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TOXICOLOGY

Comparative Effects of Methylmercury and Hg²⁺ on Human Neuronal N- and R-Type High-Voltage Activated Calcium Channels Transiently Expressed in Human Embryonic Kidney 293 Cells

Department of Pharmacology and Toxicology, Institute of Environmental Toxicology and Neuroscience Program, Michigan State University, East Lansing, Michigan

Received for publication January 22, 2003
Accepted May 23, 2003.

	Abstract

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Expression cDNA clones of _1B-1 or _1E-3 subunits coding for human neuronal N-(Ca_v2.2) or R-subtype (Ca_v2.3) Ca²⁺ channels, respectively, was combined with _2-b and _3-a Ca²⁺ channel subunits, and transfected into human embryonic kidney cells for transient expression to determine whether specific types of neuronal voltage-sensitive Ca²⁺ channels are affected differentially by methylmercury (MeHg) and Hg²⁺. For both Ca²⁺ channel subtypes, MeHg (0.125-5.0 µM) or Hg²⁺ (0.1-5 µM) caused a time- and concentration-dependent reduction of current. MeHg caused an initial, rapid component and a subsequent more gradual component of inhibition. The rapid component of block was completed between 100 and 150 s after beginning treatment. At 0.125 to 1.25 µM, MeHg caused a more gradual decline in current. Apparent IC₅₀ values were 1.3 and 1.1 µM for MeHg, and 2.2 and 0.7 µM for Hg²⁺ on N- and R-types, respectively. For N-type current, effects of Hg²⁺ were initially greater on the peak current than on the sustained current remaining at the end of a test pulse; subsequently, Hg²⁺ blocked both components of current. For R-type current, Hg²⁺ affected peak and sustained current approximately equally. Kinetics of inactivation also seemed to be affected by Hg²⁺ in cells expressing N-type but not R-type current. Washing with MeHg-free solution could not reverse effects of MeHg on either type of current. The effect of Hg²⁺ on N- but not R-type current was partially reversed by Hg²⁺-free wash solution. Therefore, different types of Ca²⁺ channels have differential susceptibility to neurotoxic mercurials even when expressed in the same cell type.

Methylmercury (MeHg) and inorganic mercury (Hg²⁺) are environmental neurotoxicants that differ chemically in ionic charge, ionic radii, and lipophilicity. Together, these factors can impact the manner in which these mercurials affect a given cellular function. Neurotoxic mercurials act on a number of cellular targets. In several neuronal systems, cellular effects of MeHg and Hg²⁺ are similar, yet distinct (Atchison et al., 1986; Hare and Atchison, 1992; Hewett and Atchison, 1992; Yuan and Atchison, 1994). The exact mechanisms by which these mercurials exert neurotoxicity are not known with certainty.

Disruption of function of voltage-sensitive Ca²⁺ channels is a prominent effect of acute exposure to low concentrations of both MeHg (Shafer and Atchison, 1991; Leonhardt et al., 1996; Sirois and Atchison, 1996, 2000; Shafer, 1998) and Hg²⁺ (Büsselberg et al., 1991; Weisenberg et al., 1995). MeHg blocks Ba²⁺ currents (I_Ba) carried through multiple subtypes of Ca²⁺ channels in primary cultures of cerebellar granule cells and in rat pheochromocytoma (PC12) cells (Shafer and Atchison, 1991; Sirois and Atchison, 2000). Hg²⁺ also alters function of several types of Ca²⁺ channels at low micromolar concentrations (Büsselberg et al., 1994; Leonhardt et al., 1996; Szucs et al., 1997). However, the actions of mercurials on Ca²⁺ channels may be more complex than mere block of function. In PC12 cells, very low concentrations of Hg²⁺ increase amplitude of current carried through voltage-sensitive Ca²⁺ channels (Rossi et al., 1993), whereas in cerebellar granule cells and NG108-15 cells, MeHg causes an increase in fura-2 fluorescence, which is dependent, at least in part, on extracellular Ca²⁺, and which is delayed by nifedipine, -conotoxin GVIA, and Ni²⁺ (Hare and Atchison, 1995; Marty and Atchison, 1997). Moreover, treatment of rodents with Ca²⁺ channel blockers prevents the toxic effects of MeHg (Sakamoto et al., 1996), and Ca²⁺ channel blockers delay the onset of cerebellar granule cell death with MeHg (Marty and Atchison, 1997; Gasso et al., 2001). Finally, in cells lacking Ca²⁺ channels, the onset of intracellular action of MeHg is delayed, suggesting that Ca²⁺ channels provide a path of entry for MeHg into the cell (Edwards et al., 2002). Therefore, mercurials seem to interact with voltage-sensitive Ca²⁺ channels in a complex manner.

Because of the numerous and important roles that voltage-sensitive Ca²⁺ channels play in neuronal function, disruption of function of voltage-sensitive Ca²⁺ channels may be a significant contributory factor in mercurial-induced neurotoxicity. There are few published reports comparing the effects of different mercurials on function of voltage-sensitive Ca²⁺ channels (Hewett and Atchison, 1992; Szucs et al., 1997; Schirrmacher et al., 1998), and no comparative study on the effects of these two forms of mercury on defined types of voltage-sensitive Ca²⁺ channels exists.

The goal of the present study was to determine whether specific types of voltage-sensitive Ca²⁺ channels were affected differentially by MeHg or Hg²⁺. We compared the effect of MeHg and Hg²⁺ on N-(Ca_v2.2) and R-(Ca_v2.3) types of voltage-sensitive Ca²⁺ channels expressed using cDNA copies of their genes transferred into human embryonic kidney cells (HEK293). These cells are nonexcitable and commonly used for heterologous expression of membrane proteins, including voltage-dependent Ca²⁺ channels (Williams et al., 1994; Perez-Garcia et al., 1995; Quefurth et al., 1998). Expression cDNA clones of _1B-1 or _1E-3 subunit were combined with _2-b and _3-a Ca²⁺ channel subunits of human neuronal origin for transient expression of N- and R-subtypes, respectively, of high-voltage activated Ca²⁺ channels. Jellyfish green fluorescent protein (GFP) was used as a cotransfection reporter.

	Materials and Methods

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Materials. HEK293 cells (ATCC CRL-1573) were purchased from American Type Culture Collection (Manassas, VA). All reagents were pure or ultrapure laboratory grade unless specifically noted otherwise. cAMP, EGTA, HEPES, ATP, -conotoxin GVIA, and tetrodotoxin were all obtained from Sigma-Aldrich (St. Louis, MO). Stock solutions (5 mM) of methylmercuric chloride (ICN Pharmaceuticals, Costa Mesa, CA) and (10 mM) HgCl₂ were prepared weekly in double-distilled water, from which test solutions were prepared daily in extracellular solution (see below). Plasmids containing expression cDNA clones of human neuronal Ca²⁺ channel subunits were generously provided by Dr. Kenneth A. Stauderman of SIBIA Neurosciences (San Diego, CA), now Merck Research Laboratories. _1E-3 (Williams et al., 1994) and _3-a subunit clones (Mark Williams, Merck Research Laboratories, personal communication) were isolated from hippocampus; _1B-1 was isolated from the IMR32 cell line (Williams et al., 1992b), and _2B was isolated from brainstem and basal ganglia (Williams et al., 1992a).

Cell Culture and Transfection. HEK293 cells were grown at 37°C in Eagle's minimal essential medium fortified with 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 10% (v/v) fetal bovine serum, and penicillin/streptomycin/amphotericin B (at a final concentration of 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate, and 0.25 µg/ml amphotericin B; Invitrogen, Carlsbad, CA) in a 5% CO₂ environment. One day before gene transfer, cells were plated at a density of 5 x 10⁵ on 35-mm culture dishes. Cells were transfected with a mixture of plasmids containing human neuronal _1B-1, or _1E-3 plus _2-b and _3-a Ca²⁺ channel subunits and a jellyfish GFP cDNA sequence using FuGENE 6 (Roche Diagnostics, Indianapolis, IN) following the manufacturer's instruction. Reactions contained a total of 3 µl of FuGENE 6 and 1 µg of plasmid DNA containing the three subunits in 1:1:1 M ratio, with GFP plasmid at 20% of the total DNA. Two days were allowed for transient expression of proteins at which time the cells were examined for GFP expression. Cells from dishes with a reasonable number of green fluorescent cells (usually 20%), were replated at a low density of sufficiently isolated individual cells to facilitate recording.

Ca²⁺ Channel Current Recording. Before recording, culture medium was removed, cells were rinsed twice with extracellular solution, and then replenished with 1 ml of extracellular recording solution. The extracellular solution contained 117 mM tetraethylammonium chloride, 20 mM BaCl₂, 1 mM MgCl₂, 25 mM D-glucose, 10 mM HEPES, and 0.001 mM tetrodotoxin; pH was adjusted to 7.2 at room temperature (23-25°C) with tetraethylammonium hydroxide. The osmolarity of the solution was 310 mOsM. Patch-clamp pipettes with resistances between 6 and 8 M were prepared from 1.5-mm i.d. glass capillaries (WPI, Sarasota, FL) using a two-stage micro-electrode puller (PP-830; Narishige, Tokyo, Japan) and fire-polished using a Narishige MF-830 microforge. Intracellular (pipette) solution contained 140 mM CsCl, 10 mM EGTA, 10 mM HEPES, 2 mM ATP-Mg, and 1 mM cAMP; pH was adjusted to 7.2 with CsOH.

The tight seal whole cell configuration of the patch-clamp technique (Hamill et al., 1981) was used on fluorescent green cells to record I_Ba through transiently expressed Ca²⁺ channels. Whole cell currents were recorded using an Axopatch-1D amplifier (Axon Instruments, Inc., Foster City, CA), sampled at 10 kHz and filtered at 2 kHz (-3 dB, four-pole Bessel filter; Axon Instruments, Inc.) and acquired on-line using the pClamp6 program (Axon Instruments, Inc.). Pipette and cell capacitance was compensated online in all experiments. Series resistance was also compensated in the range of 60 to 80%. Extracellular media were exchanged using a gravity-fed bath perfusion system (BPS-4; ALA Scientific Instruments, Westbury, NY); the flow rate was 5 x 10^-3 ml/s. The distance of the flow pipette from the cell was maintained at approximately 150 µm. All experiments were carried out at room temperature (23-25°C).

To elicit I_Ba, the membrane was depolarized by a positive voltage step from the holding potential (-90 mV). Conventional voltage clamp step protocols were used to examine the amplitude of I_Ba over a range of membrane potentials and to examine the voltage dependence of activation of I_Ba and of steady-state inactivation of I_Ba. In experiments designed to examine the effects of Ni²⁺, Cd²⁺, MeHg, Hg²⁺,or -conotoxin GVIA on I_Ba, the membrane was repeatedly (0.1 Hz) depolarized (from -90 mV) by a voltage step to 0 mV (R-type) or to 20 mV (N-type). Each depolarization was preceded by a hyperpolarizing voltage step (one-quarter the amplitude of the depolarizing step) to permit measurement of leakage current. After establishing that the current elicited by each depolarizing step was eventually constant, the test compound (modulator) was applied to the cell as mentioned above. Unless noted otherwise, in all experiments I_Ba amplitude was measured after subtraction of leak current from the whole current elicited by the activating depolarization.

Statistical Analysis. For each experiment recordings were made from a minimum of five cells, and these cells were derived from at least three independent transfections. Values are expressed as the mean ± S.E.M. The number of replicates is indicated for each experiment in the figure legend. Curve fitting of data points on graphs was performed using Origin software (OriginLab Corp., Northampton, MA).

	Results

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Characteristics of Recombinant Transiently Expressed _1B and _1E Subunit-Mediated Ca²⁺ Channel Currents in HEK293 Cells. Basic biophysical and pharmacological qualities of Ca²⁺ channels containing _1B (Ca_{v 2.2}) or _1E (Ca_{v 2.3}) subunit in combination with _2-b and _3-a subunits and expressed transiently in HEK293 cells were similar to those of N-or R-type Ca²⁺ channels, respectively, as expressed in their native environment. A constant set of _2-b and _3-a subunits was used in all experiments so that we could focus on the comparative actions of the two mercurials on the principal phenotype-defining subunit of voltage-sensitive Ca²⁺ channels, the ₁ subunit. Representative tracings of I_Ba elicited from these cells along with their current-voltage relationships and voltage dependence of steady-state inactivation and activation are shown in Fig. 1, A to C (for _1B) and Fig. 2, A to C (for _1E), respectively. For _1B-containing channels, current seemed to activate at approximately -20 mV, reached peak amplitude at about +20 mV and reversed at +60 mV. For channels containing _1E subunit, current activated at -40 mV, reached a peak amplitude at 0 mV and reversed at approximately +60 mV. The voltage for half-maximal activation (V_1/2) was 0 mV in _1B-containing channels (Fig. 1C) and -14.8 mV in _1E-containing channels (Fig. 2C), respectively. The isochronal inactivation (h) was determined using an 8-s inactivating prepulse. The inactivation curves are shown (Figs. 1C and 2C) with slope parameter (k) of 12.0 mV and V_1/2 of -64.6 mV for _1B-containing channels, and k of 9.2 mV and V_1/2 of -69.6 mV for _1E-containing channels, respectively.

View larger version (19K): [in this window] [in a new window]   Fig. 1. Biophysical characteristics of whole cell Ba2+ current mediated by recombinant N-type Ca2+ channels expressed in HEK293 cells. A, representative family of current traces (filtered at 2 kHz but not leak subtracted) elicited by voltage steps from a holding potential of -90 mV to test potentials ranging from -20 to +60 mV. Ba2+ (20 mM) was used as the charge carrier. B, current-voltage relationship for the Ba2+ current was derived from the type of experiment illustrated in A. The peak current elicited during a voltage step is plotted as a function of the test potential. C, voltage dependence of activation () and of steady-state inactivation () is depicted. To examine voltage dependence of activation, peak current elicited during a depolarizing voltage step from the holding potential to each test potential was normalized to the maximum current elicited and normalized current was plotted as a function of test potential. Data points were fitted by the curve using a Boltzmann function: I/Imax = [1 + exp{(V - V1/2)/k}]-1, with V1/2 = -64.6 mV and k = 12.0 mV. To examine the voltage dependence of steady-state inactivation, a conditioning voltage step (8 s, to potentials between -100 and 0 mV, in 10-mV increments) was applied to allow inactivation of the channel to achieve steady state, and the membrane was then stepped to +20 mV. Current elicited at +20 mV after each conditioning step was measured and normalized to amplitude elicited (at +20 mV) after a conditioning step to -100 mV. Normalized current was plotted as a function of conditioning (step) potential. Data points were fitted by the curve using a Boltzmann function: I/Imax = [1 + exp{-(V - V1/2)/k}]-1, with V1/2 = 0.0 mV and k = 3.6 mV. Ba2+ current amplitude was measured after subtraction of leak current from the whole current elicited by the activating depolarization. In B and C, values shown are the mean ± S.E.M. of 10 cells.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Biophysical characteristics of whole cell Ba2+ current mediated by recombinant R-type Ca2+ channels expressed in HEK293 cells. A, representative family of current traces (filtered at 2 kHz but not leak subtracted) elicited by voltage steps from a holding potential of -90 mV to test potentials ranging from -20 to +60 mV. Ba2+ (20 mM) was used as the charge carrier. B, current-voltage relationship for the IBa was derived from the type of experiment illustrated in A. The peak current elicited during a voltage step is plotted as a function of the test potential. C, voltage dependence of activation () and of steady-state inactivation (). To examine voltage dependence of activation, peak current elicited during a depolarizing voltage step from the holding potential to each test potential was normalized to the maximum current elicited and normalized current was plotted as a function of test potential. Data points were fitted by the curve using a Boltzmann function: I/Imax = [1 + exp{(V - V1/2)/k}]-1, with V1/2 = -69.6 mV and k = 9.2 mV. To examine the voltage dependence of steady-state inactivation, a conditioning voltage step (8 s, to potentials between -100 and 0 mV, in 10-mV increments) was applied to allow inactivation of the Ba2+ current to come to steady state, and the membrane potential was then stepped to +20 mV. Current elicited at +20 mV after each conditioning step was measured and normalized to amplitude elicited (at +20 mV) after a conditioning step to -100 mV. Normalized current was plotted as a function of conditioning (step) potential. Data points were fitted by the curve using a Boltzmann function: I/Imax = [1 + exp{-(V - V1/2)/k}]-1, with V1/2 = -14.8 mV and k = 4.4 mV. Ba2+ current amplitude was measured after subtraction of leak current from the whole current elicited by the activating depolarization. Values shown in B and C are the mean ± S.E.M. of seven cells.

Figure 3 demonstrates that I_Ba expressed in cells transfected with _1B subunit was sensitive to inhibition by -conotoxin GVIA. At 1 or 10 µM, -conotoxin GVIA caused a rapid reduction of current amplitude; the rate with which block occurred was concentration-dependent. However, ultimately, both concentrations blocked virtually all current. The effect of -conotoxin block was not reversed by washing the cell with toxin-free solution. Thus, the I_Ba recorded from HEK293 cells expressing human neuronal _1B subunits combined with a fixed complement of ₂₃ subunits has biophysical and pharmacological properties consistent with those mediated by N-type Ca²⁺ channels. We tested _1E subunit-expressing HEK293 cells with known specific antagonists (nimodipine and -agatoxin IVA) for the L- or P/Q-type channels and found the I_Ba to be resistant to block by both of these agents (data not shown), consistent with the characteristics of R-type current. Therefore, we used varying concentrations of Cd²⁺ and Ni²⁺ (both of which are known to block current carried through all known subtypes of voltage-sensitive Ca²⁺ channels) on the _1E subunit-containing cells (Fig. 4, A and B). The block of I_Ba from these cells by both divalent cations was concentration-dependent. A stepwise reduction of current was seen after addition of successively higher concentrations of Cd²⁺ or Ni²⁺; this effect seemed to plateau until the next higher concentration of metal was added. The reduction of amplitude of I_Ba was complete at 100 µM Cd²⁺ or 1 mM Ni²⁺. These two observations together indicate that the pharmacological characteristics of _1E subunit-mediated I_Ba from these cells is consistent with that of the R-type. An interesting observation at the lowest concentrations of Cd²⁺ (0.1 µM; Fig. 4A) and Ni²⁺ (1 µM; Fig. 4B) was an apparent slight increase rather than reduction in amplitude of I_Ba. This effect was more prominent with Cd²⁺.

View larger version (23K): [in this window] [in a new window]   Fig. 3. Effect of 1.0 and 10.0 µM -conotoxin GVIA (-CgTx GVIA) on peak Ba2+ current (20 mM Ba2+) amplitude in HEK293 cells expressing human neuronal 1B subunit containing N-type Ca2+ channels. At 10.0 µM, -CgTx GVIA caused an almost complete and irreversible block of Ba2+ current, because current could not be restored by washing cells with toxin-free extracellular solution. Cells were depolarized from -90 to +20 mV at a stimulation frequency of 0.1 Hz. Current responses were filtered at 2 kHz and leak current was subtracted. Values shown are the mean of five to six cells. To simplify the figure only representative S.E.M. values are shown.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Effect of different concentrations of Cd2+ (A) and Ni2+ (B) on peak Ba2+ current (20 mM Ba2+) amplitude from HEK293 cells expressing human neuronal 1E subunit mediated R-type Ca2+ channels. Notice the stepwise reduction of current amplitude with each higher concentration of Cd2+ or Ni2+ addition that seems to plateau until the next higher concentration is added. The block of Ba2+ current becomes complete at 100 µM Cd2+ and 1 mM Ni2+. Cells were depolarized from -90 to 0 mV at a stimulation frequency of 0.1 Hz. Current responses were filtered at 2 kHz and leak current was subtracted. Values shown are the mean ± S.E.M. of six to seven cells.

Perhaps this effect is due to these cations actually entering the Ca²⁺ channels based on their relatively similar ionic radii and electrical charges. These same cations become inhibitory at higher concentrations.

Time- and Concentration Dependence of MeHg Effect on I_Ba. As shown in Figs. 5A and 6A for representative traces, and in composite data from a number of preparations in Figs. 5B and 6B, MeHg caused a rapid, concentration- and time-dependent reduction in current from both _1B and _1E subunit-containing channels. As shown in Fig. 5A for cells expressing _1B-type current, the effect of 5 µM MeHg was initially greater on the peak current than on the sustained current remaining at the end of a test pulse. However, with additional time, MeHg blocked both components of current apparently equally. For cells expressing _1E subunit, the degree of block of current by 5 µM MeHg seemed to be approximately the same for both peak and sustained current (Fig. 6A). The effect of different concentrations of MeHg on whole cell I_Ba with either subunit is shown as a time course in Figs. 5B and 6B and was determined after at least 5-min stabilization of the control current. Addition of MeHg concentrations between 0.125 (or 0.25 for _1E subunit) and 5.0 µM to the extracellular solution resulted in a concentration-dependent block in the rate and magnitude of I_Ba after leak subtraction. For both kinds of channels, the block of peak current by MeHg was again concentration- and time-dependent. MeHg concentrations in excess of 1 µM caused complete block of current within 2 to 6 min. Submicromolar concentrations also caused measurable inhibition (Figs. 5B and 6B). The rate at which inhibition occurred at micromolar concentrations seemed to consist of two distinct components: one which was initially more rapid, and the other, which was more gradual (Figs. 5B and 6B). Depending upon the concentration of MeHg used, the rapid component was completed between 100 and 150 s after beginning treatment. Lower concentrations of MeHg (0.125-1.25 µM) caused a more gradual decline in current amplitude. Inhibition of current at 1.25 µM MeHg seemed ultimately to reach the same plateau as at higher concentrations. At 0.125 µM in _1B- and 0.25 µM in _1E-containing channels, inhibition never reached the same level as at higher concentrations over the 10-min recording period. Because lower concentrations of MeHg caused a slow, progressive block of current that didn't reach a plateau, we could only estimate an apparent IC₅₀ value for MeHg. This was done by comparing the percentage of block at each concentration at a set time point. At 200 s, MeHg blocked 50% of both types of current at 1.3 (N-type) and 1.1 µM (R-type).

View larger version (20K): [in this window] [in a new window]   Fig. 5. Effect of MeHg on peak Ba2+ current (20 mM Ba2+) amplitude from HEK293 cells expressing human neuronal 1B subunit-containing N-type Ca2+ channels. A, representative current traces showing the effect of 5.0 µM MeHg on peak current at different times of exposure. B, time course of block of peak current by different concentrations of MeHg. Cells were depolarized from -90 to +20 mV at a stimulation frequency of 0.1 Hz. Current responses were filtered at 2 kHz and leak current was subtracted. Values shown are the mean of five to seven cells. To simplify the figure only representative S.E.M. values are shown.

View larger version (18K): [in this window] [in a new window]   Fig. 6. Effect of MeHg on peak Ba2+ current (20 mM Ba2+) amplitude from HEK293 cells expressing human neuronal 1E subunit-containing R-type Ca2+ channels. A, representative current traces showing the effect of 5.0 µM MeHg on peak current amplitude at different times of exposure. B, time course of block of peak Ba2+ current by different concentrations of MeHg. Cells were depolarized from -90 to 0 mV at a stimulation frequency of 0.1 Hz. Current responses were filtered at 2 kHz and leak current was subtracted. Values shown are the mean of five to seven cells. To simplify the figure only representative S.E.M. values are shown.

Concentration Dependence of Hg²⁺ Effect on I_Ba. Figures 7 and 8 show the effects of Hg²⁺ on I_Ba from HEK293 cells expressing _1B and _1E subunit-containing Ca²⁺ channels, respectively. Figures 7A and 8A depict representative current traces showing effects of different concentrations of Hg²⁺ on current after 2-min exposure. Hg²⁺ caused a rapid, concentration-dependent reduction in current from both _1B and _1E subunit-expressing channels. As shown in Fig. 7A for cells expressing _1B-type current, the effect of 0.5, 1, and 5.0 µM Hg²⁺ was initially greater on the peak current than on the sustained current remaining at the end of a test pulse.

View larger version (13K): [in this window] [in a new window]   Fig. 7. Effect of Hg2+ on peak Ba2+ current (20 mM Ba2+) amplitude from HEK293 cells expressing human neuronal 1B subunit-containing N-type Ca2+ channels. A, representative current traces showing effect of different concentrations of Hg2+ on peak current amplitude after 2-min exposure. B, peak currents recorded after a 2-min exposure to different concentrations of Hg2+ were fitted using the equation I[Hg2+]/IControl = [1 + ([Hg2+]e/IC50)n]-1 with IC50 = 2.2 µM. Only a single concentration of Hg2+ was applied per cell. Cells were depolarized from -90 to +20 mV at a stimulation frequency of 0.1 Hz. Current responses were filtered at 2 kHz and leak current was subtracted. Values shown are the mean ± S.E.M. of six cells.

View larger version (13K): [in this window] [in a new window]   Fig. 8. Effect of Hg2+ on peak Ba2+ (20 mM) current amplitude from HEK293 cells expressing human neuronal 1E subunit-containing R-type Ca2+ channels. A, representative current traces showing effect of different concentrations of Hg2+ on peak current after 2-min exposure. B, peak currents recorded after a 2-min exposure to different concentrations of Hg2+ were fitted using the equation I[Hg2+]/IControl = [1 + ([Hg2+]e/IC50)n]-1 with IC50 = 0.7 µM. Only a single concentration of Hg2+ was applied per cell. Cells were depolarized from -90 to 0 mV at a stimulation frequency of 0.1 Hz. Current responses were filtered at 2 kHz and leak current was subtracted. Values shown are the mean ± S.E.M. of five cells.

However, with additional time, Hg²⁺ blocked both components of current. For cells expressing _1E subunit, the degree of block by 0.1, 0.5, and 1 µM Hg²⁺ seemed to be approximately the same for both peak and sustained current (Fig. 8A). Furthermore, the inactivation kinetics seems to be affected by Hg²⁺ in cells expressing _1B-type current. Figures 7B and 8B depict the degree of reduction of normalized I_Ba as a function of concentration of Hg²⁺. The IC₅₀ values for Hg²⁺-induced block of _1B and _1E-type current were 2.2 and 0.7 µM, respectively.

Effect of Wash with MeHg- or Hg²⁺-Free Solution on I_Ba. As shown in Fig. 9, the block by MeHg of either type of current could not be reversed by washing with MeHg-free solution. However, the Hg²⁺ induced reduction of I_Ba through _1B-mediated N-type channels was partially reversible (Fig. 10A), but the I_Ba reduction from _1E-mediated R-type channels was not reversed by washing with Hg²⁺-free solution (Fig. 10B).

View larger version (17K): [in this window] [in a new window]   Fig. 9. Irreversible block by MeHg of Ba2+ current (20 mM Ba2+) in HEK293 cells expressing 1B or 1E subunit-containing human neuronal Ca2+ channels. A 5-min treatment with 5 µM MeHg was followed by wash with MeHg-free solution. Cells were depolarized at a stimulation frequency of 0.1 Hz from -90 to +20 mV for cells with channels containing 1B and -90 to 0 mV for those containing 1E. Current responses were filtered at 2 kHz and leak current was subtracted. Values shown are the mean ± S.E.M. of five to seven cells.

View larger version (15K): [in this window] [in a new window]   Fig. 10. Effects of wash with Hg2+-free solution on block of IBa. A, block by 5 µM Hg2+ of peak Ba2+ current (20 mM Ba2+) in HEK293 cells expressing 1B subunit-containing human neuronal Ca2+ channels after a 1-min exposure is partially reversed by wash with Hg2+-free solution. B, block by 5 µM Hg2+ of peak Ba2+ current in HEK293 cells expressing 1E subunit-containing human neuronal Ca2+ channels after a 1-min exposure was not reversed by wash with Hg2+-free solution. Cells were depolarized from -90 to +20 mV for 1B and -90 to 0mV for 1E. Current responses were filtered at 2 kHz and leak current was subtracted. Values shown are the mean ± S.E.M. of five to seven cells.

	Discussion

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Previous studies have shown that MeHg (Shafer and Atchison, 1991; Marty and Atchison, 1997; Shafer, 1998; Sirois and Atchison, 2000) and Hg²⁺ (Büsselberg et al., 1994; Leonhardt et al., 1996; Szucs et al., 1997) affect the function of native Ca²⁺ channels in multiple types of cells. However, in most of these studies, the cells examined express more than one subtype of Ca²⁺ channels, and attempts to compare the actions of these metals on known subtypes of Ca²⁺ channel were incomplete and/or indirect. Until recently (Peng et al., 2002), there have been no studies of effects of mercurials on distinct subtypes of Ca²⁺ channel in isolation. The present study using transient expression of human neuronal Ca²⁺ channels is the first to characterize and compare the effects of the organomercurial MeHg and inorganic Hg²⁺ on current mediated by single, identified phenotypes (N- and R-type) of Ca²⁺ channel in isolation. Because the two types of expressed Ca²⁺ channel contained the same ₂ and subunits, differential effects of the mercurials on expressed N- and R-type channel current must be due largely to the mercurial interactions with, or action on, the ₁ pore-forming subunit of the channel. Our results support and extend several aspects of previous studies of mercurials on native N-type and R-type Ca²⁺ channels. Our results demonstrate that, first, MeHg is an irreversible and essentially equipotent inhibitor of current recorded from HEK293 cells expressing human neuronal _1B (N-type) or _1E (R-type) subunit containing Ca²⁺ channels. Second, although Hg²⁺ inhibits current mediated by the same channels, it has a more potent effect on recombinant R-type channels than N-type channels. Third, the effect of Hg²⁺ is partially reversible for recombinant N-type channels but is evidently irreversible for R-type channels.

Our recordings show that the voltage- and time-dependent characteristics of the current mediated by channels containing the _1B subunit and those containing the _1E subunit expressed in transfected cells resemble those of native N- and R-type Ca²⁺ channels, respectively, in neurons (Randall and Tsien, 1995). The different biophysical characteristics of native N-type channel current and current mediated by recombinant channels containing the _1B subunit and of native R-type channel current and current mediated by recombinant channels containing the _1E subunit may simply reflect differences of the ₂ and subunits expressed in recombinant and native channels. Alternatively, these differences may be due to differences of the human ₁ subunits expressed in cells studied here and the ₁ subunits of the native channels of nonhuman derived neurons such as those studied by Randall and Tsien (1995). Furthermore, we have demonstrated that recombinant channels containing the _1B subunit are sensitive to -conotoxin GVIA as are native N-type Ca²⁺ channels, whereas those channels expressing the _1E subunit were highly sensitive to block by Cd²⁺ and to a lesser extent Ni²⁺ as is the case for native R-type channels.

The data presented here show that recombinant expressed N-type channels were more sensitive to the effects of MeHg than were presumptive N-type native channels in PC12 cells (Shafer and Atchison, 1991; Shafer, 1998) and dorsal root ganglion cells (Leonhardt et al., 1996) but were equally sensitive compared with presumptive N-type channels of rat cerebellar granule cells (Sirois and Atchison, 2000). Such differential sensitivity of native and recombinant expressed channels has also been reported for other toxins, including calcicludine effects on L-type channels (Stotz et al., 2000), -agatoxin IVA effects on P/Q-type channels (Bourinet et al., 1999), and peptide spider toxin DW13.3 effects on N-type channels (Sutton et al., 1998). These differential effects of mercurials and other toxins on recombinant and native channels may reflect differences between human and nonhuman ₁ subunits (present in studies of recombinant channels and native channels, respectively), or between the accessory (₂ and ) subunits of various native channels and recombinant channels.

The present study of mercurials on recombinant Ca²⁺ channels also confirms previous observations concerning several aspects of the effect of MeHg on native channels. First, as has been observed for native channels in cerebellar granule cells (Sirois and Atchison, 2000) and PC12 cells (Shafer and Atchison, 1991), the effect of MeHg on recombinant _1B-(N-type) and _1E (R-type)-containing channels was not voltage-dependent. Second, the reduction of current during exposure to MeHg was irreversible regardless of whether the current was mediated by channels containing the _1B (N-type) or _1E (R-type) subunit or by various types of native Ca²⁺ channel. Third, it is also noteworthy that the effect of MeHg proceeds to complete reduction of the current mediated by channels containing the _1B (N-type) or _1E (R-type) subunit and by at least some native channels (given a sufficient duration of exposure to MeHg), although this effect was not observed for MeHg effects on recombinant _1C (L-type) subunit-containing channels (Peng et al., 2002). Fourth, the effect of Hg²⁺ on native channels, which has variously been reported to be reversible or irreversible (Leonhardt et al., 1996), was found in the present study to be partially reversible for channels containing the _1B (N-type) subunit, but evidently irreversible, for channels containing the _1E (R-type) subunit. Although mechanisms associated with reversibility were not examined, this observation may help to explain the variability of reversal seen in studies with Hg²⁺ of native channels, inasmuch as, depending upon the complement of channel phenotype in the preparation being studied, the actions of Hg²⁺ may be more or less reversible. In the case of the irreversible effect of MeHg or Hg²⁺, it is possible that the mercurial binds covalently to the channel (so that the mercurial cannot be removed) or interacts in some way with the channel that is irreversible (even after removal of the mercurial). Again, differences concerning the reversibility of a mercurial effect on native channels and on recombinant channels may reflect differences between the ₁ subunit or the accessory units of the native and recombinant Ca²⁺ channels. Regardless of the reversibility of the interaction of Hg²⁺ or MeHg with the channel, the specific site of interaction between mercurial and channel is unclear. Hg²⁺ or MeHg could act directly in the channel pore, have nonspecific or specific interactions with negatively charged groups on the cell surface, or act at a regulatory or allosteric binding site on the intra- or extracellular surface of the channel. However, it should be noted that Shafer (1998) reported that intracellular application of MeHg did not affect Ca²⁺ channel current in PC12 cells. Another difference between the effects of Hg²⁺ on recombinant and native channels is that Hg²⁺ seems to increase Ca²⁺ current through native channels at low concentration (Manalis and Cooper, 1975; Rossi et al., 1993); this phenomenon was not observed in our study of recombinant channels.

In summary, both MeHg and Hg²⁺ perturb the function of heterologously expressed, recombinant human neuronal N-type and R-type Ca²⁺ channels at low micromolar concentrations, well within the range of concentrations known to cause disruption of function of corresponding native channels as well as toxicity from these agents in vivo. MeHg was an equipotent inhibitor of human neuronal N-type and R-type Ca²⁺ channels expressed in HEK293 cells. However, there seem to be subtle differences on the effects of Hg²⁺, which vary somewhat depending on the type of ₁ subunit.

	Acknowledgements

 
We acknowledge the generous contribution of the Ca²⁺ channel cDNA clones by SIBIA Neurosciences (now Merck Research Laboratories, San Diego, CA). Thanks are also due to Dr. Peter J. R. Cobbett (Department of Pharmacology and Toxicology, Michigan State University) for critical reading of the manuscript and valuable discussion. The excellent secretarial assistance of Mallory Koglin and Kelly O'Brien is especially appreciated.

	Footnotes

 
This study was supported by National Institutes of Health Grants R01ES03299 and R01ES05822 (to W.D.A.). A preliminary report of these findings was presented at the 2001 Annual Meeting of the Society of Toxicology in San Francisco, CA and published in Toxicologist 60:185.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

DOI: 10.1124/jpet.103.049429.

ABBREVIATIONS: MeHg, methylmercury; I_Ba, Ba²⁺ current; HEK, human embryonic kidney; GFP, green fluorescent protein; V_1/2, voltage for half-maximal activation.

¹ R.K.H. and S.-Q.P. contributed equally to this study.

Address correspondence to: Dr. Bill Atchison, Department of Pharmacology and Toxicology, Michigan State University, B-331 Life Sciences Bldg., East Lansing, MI 48824-1317. E-mail: atchiso1{at}msu.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Atchison WD (2003) Effects of toxic environmental contaminants on voltage-gated calcium channel function: from past to present. J Bioenerg Biomembr, in press.
Atchison WD, Joshi U, and Thornburg JE (1986) Irreversible suppression of calcium entry into nerve terminals by methylmercury. J Pharmacol Exp Ther 238: 618-624.[Abstract/Free Full Text]
Bourinet E, Soong TW, Sutton K, Slaymaker S, Mathews E, Monteil A, Zamponi GW, Nargeot J, and Snutch TP (1999) Splicing of _1A subunit gene generates phenotypic variants of P- and Q-type calcium channels. Nat Neurosci 2: 407-414.[CrossRef][Medline]
Brust PF, Simerson S, McCue AF, Deal CR, Schoonmarker S, Williams ME, Velicelebi G, Johnson EC, Harpold MM, and Ellis SB (1993) Human neuronal voltage-dependent calcium channels: studies on subunit structure and role in channel assembly. Neuropharmacology 32: 1089-1102.[CrossRef][Medline]
Büsselberg D, Pekel M, Michael D, and Platt B (1994) Mercury and zinc: two divalent cations with different actions on voltage-activated calcium channel currents. Cell Mol Neurobiol 14: 675-687.[CrossRef][Medline]
Büsselberg D, Vans M, Rahmann H, and Carpenter D (1991) Effects of inorganic and triethyl lead and inorganic mercury on the voltage activated calcium channels of Aplysia neurons. Neurotoxicology 12: 733-744.[Medline]
Catterall WA (1998) Structure and function of neuronal Ca²⁺ channels and their role in neurotransmitter release. Cell Calcium 24: 307-323.[CrossRef][Medline]
Catterall WA (2000) Structure and regulation of voltage-gated Ca²⁺ channels. Annu Rev Cell Dev Biol 16: 521-555.[CrossRef][Medline]
DeWaard M and Campbell KP (1995) Subunit regulation of the neuronal _1A Ca²⁺ channel expressed in Xenopus oocytes. J Physiol (Lond) 485: 619-634.[Abstract/Free Full Text]
Edwards JR, Cobbett P, and Atchison WD (2002) Comparative disruption of intracellular calcium regulation by methylmercury in PC12 and PC18 cells. Soc Neurosci Abstr, Abstracts Viewer/Itinerary Planner 833.5.
Gasso S, Cristofol RM, Selema G, Rosa R, Rodriguez-Farre E, and Sanfeliu C (2001) Antioxidant compounds and Ca²⁺ pathway blockers differentially protect against methylmercury and mercuric chloride neurotoxicity. J Neurosci Res 66: 135-145.[CrossRef][Medline]
Hamill OP, Marty A, Neher E, Sakmann B, and Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfluegers Arch 391: 85-100.[CrossRef][Medline]
Hare MF and Atchison WD (1992) Comparative action of methylmercury and divalent inorganic mercury on nerve terminal and intraterminal mitochondrial membrane potentials. J Pharmacol Exp Ther 261: 166-172.[Abstract/Free Full Text]
Hare MF and Atchison WD (1995) Nifedipine and tetrodotoxin delay the onset of methylmercury induced increase in [Ca²⁺]_i in NG108-15 cells. Toxicol Appl Pharmacol 135: 299-307.[CrossRef][Medline]
Hewett SJ and Atchison WD (1992) Effects of charge and lipophilicity on mercurial-induced reduction of ⁴⁵Ca²⁺ uptake in isolated nerve terminals of the rat. Toxicol Appl Pharmacol 113: 267-273.[CrossRef][Medline]
Kiss T and Osipenko ON (1994) Toxic effects of heavy metals on ionic channels. Pharmacol Rev 46: 245-267.[Medline]
Leonhardt R, Pekel M, Platt B, Haas HL, and Büsselberg D (1996) Voltage-activated calcium channel currents of rat DRG neurons are reduced by mercuric chloride (HgCl₂) and methylmercury (CH₃HgCl). Neurotoxicology 17: 85-92.[Medline]
Manalis RS and Cooper GP (1975) Evoked transmitter release increased by inorganic mercury at frog neuromuscular junction. Nature (Lond) 257: 690-691.[CrossRef][Medline]
Marty MS and Atchison WD (1997) Pathways mediating Ca²⁺ entry in rat cerebellar granule cells following in vitro exposure to methyl mercury. Toxicol Appl Pharmacol 147: 319-330.[CrossRef][Medline]
McEnery MW, Vance CL, Begg CM, Lee WL, Choi Y, and Dubel SJ (1998) Differential expression and association of calcium channel subunits in development and disease. J Bioenerg Biomembr 30: 409-418.[CrossRef][Medline]
Meir A and Dolphin AC (2002) Subunit interactions and channelopathies in Ca_v channels. Modulator (Alamone Labs, Israel) 16: 4-13.
Pan JQ and Lipscombe D (2000) Alternative splicing in the cytoplasmic II-III loop of the N-type Ca²⁺ channel _1B subunit: functional differences are subunit-specific. J Neurosci 20: 4769-4775.[Abstract/Free Full Text]
Peng S-Q, Hajela RK, and Atchison WD (2002) Effects of methylmercury on human neuronal L-type calcium channels transiently expressed in human embryonic kidney cells (HEK293). J Pharmacol Exp Ther 302: 424-432.[Abstract/Free Full Text]
Perez-Garcia MT, Kamp TJ, and Marban E (1995) Functional properties of cardiac L-type calcium channels transiently expressed in HEK293 cells. Roles of ₁ and subunits. J Gen Physiol 105: 289-305.[Abstract/Free Full Text]
Quefurth HW, Haughey NJ, Greenway SC, Yacono PW, Golan DE, and Geiger JD (1998) Expression of ryanodine receptors in human embryonic kidney (HEK293) cells. Biochem J 334: 79-86.
Randall A and Tsien RW (1995) Pharmacological dissection of multiple types of Ca²⁺ channel current in rat cerebellar granule neurons. J Neurosci 15: 2995-3012.[Abstract]
Rossi AD, Larsson O, Manzo L, Orrenius S, Vahter M, Berggren PO, and Nicotera P (1993) Modifications of Ca²⁺ signaling by inorganic mercury in PC12 cells. FASEB J 7: 1507-1514.[Abstract]
Sakamoto M, Ikegami N, and Nakano A (1996) Protective effects of Ca²⁺ channel blockers against methylmercury toxicity. Pharmacol Toxicol 78: 193-199.[Medline]
Schirrmacher K, Wiemann M, Bingmann D, and Büsselberg D (1998) Effects of lead, mercury and methylmercury on gap junctions and [Ca²⁺]_i in bone cells. Calcif Tissue Int 63: 134-139.[CrossRef][Medline]
Shafer TJ (1998) Effects of Cd²⁺, Pb²⁺and CH₃Hg⁺ on high voltage-activated calcium currents in pheochromocytoma (PC12) cells: potency, reversibility, interactions with extracellular Ca²⁺ and mechanisms of block. Toxicol Lett 99: 207-221.[CrossRef][Medline]
Shafer TJ and Atchison WD (1991) Methylmercury blocks N- and L-type Ca²⁺ channels in nerve growth factor differentiated pheochromocytoma cells. J Pharmacol Exp Ther 258: 697-702.
Sirois JE and Atchison WD (1996) Effects of mercurials on ligand- and voltage-gated ion channels: a review. Neurotoxicology 17: 63-84.[Medline]
Sirois JE and Atchison WD (2000) Methylmercury affects multiple subtypes of calcium channels in rat cerebellar granule cells. Toxicol Appl Pharmacol 167: 1-11.[CrossRef][Medline]
Stotz SC, Spaetgens RL, and Zamponi GW (2000) Block of voltage-dependent calcium channels by the green mamba toxin calcicludine. J Membr Biol 174: 157-165.[CrossRef][Medline]
Sutton KG, Siok C, Stea A, Zamponi G, Heck SD, Volkmann RA, Ahlijanian MK, and Snutch TP (1998) Inhibition of neuronal calcium channels by a novel peptide spider toxin, DW13.3. Mol Pharmacol 54: 407-418.[Abstract/Free Full Text]
Szucs A, Angiello C, Salanki J, and Carpenter DO (1997) Effects of inorganic and methylmercury on the ionic currents of cultured rat hippocampal neurons. Cell Mol Neurobiol 17: 273-288.[CrossRef][Medline]
Tsien RW, Lipscombe D, Madison D, Bley K, and Fox A (1995) Reflections on calcium channel diversity, 1988 -1994. Trends Neurosci 18: 52-54.[CrossRef][Medline]
Weisenberg F, Bickmeyer U, and Wiegand H (1995) Effects of inorganic mercury on calcium channel currents and catecholamine release from bovine chromaffin cells. Arch Toxicol 69: 191-196.[CrossRef][Medline]
Williams ME, Brust PF, Feldman DH, Patthi S, Simerson S, Maroufi A, McCue AF, Velicelebi G, Ellis SB, and Harpold MM (1992b) Structure and functional expression of an -conotoxin sensitive human N-type calcium channel. Science (Wash DC) 257: 389-395.[Abstract/Free Full Text]
Williams ME, Feldman DH, McCue AF, Brenner R, Velicelebi G, Ellis SB, and Harpold MM (1992a) Structure and functional expression of ₁, ₂ and subunits of a novel human neuronal calcium channel subtype. Neuron 8: 71-84.[CrossRef][Medline]
Williams ME, Marubio LM, Deal CR, Hans M, Brust PF, Philipson LH, Miller RJ, Harpold MM, and Ellis SB (1994) Structure and functional characterization of neuronal _1E calcium channel subtypes. J Biol Chem 269: 22347-22357.[Abstract/Free Full Text]
Yuan Y and Atchison WD (1994) Comparative effects of divalent mercury, methyl-mercury and phenylmercury on membrane excitability and synaptic transmission of CA1 neurons in hippocampal slices of the rat. Neurotoxicology 15: 403-412.[Medline]

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