h) is the average membrane current measured during the holding potential of –100 mV, and V_membr is the membrane potential.

Concentration dependencies were fitted by the Hill eq. 2:

where I and I₀ are calcium current amplitudes measured in the presence and absence of mercury, respectively, [mercury] is the concentration of particular form of mercury, IC₅₀ is the half-maximal inhibitory concentration, and n is the Hill coefficient. Significance of the observed effects was assessed by paired or nonpaired Student's t test, as appropriate. Values of p < 0.05 were considered to be significant. All experimental values are expressed as mean ± S.E.M.

	Results

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Background Current Activated by Inorganic Mercury. During initial experiments with Hg²⁺, rapid increase of a background current in the presence of micromolar mercury concentration was observed. For clear definition of experimental conditions, an analysis of this phenomenon was necessary. Nontransfected HEK 293 cells were used for these experiments. Amplitude of the leak current was concentration dependent and started to increase immediately after application of suprathreshold concentrations of Hg²⁺. The current was activated by the ramp protocol. Small constant leak current with an average amplitude bellow 5 pA/pF at a membrane potential of –80 mV was observable in cells exposed to constant flow of the control solution (data not shown) or to 10 nM Hg²⁺ (Fig. 1a). The threshold concentration for an activation of the Hg²⁺-dependent background current was 100 nM. This concentration caused gradual increase in the membrane current amplitude, which started almost immediately after switching to perfusion by Hg²⁺-containing solution and saturated after 200 s. At higher concentrations, no saturation of the background current amplitude was reached during 6-min-long application. I-V relation measured by voltage ramp protocol (Fig. 1b) revealed a purely linear character of the membrane current measured under the control conditions. The linear I-V is characteristic for a nonspecific leak current. In the presence of 1 µM Hg²⁺, the I-V relation was strongly outwardly rectifying. Such voltage course is characteristic for current carried through voltage-dependent ion channels. Nevertheless, the I-V relation in the presence of Hg²⁺ retained linear voltage course for membrane voltages between –80 and –20 mV. Background currents were essentially time-independent under the control conditions and in the presence of Hg²⁺ (Fig. 1, c and d). Therefore, it was justifiable to use the linear leak subtraction method for the analysis of currents activated by depolarizing pulses to –30 mV.

View larger version (35K): [in this window] [in a new window]   Fig. 1. Background current induced by inorganic mercury in nontransfected HEK 293 cells. The current was activated by 100-ms-long voltage ramps from the membrane potential of –80 to +80 mV applied each 3 s. a, current amplitudes were evaluated from voltage ramps at a potential of –80 mV. Hg2+ application is marked by an arrow. The following number of cells was averaged for individual Hg2+ concentrations: 10 nM, n = 3; 100 nM, n = 5; 1 µM, n = 8; 10 µM, n = 4. b, I-V relations measured by voltage ramp protocol under the control conditions and after 200 s of presence of 1 µM Hg2+. c, current traces measured in the control bath solution by a series of 100-ms-long depolarizing pulses from an HP of –100 mV to voltages between –70 and +50 mV. d, current traces measured after 200 s of presence of 1 µM Hg2+ by the same voltage protocol as in c.

Hg²⁺ Inhibits Calcium Current through the Ca_v3.1 Channel in a Concentration-Dependent Manner. Inorganic mercury in concentrations ranging from 10 nM to 100 µM inhibited the calcium current through the expressed Ca_v3.1 channels (Fig. 2, a–d). All analyses of experimental data were performed on recordings corrected for linear leak component. Effect of 10 nM Hg²⁺ was negligible. Inhibition of the current amplitude increased with increasing Hg²⁺ concentration and was nearly complete at a concentration of 100 µM. The block was virtually irreversible at low concentrations but was partly reversible at higher concentrations. At a concentration of 100 µM, the reversibility of Hg²⁺ effect was not tested because of rapid increase in background current, which eventually led to loss of the proper whole-cell clamp. Fit of experimental data by Hill equation resulted in an IC₅₀ = 0.63 ± 0.11 µM and a Hill coefficient of n = 0.73 ± 0.08.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Inhibition of the Cav3.1 calcium channel by inorganic mercury. The current was activated by a train of depolarizing pulses applied from an HP of –100 to –30 mV with a frequency of 0.2 Hz. Hg2+ application is marked by filled symbols and an arrow. Starts of Hg2+ washout are marked by filled symbols. Numbers of cells averaged for individual Hg2+ concentrations are as follows: a, 10 nM, n = 4; 100 nM, n = 6; 1 µM, n = 8; b, 10 µM, n = 5; 100 µM, n = 6. c, examples of current traces measured during pulses marked in a and b by filled symbols in the absence (solid line) or presence (dashed line) of Hg2+. The linear component of the leak current is subtracted from the raw data. d, concentration dependence of the inhibition of current amplitude. Solid line represents a fit of experimental data by the Hill equation with an IC50 of 0.63 ± 0.11 µM and a Hill coefficient of 0.73 ± 0.08.

Hg²⁺ Slows Kinetics of the Calcium Current through the Ca_v3.1 Channel and Alters Its Voltage Dependence. Visual inspection of current recordings presented in Fig. 2c suggested that, in addition to amplitude inhibition, Hg²⁺ slowed all processes underlying the gating of the Ca_v3.1 channel, i.e., activation, inactivation, and deactivation. To quantify this phenomenon, current trace was fitted by Hodgkin-Huxley equation in the m2h form. The threshold concentration for all changes in channel kinetics was 100 nM. The activation time constant increased with increasing Hg²⁺ concentration (Fig. 3a). The inactivation time constant increased to such an extent that, at the concentration of 10 µM, the Hg²⁺ current was virtually noninactivating, and current traces could not be satisfyingly fitted (Figs. 2c and 3b). A single exponential curve fit the time course of the tail current. In contrast to activation and inactivation time constants, an increase in time constants of current deactivation saturated at the concentration of 1 µM Hg²⁺ (Fig. 3c).

View larger version (21K): [in this window] [in a new window]   Fig. 3. Hg2+ slowed the kinetics of the currents through the Cav3.1 channel. Time constants were evaluated from the data presented in Fig. 2. Open bars represent time constant calculated from the current traces recorded just before Hg2+ application. Filled bars correspond to the time constants calculated from the current traces recorded just before the start of Hg2+ washout. Time constants of current activation and inactivation were evaluated from the fit of current traces by m2h form of the Hodgkin-Huxley equation. a, time constants of current activation (act). b, time constants of current inactivation (inact). c, deactivation time constants (deact) were estimated from monoexponential fits of tail currents. The significance of difference between two data sets (control versus Hg2+) was evaluated by the paired Student's t test. **, p < 0.01; ***, p < 0.001.

Effect of Hg²⁺ on the position of the peak of I-V relation and on the shape of I-V relation was investigated using a 100-ms-long voltage ramp. Ramp protocol was preferred to a series of depolarizing pulses in these experiments. As demonstrated in Fig. 1, higher concentrations of Hg²⁺ caused a rapid increase in the background current; therefore, subtraction of the linear leak component provided more credible results when applied to rapid ramp protocol. Hg²⁺ (100 nM) did not shift the peak or altered the shape of I-V relation (data not shown). Concentration of 1 µM shifted the peak of I-V to more depolarized voltages and increased its width (Fig. 4, a and b). At higher Hg²⁺ concentrations, rapid increase in the background current precluded analysis of the I-V relation.

View larger version (11K): [in this window] [in a new window]   Fig. 4. Hg2+ shifted the I-V relation of the current through the Cav3.1 channel. I-V relations were measured by a series of 100-ms-long voltage ramps from the membrane potential of –80 to +20 mV. a, currents recorded just before application of 1 µM Hg2+ (solid line) and after 150 s of its presence (dashed line). b, for a better comparison, traces from a were normalized to the maximal current amplitude.

View larger version (32K): [in this window] [in a new window]   Fig. 5. Inhibition and potentiation of the current through the Cav3.1 channel by MeHg. The current was activated by a train of depolarizing pulses applied from an HP of –100 to –30 mV with a frequency of 0.2 Hz. a, time course of the inhibition of current amplitude by intermediate to high MeHg concentrations. An arrow marks the moment of Hg2+ application. Numbers of cells averaged for individual Hg2+ concentrations are as follows: 100 nM, n = 10; 1 µM, n = 11; 10 µM, n = 6; 100 µM, n = 6. b, examples of current traces measured during a train of depolarizing pulses in the absence (solid line) or presence (dashed line) of each concentration of MeHg. c, an example of the current inhibition by 100 pM MeHg. Solid line indicates the presence of MeHg in the bath solution. The moments of MeHg application and washout are indicated by filled symbols. Examples of current traces are shown in the inset, and number indicates the times when they were recorded. d, an example of the current potentiation by 100 pM MeHg. Solid line indicates the presence of MeHg in the bath solution. The moments of MeHg application and washout are indicated by filled symbols. Examples of current traces are shown in the inset, and numbers indicate the time points when they were recorded. e, an example of a dual effect of the low concentration of MeHg on the current amplitude. Solid line indicates the presence of MeHg in the bath solution. The points of MeHg application and washout are indicated by filled symbols. Examples of current traces are shown in the inset, and numbers indicate the time points when they were recorded. f, concentration dependence of the inhibition and potentiation of current amplitude. Open symbols represent positive effect of MeHg on the calcium current amplitude, and filled symbols represent current inhibition. For 100 pM MeHg, activation and inhibition were evaluated for distinct cells, and for 10 pM and 1 nM, both effects were estimated on the same cells. Numbers written next to each point represent the numbers of cells averaged. Dashed lines are simple connectors of experimental data. Solid line represents a fit of inhibition data by the Hill equation. Scale bars in all panels represent 10 ms (horizontal) and 500 pA (vertical).

View larger version (23K): [in this window] [in a new window]   Fig. 6. MeHg slowed the kinetics of the current activation and accelerated its inactivation and deactivation. Time constants were calculated from the data presented in Fig. 5a just before MeHg application (open bars) and just before the start of MeHg washout (filled bars). a, time constants of current activation (act) were evaluated from the fit of current traces by m2h form of the Hodgkin-Huxley equation. b, time constants of current inactivation (inact) were evaluated by the same method as in a. c, deactivation time constants (deact) were estimated from monoexponential fits of tail currents. The significance of difference between two data sets was evaluated by the paired Student's t test. **, p < 0.01; ***, p < 0.001.

View larger version (12K): [in this window] [in a new window]   Fig. 7. MeHg does not affect the shape of I-V relation. a, I-V relations were measured by series of depolarizing pulses from an HP of –100 mV to indicated voltages before (open symbols, control conditions, n = 10) and after (filled symbols, 10 µM MeHg, n = 10) pulse protocols presented in Fig. 5a. b, data from a were normalized to a peak amplitude of each I-V relation to facilitate the comparison.

View larger version (20K): [in this window] [in a new window]   Fig. 8. Chronic application of MeHg has minor effects on the current through the Cav3.1 calcium channel. a, I-V relations were measured by series of depolarizing pulses from an HP of –100 mV to indicated voltages. Open symbols represent an averaged I-V from 15 cells cultured for 72 h under control conditions. Filled symbols represent an averaged I-V from 15 cells cultured for 72 h in the presence of 1 nM MeHg. b, data from a were normalized to peak amplitude of each I-V relation to facilitate the comparison. c, averaged current densities measured from 15 cells during depolarizing pulses from HP of –100 to –30 mV (the peaks of I-V relations shown in a and b). Cells were cultured in parallel in Dulbecco's modified Eagle's medium (open bars) and in the presence of 1 (hatched bars) or 10 (filled bars) nM MeHg in Dulbecco's modified Eagle's medium. The significance of difference between two data sets was evaluated by the unpaired Student's t test. **, p < 0.01.

Effects of Hg²⁺ and MeHg on Cell Viability. Rapid increase in background current observed upon cell exposure to Hg²⁺ could be explained by cytotoxic effects of mercury, leading to an increase in membrane permeability and eventually to the cell death. Viability of HEK 293 cells stably expressing the Ca_v3.1 channel was determined using the colorimetric MTT test. As shown in Fig. 9, 10-min-long treatment with 10 µM MeHg or 1 µM Hg²⁺ did not cause significant increase in cell death. After 4 h of incubation, a significant (p < 0.001) increase in the cell death was observed in the cells treated with 1 µM Hg²⁺ but not in those treated with 10 µM MeHg.

View larger version (52K): [in this window] [in a new window]   Fig. 9. Effect of Hg2+ and MeHg on the viability of HEK 293 cells. HEK 293 cells stably transfecting the Cav3.1 channel were treated with the control MEM, 1 µM Hg2+, and 10 µM MeHg for 10 min and 4 h, and the viability was assessed by MTT assay. Results are expressed as an absorbance measured at 540 nM in arbitrary units and represent the mean ± S.E.M. of six measurements. The significance of difference between two data sets was evaluated by the unpaired Student's t test. ***, p < 0.001.

View larger version (56K): [in this window] [in a new window]   Fig. 10. Hg2+ and MeHg do not activate apoptosis or necrosis of HEK 293 cells. FACS dot plots of cells maintained in MEM (a), cells exposed to MEM with 1 µM Hg2+ (b), and cells exposed to MEM with 10 µM MeHg (c) for 10 min (left column) or for 4 h (right column). Apoptotic cells were stained by annexin V-FITC conjugate (y-axes) and necrotic cells by propidium iodide (x-axes). Each panel was divided into four quadrants: bottom left, viable cells; bottom right, necrosis; top right, early apoptosis; top right, late apoptosis and necrosis.

	Discussion

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Both mercurials inhibited the current through the expressed Ca_v3.1 calcium channel in concentrations above 10 nM. Inorganic mercury was an approximately 20-fold more potent inhibitor of T-type calcium current than methylmercury. An IC₅₀ of 0.63 µM found in our study for Hg²⁺ corresponds to that reported by the groups of Pekel and Leonhardt (Pekel et al., 1993; Leonhardt et al., 1996b) for T-type current in rat DRG neurons. IC₅₀ close to 1 µM Hg²⁺ is also similar to those reported for HVA calcium channels (for review, see Atchison, 2003). In contrast, Szücs et al. (1997) found dual effect of Hg²⁺ on low-voltage-activated calcium channel in cultured rat hippocampal neurons. His study identified the type of observed calcium channel solely on the basis of its activation potential. The potentiating effect of Hg²⁺ in hippocampal cells might have been mediated by another subtype of LVA calcium channel, i.e., Ca_v3.2 or Ca_v3.3, whose expression level in rat hippocampus is high (for review, see Lacinova, 2004). Alternatively, potentiation seen by these authors may be mediated by HVA calcium channels, e.g., Ca_v2.3 or Ca_v1.3, which could be activated at voltages of –20 and –10 mV, used in Szücs' experiments. This interpretation is supported by the report of Rossi et al. (1993), who found an enhancement of calcium current by Hg²⁺ in this depolarization range in PC12 cells and attributed it to the effect on L-type calcium channel.

In our study, only the MeHg had dual effect on calcium current through the Ca_v3.1 channel. This effect was observed at picomolar concentrations and was combined with an inhibitory effect. Similar observation was made by Szücs in hippocampal neurons. In these experiments, a micromolar concentration of MeHg was used (Szücs et al., 1997). The shift in concentration dependence of MeHg effect could be explained, as we suggested in the case of Hg²⁺ effect, by different subtype of LVA calcium channel. Additionally, 10 mM Ca²⁺ was used in Szücs' experiments, whereas we have used 2 mM Ca²⁺. If calcium competes with mercury for binding site in the channel, enhanced calcium concentration would shift the dose-response curve toward higher concentrations. Increase in MeHg concentration at which current potentiation could be seen (1000-fold) is too big to consider solely this effect as an explanation. Therefore, subtype specificity may be a more important factor.

Increase of calcium current in the presence of low concentrations of MeHg is unique to LVA calcium channels. No such effect was observed in HVA calcium channels (Atchison, 2003). Because of its transient nature during acute application, this effect may be of importance during chronic environmental exposure rather than during acute poisoning. Indeed, 72-h-long exposure to 1 nM MeHg enhanced significantly the averaged current amplitude. This result support and extends our observation that low concentrations of MeHg caused dual acute effect on the current amplitude. Apparently, in chronic experiments, current potentiation by 1 nM MeHg overwhelmed the current inhibition. When concentration was increased to 10 nM, current inhibition was more potent. The observed decrease of the calcium current amplitude was in agreement with the study made by Shafer et al. (2002) on HVA calcium channels in PC12 cells. Nevertheless, the decrease in current amplitude observed in our experiments was not significant. Furthermore, IC₅₀ for acute current inhibition by MeHg was 10- to 20-fold higher than those found for HVA calcium channels in native tissue (Shafer and Atchison, 1991; Leonhardt et al., 1996a,b; Sirois and Atchison, 2000) or in an expression system (Peng et al., 2002; Hajela et al., 2003). Therefore, we may conclude that the LVA Ca_v3.1 calcium channel is generally less sensitive to MeHg than HVA calcium channels are. Leonhardt et al. (1996b) reported similar sensitivity of LVA and HVA calcium channels in DRG neurons to MeHg; however, in that work, they did not distinguish between channel subtypes.

In agreement with reports on native or expressed HVA or LVA calcium channels, the inhibition of the Ca_v3.1 calcium channel by MeHg was irreversible. In contrast, activation of the current by low MeHg concentration reversed readily upon washout. This observation suggests that both effects are mediated by interaction with different interaction sites and/or by different mechanisms. Existence of two interaction sites for MeHg is supported also by dual effect of its picomolar concentrations on the current amplitude and by low steepness of dose-response relationship. Inhibition by Hg²⁺ was partly reversible at higher mercury concentrations. Reversibility may be highly selective for channel subtypes because it was observed for Ca_v2.2 but not for Ca_v2.3 channels expressed in HEK 293 cells (Hajela et al., 2003).

Reports on voltage dependence of interaction of mercurials with calcium channels are controversial. Most authors did not find any voltage dependence for Hg²⁺ (Busselberg et al., 1994) or MeHg (Sirois and Atchison, 2000; Peng et al., 2002; Hajela et al., 2003) interaction with HVA calcium channels. In contrast, Leonhardt et al. (1996b) found a shift of I-V relation toward more depolarized membrane potentials upon exposure of DRG cells to both Hg²⁺ and MeHg. The calcium current measured from DRG neurons does include LVA calcium current. In our study, a similar shift of I-V was found for Hg²⁺ only. Additionally, the I-V relation was not only shifted but also widened, or, in other words, the slope factor for dependence of current amplitude on membrane depolarization was enhanced. This observation suggests that Hg²⁺ interferes with the channel activation and/or inactivation gate. In agreement with this suggestion, Hg²⁺ slowed down significantly kinetics of channel activation, inactivation, and deactivation.

MeHg altered channel kinetics in concentrations, which caused inhibition of the calcium current. These effects were partly opposite to the effects of Hg²⁺ on the calcium current. MeHg accelerated channel inactivation and deactivation, whereas it slowed down the channel activation in parallel to the effect of Hg²⁺ on the activation. Such effects suggest an interaction of MeHg with an open channel state. This explanation is in line with findings that inhibition of calcium current by MeHg is frequency-dependent (Sirois and Atchison, 2000; Peng et al., 2002).

The finding that Hg²⁺ activates long-lasting inward current at higher concentrations corresponds with reports on DRG neurons (Pekel et al., 1993; Leonhardt et al., 1996b). This current is specific for certain cell types because it was not observed in Aplysia neurons (Pekel et al., 1993). The channel responsible for this current was not identified, but we may hypothesize that it is a chloride-permeable channel because the current activation was not affected by replacement of NaCl by NMDG-Cl in extracellular solution (L. Lacinova, M. Kurejova, and M. Drabova, unpublished data).

An alternative explanation for the increased background conductance may be an increase in the permeability of the cell membrane because of cytotoxic effects of Hg²⁺. We have tested for apoptotic and necrotic cell death using MTT assay and flow cytometry. No cytotoxic effects of any form of mercury were detected after 10-min-long treatment, corresponding to the typical length of electrophysiological experiment. Minor but significant cell death most probably due to necrosis was observed after 4-h treatment with 1 µM Hg²⁺. This could not interfere with electrophysiological experiments and their interpretations.

In conclusion, both MeHg and Hg²⁺ inhibited current through the Ca_v3.1 calcium channel at low micromolar concentration. Therefore, this interaction may significantly contribute to pathology of acute mercury poisoning. T-type calcium channels may generate a low-threshold calcium spike, which plays an important role in the genesis of burst firing (Perez-Reyes, 2003). Because these channels are preferentially localized to dendrites, their inhibition may interfere with dendritic signal amplification. In thalamic neurons, T-type currents play an important role in oscillatory behavior (for review, see Perez-Reyes, 2003). Therefore, their suppression may lead to inappropriate oscillations of these circuits or thalamocortical dysrhythmias. A prolonged exposure to nanomolar concentrations of MeHg perturbs the channel function. These effects may increase Ca²⁺ entry through the T-type channels, contribute to spike repolarization and after-hyperpolarizations, and eventually might lead to overexcitability in various neuronal tissues. A long exposure to low mercury concentrations may thus contribute to pathology of chronic poisoning.

	Acknowledgements

 
We thank Stanislava Manová, Mário ere, and Lenka Gibalová for skillful technical assistance, Ján Sedlák and Branislav Uhrík for valuable discussion, and Anthony Gioio for helpful comments on the manuscript.

	Footnotes

 
This work was supported by Volkswagen Stiftung Grants VEGA 2/4009 and APVT-51-027404.

doi:10.1124/jpet.105.095463.

ABBREVIATIONS: MeHg, methylmercury; Hg²⁺, inorganic mercury; HVA, high-voltage-activated; LVA, low-voltage-activated; DRG, dorsal root ganglion; I-V, current-voltage; HEK, human embryonic kidney; MEM, minimal essential medium; HP, holding potential; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Address correspondence to: L'ubica Lacinová, Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Vlárska 5, 833 34 Bratislava, Slovakia. E-mail: lubica.lacinova{at}savba.sk

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