Introduction

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Lead continues to be an environmental hazard, contaminating food, air and water supplies (Davis et al., 1993). This raises public concern because what were once considered safe levels have been proved to cause neurotoxicity, especially in children (Angle, 1993). The threshold for toxicity is now considered to be when blood levels are > 10 µg/dl (0.5 µM) (Cory-Slechta, 1995; Davis et al., 1993). Lead toxicity can be successfully treated with chelators such as EDTA, DMSA and DMPS (Angle, 1993; Aposhian, 1983); DMSA appears to be the drug of choice due to its effectiveness and lower toxicity (Aposhian et al., 1995; Graziano et al., 1992).

The mechanism or mechanisms by which Pb⁺⁺ causes neurotoxicity are poorly understood. Pb⁺⁺ does not participate in redox reactions, but it can form complexes with sulfhydryl, amine and carboxyl groups, thereby allowing for high-affinity binding to proteins (Goering, 1993). Pb⁺⁺ can bind to the metal binding sites of metallothioneins and to proteins with E-F hand motifs such as calmodulin. In many cases, Pb⁺⁺ can mimic the action of Ca⁺⁺ ; Pb⁺⁺ can activate Ca⁺⁺ -dependent protein kinase C (Markovac and Goldstein, 1988), troponin C (Chao et al., 1990) and Ca⁺⁺ -activated K⁺ channels (Oortgiesen et al., 1993). Pb⁺⁺ can also act as an antagonist, blocking Ca⁺⁺ permeation through voltage-gated Ca⁺⁺ channels. In contrast, Pb⁺⁺ does not block Na⁺ or K⁺ channels (Büsselberg et al., 1991; Reuveny and Narahashi, 1991). Because Ca⁺⁺ is a key second messenger, it seems likely that Pb⁺⁺ causes neurotoxicity by disrupting Ca⁺⁺ homeostasis (Simons, 1993).

Lead block of voltage-gated Ca⁺⁺ channels has been studied in a number of cell types (for a review, see Audesirk, 1993). Voltage-gated Ca⁺⁺ channels are multisubunit complexes (Perez-Reyes and Schneider, 1994). The alpha-1 subunit contains the pore, the voltage-sensor, and most of the drug binding sites. The genes for six alpha-1 subunits have been cloned. On the basis of sequence identity, these alpha-1 subunits can be divided into two subfamilies: (1) L-type, alpha-1S (skeletal), alpha-1C (cardiac and brain) and alpha-1D (neuroendocrine); and (2) non-L-type, alpha-1A (brain P/Q-type), alpha-1B (brain N-type) and alpha-1E (brain R-type). Purification studies have shown that skeletal and cardiac muscle L-type and neuronal N-type channels also possess common alpha-2-delta subunits and a specific beta subunit. Four beta subunit genes have been cloned (Perez-Reyes and Schneider, 1994). Coexpression studies have shown that beta subunits can increase the functional expression of alpha-1 and modulate the biophysical properties of the channel (Perez-Reyes and Schneider, 1994). Coexpression with alpha-2-delta also increases functional expression and modifies the pharmacological properties of the cloned channel (Mikami et al., 1989; Shistik et al., 1995; Wei et al., 1995).

Previous studies have established that voltage-gated Ca⁺⁺ channels can be blocked by low (µM) concentrations of Pb⁺⁺. Washout-resistant block, termed "irreversible inhibition" (Audesirk, 1993), has been noted before but not studied in detail. Because Ca⁺⁺ channels are highly regulated by protein kinases and Pb⁺⁺ is capable of activating protein kinase C at very low concentrations (nM), it is possible that the observed block was not due to a direct effect of Pb⁺⁺ on the channel. Because the cloned L-type Ca⁺⁺ channel is not regulated by protein kinases (Zong et al., 1995), we were able to study the direct effects of Pb⁺⁺ on Ca⁺⁺ channel activity. We describe the toxicological effect of Pb⁺⁺ on L-type Ca⁺⁺ channels, pharmacological relief of block and some biophysical properties of Pb⁺⁺ block and unblock and show that there are two types of block: one is reversed by washing with lead-free solutions, whereas the second type requires treatment with lead antidotes such as DMSA or EDTA.

	Materials and Methods

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Materials. High purity lead acetate (99.999%) was purchased from Aldrich Chemical Co. (Milwaukee, WI). It was dissolved in water at a concentration of 10 mM. Serial dilutions were freshly prepared and then diluted 1:100 in the bath solution to give the indicated concentration. In contrast to lead chloride (Matthews et al., 1993), no white precipitate was observed in any of the lead acetate solutions. The concentration of free Pb⁺⁺ in the bath solution was not determined, but it may either be lower than expected due to binding to ligands in the bath solution or higher due to Pb⁺⁺ contamination of the reagents (Matthews et al., 1993; Simons, 1993). All other chemicals were purchased from Aldrich or Sigma (St. Louis, MO).

Oocyte expression. Capped cRNAs were synthesized in vitro using T7 RNA polymerase and the mMESSAGE mMACHINE kit (Ambion, Austin, TX). The concentration of cRNA was measured spectrophotometrically. The production of full-length cRNA transcripts was verified after electrophoretic separation on a denaturing agarose gel.

Electrophysiological analysis of injected oocytes. Oocytes were voltage-clamped using a two-microelectrode voltage-clamp amplifier (OC-725B, Warner Instrument Corp., Hampden, CT). Pipettes were made from glass capillaries (#6010, A-M Systems, Everett, WA), using a Model P-97 Flaming-Brown pipette puller (Sutter Instrument Co., Novato, CA). Voltage and current electrodes (1.8-2.6-M tip resistance) were filled with 3 M KCl. The oocytes were impaled in SOS solution, and then the bath solution was exchanged with a solution of 10 mM Ba(OH)₂, 80 mM NaOH, 1 mM KOH and 5 mM HEPES, adjusted to pH 7.4 with methanesulfonate (Lory et al., 1990). Because methanesulfonic acid may bind Pb⁺⁺, experiments were repeated using 10 mM BaCl₂, 90 mM NaCl, 2 mM KCl and 5 mM HEPES, pH 7.4. Similar results were obtained, and the data were pooled. Data were acquired at 2000 Hz using the pCLAMP system (Digidata 1200 and pCLAMP 6.0, Axon Instruments, Foster City, CA) and filtered at 400 or 1000 Hz (#902 Frequency Devices, Haverhill, MA).

Generation of a stably transfected HEK 293 cell. The cDNA insert encoding the alpha-1 subunit was subcloned into the expression vector pRC/CMV (InVitrogen, San Diego, CA). This vector contains a neomycin resistance gene, which allows for selection of transfected cells with geneticin (G418; GIBCO, Grand Island, NY). HEK 293 cells (1 × 10⁶ cells in a 100-mm culture dish) were transfected with 10 µg of alpha-1 cDNA using lipofectamine (GIBCO). At 24 hr after transfection, the cells were suspended in Dulbecco's modified Eagle's medium supplemented with G418 (0.4 g/L) and fetal bovine serum (10%). Individual colonies were isolated and plated onto 24-well plates. The clones were expanded and then analyzed for alpha-1 protein expression using dihydropyridine binding assays with the ligand (+)-[³H]PN200-110 (Amersham, Arlington Heights, IL). Single-point assays were done as described previously (Perez-Reyes et al., 1992). Cell clone A20, which expressed 100 fmol of binding sites/mg of membrane protein, was selected for further study. Results were also obtained using cell clone LCa10; this cell line is a subclone of HCa₁₂-2 (Perez-Reyes et al., 1994). During passage in culture, HCa₁₂-2 lost expression of beta-2. A single cell was recloned, expanded and characterized. Polymerase chain reaction using beta-specific primers confirmed the lack of beta-2 mRNA expression. Results using the two alpha-1-transfected cell lines were identical and have been pooled.

Electrophysiological analysis of HEK 293-transfected cells. HEK 293 cells were plated onto coverslips and cultured for 1 to 5 days before electrophysiological studies. Cells were grown in Dulbecco's modified Eagle's medium supplemented with 0.4 g/L G418, 10% fetal bovine serum, 100 units/ml penicillin and 100 µg/ml streptomycin. To minimize dialysis of the cell and Ca⁺⁺ channel rundown, we used the perforated patch technique with amphotericin B (Rae et al., 1991). Amphotericin B was freshly dissolved (30 mg/ml) in dimethylsulfoxide. This stock was diluted to 0.24 mg/ml into a typical internal pipette solution that contained 55 mM CsCl, 75 mM CsSO₄, 10 mM MgCl₂, 0.1 mM EGTA and 10 mM HEPES, pH adjusted to 7.2 with CsOH (Perez-Reyes et al., 1994). Pipette tips were briefly dipped into this internal solution and then backfilled with the same solution plus amphotericin B. The external Tyrode solution contained 140 mM NaCl, 6 mM KCl, 2 mM CaCl₂, 10 mM glucose and 5 mM HEPES, pH 7.4. The recording solution contained 10 mM BaCl₂ solution (or 2 mM CaCl₂ for ventricular myocytes), 140 mM TEA chloride, 5 mM CsCl, 1 mM MgCl₂, 5 mM glucose and 10 mM HEPES, pH adjusted to 7.4 with TEA-OH (Perez-Reyes et al., 1994). Preliminary experiments used the following recording solution: 10 mM BaCl₂, 130 mM aspartic acid, 130 mM NMG, 10 mM aminopyridine, 1 mM MgCl₂, 10 mM glucose and 10 mM HEPES, pH adjusted to 7.3 with aspartic acid (de Leon et al., 1995).

Data analysis. All experiments began by recording of control currents (~5 min). Only cells with stable currents were used for analysis. All test compounds were diluted in external solution and then perfused into the bath at a rate of 2 to 4 ml/min. The perfusion rate varied between experiments but was similar between solutions in any single experiment. The bath chamber had a volume of .15 ml. The bath was continuously perfused throughout the experiment. Experiments were conducted at room temperature, 22° to 24°C.

	Results

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Figure 1 shows current traces recorded from cells before and after exposure to 1 and 10 µM Pb⁺⁺ and then after washout. Inward Ca⁺⁺ currents in rat ventricular myocytes are blocked by low concentrations of Pb⁺⁺ (fig. 1A). This current is through L-type Ca⁺⁺ channels (Bean, 1989). The minimum subunit composition of these channels is alpha-1C, alpha-2 and beta-2 (Perez-Reyes and Schneider, 1994). Expression of the cloned alpha-1C subunit alone induces dihydropyridine-sensitive currents that can be measured with Ba⁺⁺ as the charge carrier (Mikami et al., 1989). Pb⁺⁺ also blocks inward Ba⁺⁺ currents through channels composed of alpha-1C alone, expressed in either X. laevis oocytes (fig. 1B) or HEK 293 cells (fig. 1C). Because it has been previously shown that currents from the cloned channel are not regulated by protein kinases (Zong et al., 1995), and hence more stable, we selected these for further study.

View larger version (K): [in this window] [in a new window]   Fig. 1.   Pb++ block of L-type Ca++ channel currents. Inward currents were elicited by test depolarizations to +10 mV (A and C) or +20 mV (B) from a holding potential of 80 mV. Traces were recorded before (control), after exposure to bath solution containing the indicated concentrations (µM) of lead and after washout with lead-free solution. A, Pb++ block of native L-type Ca++ channels in rat ventricular myocytes was measured using 2 mM Ca++ as charge carrier. Currents were recorded using the ruptured patch-clamp technique. B, Pb++ block of cloned L-type Ca++ channels in X. laevis oocytes injected with alpha-1C and beta-2. Currents were recorded with a two-microelectrode voltage-clamp using solutions containing 10 mM Ba++ as charge carrier. C, Pb++ block of a cloned L-type Ca++ channel composed of alpha-1C alone expressed in HEK 293 cell line LCa10. Currents were recorded using the perforated patch-clamp technique. The extracellular solution contained 10 mM Ba++ as charge carrier. Residual capacitative transients were deleted.

Lead dose response was measured by adding lead acetate to the bath solution and then measuring the block of cloned L-type currents expressed in oocytes (fig. 2A). Preliminary experiments suggested that block of alpha-1C expressed alone in HEK 293 cells was less sensitive to block than alpha-1C-beta currents in oocytes (fig. 2B). Because beta subunits alter channel gating (Perez-Reyes and Schneider, 1994), we tested the hypothesis that subunit composition may alter Pb⁺⁺ block. Oocytes were injected with cRNA for alpha-1C, alpha-1C-beta-2, alpha-1C-beta-4 or alpha-1C-alpha-2-delta-beta-2. Despite large differences in the resulting current amplitudes (Perez-Reyes et al., 1992; Wei et al., 1995), Pb⁺⁺ blocked the currents with equal potency; therefore, pooled data were used for dose-response analysis (fig. 2A; IC₅₀ = 152 ± 87 nM). L-type currents recorded from ventricular myocytes were also blocked to a similar extent.

View larger version (K): [in this window] [in a new window]   Fig. 2.   Dose dependence of Pb++ block. Inhibition of L-type Ca++ channel activity by Pb++ was measured in (A) oocytes and myocytes or (B) HEK 293 cells. Currents were elicited using the voltage protocols described in the legend to figure 1. A, Oocytes were injected with alpha-1C alone (), alpha-1C-beta-2 () and alpha-1C-beta-2-alpha-2-delta (). , Percent block of rat ventricular myocyte currents at 1 µM Pb++. The number of observations varies between points but is greater >4 (range, 4-25; recorded from 76 oocytes). Because there was no difference between the subunit combinations, the data were pooled and then fit (smooth line) using a sigmoidal dose-response equation (R2 = 0.98, Hill slope = 0.5). B, Pb++ block of alpha-1C currents in HEK 293 cells was measured using two external bath solutions that contained either 140 mM TEA () or 130 mM NMG and 130 mM aspartic acid (). The smooth curves were generated from fits to the data (for both R2 = 0.97, Hill slope = 0.5).

Figure 2B shows Pb⁺⁺ block of alpha-1-induced currents in HEK 293 cells. We tested the hypothesis that the bath solution used for HEK 293 cell experiments was chelating Pb⁺⁺. Lead was a much more potent blocker in 10 mM BaCl₂ solutions containing TEA (IC₅₀ = 169 nM) than in solutions containing both NMG and aspartic acid (IC₅₀ = 23 µM). Therefore, low concentrations of Pb⁺⁺ blocked the cloned L-type channel in both expression systems and L-type channels in situ.

Two protocols were used to measure Pb⁺⁺ dose responses: (1) where increasing concentrations of Pb⁺⁺ were tested sequentially and (2) where there was a washout with control solution between Pb⁺⁺ exposures (fig. 3). Figure 3 shows that Pb⁺⁺ block was fast and quickly reversed by washout, allowing for many concentrations to be tested. It is common for L-type Ca⁺⁺ channel currents to rundown (McDonald et al., 1994). In contrast, currents recorded from alpha-1C-injected oocytes displayed very little rundown (0.23% per minute, n = 22; see figs. 5 and 6). We also noticed that the extent of recovery was dependent on Pb⁺⁺ concentration. Thus, a component of Pb⁺⁺ block was resistant to washout, or "irreversible" under the time scale of these experiments (Audesirk, 1993). To quantify this component, we compared the currents after washout to the original pre-Pb⁺⁺ control and then calculated the amount of washout resistant block (fig. 3B). Significant washout-resistant block could be measured after exposure to concentrations as low as 10 nM Pb⁺⁺ (8.7 ± 2.7, n = 8).

View larger version (K): [in this window] [in a new window]   Fig. 3.   Pb++ inhibition is not totally reversed by washout. A, L-type Ca++ channel currents were measured from a single oocyte injected with alpha-1C-beta-2. Currents were elicited every 15 sec with a test pulse to +20 mV from a holding potential of 80 mV. The concentration (µM) and time of Pb++ application are indicated at the top. Between Pb++ exposures, the oocyte was washed with control solution (flow, 2-4 ml). B, Dose dependence of the washout-resistant block. Currents recovered after extensive washing with control solution were compared with the currents measured before the cell was exposed to Pb++. The effects of rundown were minimized by excluding experiments in which rundown exceeded 1% per minute (average, 0.23 ± 0.04%/min, n = 20) and by including results from other experimental protocols in which only one dose of Pb++ was tested (e.g., figs. 4 and 6). Values represent the mean ± S.E.M. (n, 7-25). The smooth curve was generated from a fit of the data using a sigmoidal dose-response equation (R2 = 0.99, Hill slope = 0.4).

View larger version (K): [in this window] [in a new window]   Fig. 5.   Full reversal of Pb++ block requires treatment. Currents were recorded from oocytes injected with alpha-1C-beta-2-alpha-2-delta. Average time course (n = 4) shows block by 100 µM Pb++, washout and then treatment with 100 µM Pb++ plus 200 µM EDTA. The control currents averaged 2.5 µA.

View larger version (K): [in this window] [in a new window]   Fig. 6.   Requirement for DMSA treatment to restore channel activity after exposure to 100 nM Pb++. A, Representative current traces from an experiment B recorded from an oocyte injected with alpha-1C-beta-2. Currents were measured during test pulses to +20 mV every 30 sec. Currents were recorded without leak subtraction. The current recorded in 100 nM Pb++ was blocked with a time constant of 7 msec. The control bath solution was 10 mM BaCl2 (see text for composition) supplemented with 10 µM DMSA or 100 nM lead acetate. C, Averaged response to the protocol shown in B. Currents were normalized to the value measured during control-1 and then averaged (n = 6). Similar results were obtained after 5- or 10-min exposure to 100 nM Pb++, so the data were pooled. Control-2 is significantly different from control-1 (P = .012). The decrease in current amplitude between DMSA-1 and DMSA-2 was used to calculate the apparent rundown rate (7% in 37 min). A similar decrease was observed between control-1 and control-3 (9%).

Experiments were performed to test the efficacy of heavy metal poisoning antidotes, such as EDTA or DMSA (Aposhian, 1983; Angle, 1993; Aposhian et al., 1995), to reverse the Pb⁺⁺ block of L-type channels. To mimic the clinical situation, we first induced Pb⁺⁺ block and then tried to reverse block using a solution containing both antidote and Pb⁺⁺. Figure 4 shows that DMSA, DMPS and EDTA could fully reverse Pb⁺⁺ block, even when very high (>10 µM) concentrations of Pb⁺⁺ were used. All three drugs caused a small stimulation relative to control activity (DMSA, 112 ± 2%, n = 14; EDTA, 117% ± 3%, n = 7; and DMSA, 118%, n = 2). This stimulation may be due to the presence of Pb⁺⁺ in the reagents used (Simons, 1993).

View larger version (K): [in this window] [in a new window]   Fig. 4.   Heavy metal poisoning antidotes completely reverse Pb++ block. L-type Ca++ channel currents were measured in oocytes injected with alpha-1C-beta-2-alpha-2-delta. Peak current amplitude was normalized to the original control current. Oocytes were first exposed to a saturating concentration of Pb++ (10 or 100 µM), followed by treatment with Pb++ plus a 2-fold molar excess of DMSA (n = 3), DMPS (n = 6) or EDTA (n = 4).

We also tested the effect of the antidotes on the washout-resistant component of Pb⁺⁺ block. Oocytes were treated with a high concentration (100 µM) of Pb⁺⁺, washed with control solution and then treated with Pb⁺⁺ plus EDTA (fig. 5). As shown above, washout alone did not completely restore the currents to control levels; however, treatment with Pb⁺⁺ plus EDTA did. The concentration of Pb⁺⁺ used in these experiments was >600-fold higher than the apparent IC₅₀ value. This raises the possibility that a small contamination could be causing the observed block. For example, Pb⁺⁺ may have accumulated in the cell, or on the recording chamber, and then slowly released during the control wash. To rule out these possibilities, we conducted experiments using 100 nM Pb⁺⁺ (fig. 6). Representative current traces are shown in figure 6A. Because DMSA causes a small stimulation of currents, this experiment contains three control washes (representative experiment is shown in fig. 6B). The first control was used to establish the base-line current and to measure the stimulation by DMSA over control. The second control is the washout of Pb⁺⁺, and the steady-state value was compared with control-1 to determine the washout-resistant inhibition. The third control is the washout after a short treatment (5 min) with 10 µM DMSA. If Pb⁺⁺ accumulation, or contamination, was causing the washout-resistant inhibition, then this third control would have returned to the value measured during control-2 (75 ± 4%, n = 4, fig. 6C). This was not the case; control-3 activity returned to 91 ± 3% of control-1. The deviation from 100% is presumably due to channel rundown.

Figure 7 shows average current-voltage relationships measured in oocytes (fig. 7A) and transfected HEK 293 cells (fig. 7B) in either the absence or presence of Pb⁺⁺. The percent inhibition observed at each test potential was averaged and then plotted (fig. 7, C and D). Very little block by 1 µM Pb⁺⁺ was observed when the test potential was near threshold (20 mV). Pb⁺⁺ was a more effective blocker during pulses to higher potentials. Although less pronounced, voltage-dependent block was also observed at higher Pb⁺⁺ concentrations (10 µM; fig. 7C). Similar voltage-dependent block was observed in HEK 293 cells (fig. 7D).

View larger version (K): [in this window] [in a new window]   Fig. 7.   Voltage dependence of Pb++ block. Plateau currents are plotted as a function of test potential. A, Currents were recorded from oocytes injected with alpha-1C-beta-2 (n = 3), in either the absence () or presence () of 1 µM Pb++. B, Average (n = 8) current-voltage relationships measured from alpha-1C-transfected HEK 293 cells in the absence () or presence () of 30 µM Pb++. These experiments were performed using the 10 mM Ba++ solution that also contained NMG and aspartic acid. C, Percent inhibition measured in oocytes by either 1 () or 10 () µM Pb++. D, Percent inhibition measured in HEK 293 cells by 30 µM Pb++ ().

Strong depolarizations have been shown to enhance L-type channel gating (Pietrobon and Hess, 1990) and to reverse Cd⁺⁺ block of Ca⁺⁺ currents from frog sympathetic neurons (Thévenod and Jones, 1992). Our hypothesis was that Pb⁺⁺ was an open channel blocker, similar to Cd⁺⁺, and that strong depolarizations may remove block. Voltage protocols contained the following three depolarizations: (1) a control test pulse to +20 mV, (2) a depolarizing pulse of varying amplitude and (3) a second test pulse. Between the depolarizations was a short (10 msec) repolarization to 80 mV. Due to the large oocyte capacitance, these experiments were performed using only transfected HEK 293 cells. Figure 8A shows that the middle pulse had little (slight inhibition) or no effect on control currents. Figure 8B shows the currents recorded in the presence of Pb⁺⁺. Larger currents were measured in the second test pulse when the interpulse potential was >50 mV. To quantify this increase, peak currents were measured in the second test pulse and then divided by the current remaining at the end of the first test pulse. Figure 8C plots this postpulse-to-prepulse current ratio as a function of the depolarizing test potential.

View larger version (K): [in this window] [in a new window]   Fig. 8.   Strong depolarizations unblock the channel. Currents were measured in alpha-1C-transfected HEK 293 cells. The voltage protocol contained three depolarizations: pulse 1 was a step to +20 mV (prepulse), pulse 2 was an interpulse to varying potentials and pulse 3 was a step to +20 mV (postpulse). Currents were measured in the absence (A) or presence (B) of 30 µM Pb++. This experiment was performed using the 10 mM Ba++ solution that also contained NMG and aspartic acid. Capacitative transients and leak currents were not subtracted. The large outward currents elicited during the interpulse are not shown. Unblock was measured by dividing the peak current in the postpulse by the plateau current in the prepulse. C, Ratio of postpulse to prepulse current was plotted as a function of the interpulse potential. Same data shown in (A) control () and (B) Pb++ (). D, Currents were enlarged and superimposed to show the kinetics of Pb++ block. The control trace is taken from the first pulse in episode. The Pb++ traces are taken from the third pulse in episodes 1 (interpulse to +40 mV) and 13 (interpulse to +160 mV).

The ability of strong depolarizations to remove Pb⁺⁺ block allows for measurement of Pb⁺⁺ blocking kinetics during the second test pulse. Figure 8D shows an overlay of currents measured during control (first test pulse, episode 1), in the presence of Pb⁺⁺ (second test pulse, after +40 mV depolarization, episode 1) and after a strong depolarization in the presence of Pb⁺⁺ (second test pulse, after +160 mV depolarization, episode 13). Control currents displayed very little decay over this time period. In contrast, currents taken in the presence of Pb⁺⁺ did decay (currents in first test pulse, fig. 7B). This decay was greater after a strong depolarization. In analogy to Thévenod and Jones, we interpret this decay as due to Pb⁺⁺ blocking of open channels. Exponential fits to the data indicate that Pb⁺⁺ reblocks with a  value of 11 msec. This rate was both voltage (during a 0- mV test pulse  = 17 msec) and concentration dependent (at a 3-fold lower concentration of Pb⁺⁺, the reblock  was 17 msec at +20 mV and 42 msec at 0 mV).

	Discussion

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Lead is a potent blocker of L-type Ca⁺⁺ channels, inhibiting both native and cloned channels with an IC₅₀ of 150 nM. Similar total lead concentrations (500 nM) in blood are considered to be the threshold for toxicity (Cory-Slechta, 1995; Davis et al., 1993). It has been noted that the concentration of free Pb⁺⁺ in plasma is much lower than the total blood concentration (Audesirk, 1993). Similarly, free Pb⁺⁺ concentrations in vitro may be either much lower than expected due to binding to buffer components or higher due to contamination of reagents (Matthews et al., 1993; Simons, 1993). For example, we found that the potency of Pb⁺⁺ block depended on the composition of the bath solution. Both solutions contained 10 mM BaCl₂, but one contained TEA and the other contained NMG and aspartic acid. It is likely that Pb⁺⁺ was buffered by aspartic acid. Despite these uncertainties in the free Pb⁺⁺ concentration, our results clearly show that Pb⁺⁺ has a very high affinity for L-type channels.

Ca⁺⁺ influx via voltage-gated Ca⁺⁺ channels is a highly regulated event. Cardiac L-type channels are regulated by both cAMP-dependent protein kinase and protein kinase C (McDonald et al., 1994). Pb⁺⁺ can stimulate protein kinase activity (Markovac and Goldstein, 1988); therefore, direct effects of Pb⁺⁺ on the channel can be obscured by its effects on kinases. We avoided this problem by using the cloned alpha-1C, which is not regulated by protein kinases as observed in situ (Zong et al., 1995).²

Simple washout of Pb⁺⁺ did not completely reverse the block of L-type Ca⁺⁺ channels. During our dose-response analysis, we noticed a difference between protocols in which Pb⁺⁺ was added sequentially and protocols that included a wash. Washout-resistant block has been reported previously in studies using rat dorsal root ganglion neurons (Büsselberg et al., 1994) and snail neurons (Audesirk, 1993). This lack of recovery was termed "irreversible inhibition" and surprisingly was not dose dependent (Audesirk, 1993). In contrast, we found that washout-resistant block was dose dependent, following a similar dose response as the total block. We show for the first time that complete recovery of currents required treatment with heavy metal antidotes, such as DMSA, DMPS and EDTA. The ability of these antidotes to recover 100% of the original channel activity indicates that this washout-resistant inhibition is not simply due to channel rundown. A clinical implication of these findings is that some Pb⁺⁺ targets will continue to be blocked after removal of Pb⁺⁺ and that full reversal of lead poisoning requires treatment.

Numerous heavy metals, including Pb⁺⁺, block voltage-gated Ca⁺⁺ channels (Audesirk, 1993; McDonald et al., 1994). Heavy metal block has been studied because it provides (1) a toxicological description of heavy metal action, (2) a basis for classifying channel subtypes and (3) clues on the structure-function relationships of the channel. In many studies, the blocked currents are subtracted from the control currents to infer block of channel subtypes that differ in their inactivation rates (Audesirk and Audesirk, 1993). Our studies indicate that Pb⁺⁺ block occurs during the pulse, thereby complicating this type of analysis. Studies with Pb⁺⁺ have established that it is selective for voltage-gated Ca⁺⁺ channels, with little effect on Na⁺ or K⁺ channels (Büsselberg et al., 1991; Reuveny and Narahashi, 1991). Unlike Ni⁺⁺, Pb⁺⁺ does not appear to be very selective among the various voltage-gated Ca⁺⁺ channels, blocking low- and high-threshold channels with similar potency (Audesirk, 1993).

The mechanism by which Pb⁺⁺ blocks Ca⁺⁺ channels is poorly understood. Largely based on the observation that increasing extracellular Ca⁺⁺ decreased Pb⁺⁺ block, it was suggested that Pb⁺⁺ was an open channel blocker (Büsselberg et al., 1991). Our results indicate that Pb⁺⁺ block is very similar to the open channel blocker Cd⁺⁺; block is voltage dependent (Chow, 1991; Swandulla and Armstrong, 1989) and can be relieved by strong depolarizations (Thévenod and Jones, 1992). Single-channel studies have allowed direct measurement of the rate of Cd⁺⁺ block and unblock (Lansman et al., 1986). These studies found that the rate of unblock was voltage dependent, whereas the rate of block was largely voltage independent. Because these changes occurred in the same voltage range as channel activation, we can infer that different states of the channel have different affinity for blocker. By analogy, we predict that the rate of Pb⁺⁺ unblock is voltage dependent. At potentials of <20 mV, when most channels are in the closed state, we predict that Pb⁺⁺ unblock is faster than block, resulting in little tonic block. At potentials where channel opening is favored (>+20 mV), we predict that Pb⁺⁺ block is faster than unblock, resulting in a time-dependent block. This would also explain the observed voltage dependence of Pb⁺⁺ block. Similar voltage-dependent block was observed in studies using neurons from Aplysia and rat dorsal root ganglion (Büsselberg et al., 1991) but not rat hippocampus (Audesirk and Audesirk, 1993). Perhaps the voltage dependence of block was obscured by the fact that neurons contain multiple subtypes of Ca⁺⁺ channels that differ in their voltage dependence.

Surprisingly, stronger depolarizations (>50 mV) also caused unblock. We took advantage of this unblock phenomenon to measure reblock kinetics. We found that the Pb⁺⁺ block of open channels was fast ( = 11 msec) and voltage dependent. The mechanism by which strong depolarizations reduce divalent cation block are not understood. Strong depolarizations have been shown to alter L-type channel gating (Pietrobon and Hess, 1990). Possibly, this depolarization is inducing a channel conformation with lower affinity for Pb⁺⁺. A second possibility is that reverse current flow through the channel knocks Pb⁺⁺ off its binding site in the pore. An implication of this result is that facilitation may be due to unblock of channels by contaminating divalent cations. In addition, these results provide another example of how the cloned channel is not regulated in the same manner as in situ channels.

In addition to blocking Ca⁺⁺ channels, Pb⁺⁺ and Cd⁺⁺ may permeate these channels (Chow, 1991; Tomsig and Suszkiw, 1991). Perhaps Pb⁺⁺ permeates the channel at negative test potentials where the driving force for Ba⁺⁺ is high (E_rev = +60).

Ca⁺⁺ channels are multisubunit complexes that contain a large alpha-1 subunit that forms the pore and several auxiliary subunits (Perez-Reyes and Schneider, 1994). Molecular cloning has revealed that there is a family of at least six related alpha-1 subunits. One region that is particularly well conserved among these subtypes is the pore-lining region (Perez-Reyes and Schneider, 1994). This region contains glutamate residues that are thought to form a ring of negative charge. Mutagenesis studies have shown that mutation of these glutamates alters divalent cation permeation and block (Parent and Gopalakrishnan, 1995; Tang et al., 1993; Yang et al., 1993). Due to sequence conservation between the cloned alpha-1 subunits, we predict that Pb⁺⁺ block will be very similar between these Ca⁺⁺ channel subtypes. We suggest that the quickly reversible block represents Pb⁺⁺ binding to this site. In contrast, washout-resistant block may be due to Pb⁺⁺ binding to a second site from which it only slowly dissociates. Pb⁺⁺ can be removed from the second site by chelators, indicating that this site is accessible from the extracellular medium. Recent mutagenesis studies support the hypothesis that there are two divalent cation binding sites in the pore (Parent and Gopalakrishnan, 1995).

In addition to heart, L-type Ca⁺⁺ channels are distributed throughout the brain. In particular, in situ hybridization has shown that alpha-1C is abundantly expressed in the hippocampus (Tanaka et al., 1995). Because our studies show that subunit composition does not affect Pb⁺⁺ block, these results should be applicable to different neuronal subtypes. In conclusion, L-type Ca⁺⁺ channels are so susceptible to Pb⁺⁺ that they may be blocked during lead poisoning, contributing to the observed neurotoxicity.