Introduction

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Voltage-sensitive Ca²⁺ channels regulate a number of critical cellular functions in the nervous system, such as synaptic transmission (Catterall, 2000). Several distinct subtypes of neuronal "high-voltage-activated" Ca²⁺ channels (L, N, P/Q, and R) have been identified based on their biophysical and pharmacological properties (Tsien et al., 1995). These channels consist of four subunits: ₁, , ₂, and ; the ₁ subunit is the pore-forming, voltage-sensing, ligand-binding, and subtype-determining moiety (Hofmann et al., 1999). At least six distinct ₁ subunits have been cloned for high-voltage-activated Ca²⁺ channels, encoding _1A, _1B, _1C, _{1D, 1E}, and _1S phenotypes. Expression studies have shown that _1C, _1D, and _1S phenotypes encode dihydropyridine-sensitive Ca²⁺ channels (L-type) (Williams et al., 1992a; Tomlinson et al., 1993), _1B encodes -conotoxin GVIA-sensitive Ca²⁺ channels (N-type) (Williams et al., 1992b; Cahill et al., 2000), _1A encodes -agatoxin IVA-sensitive Ca²⁺ channels (P/Q-type) (Mori et al., 1991; Stea et al., 1994), and _1E encodes Ca²⁺ channels resistant to most currently known specific inhibitors (R-type) (Williams et al., 1994; Bourinet et al., 1996), but partially susceptible to a tarantula spider toxin SNX 482 (Bourinet et al., 2001) and evidently also susceptible to block by Cd²⁺. Four different  subunits₁ to ₄and two different ₂ subunits serve to regulate assembly and modulate the kinetic parameters of the channels. Each of these subunit types can also have various isoforms and splice variants, further complicating the functional expression characteristics and classification (Brust et al., 1993; De Waard and Campbell, 1995; McEnery et al., 1998; Pan and Lipscombe, 2000). Because of their portal location within the plasma membrane, Ca²⁺ channels are readily exposed to toxicants, and are potentially early targets of the actions of a number of toxicants (Kiss and Osipenko, 1994). In view of the crucial roles that Ca²⁺ channels play in key cellular functions, toxicant effects on Ca²⁺ channels could have significant deleterious consequences for cell function.

Lead (Pb²⁺) is a commonly occurring and persistent environmental neurotoxicant that causes block of function of voltage-activated Ca²⁺ channels (Audesirk and Audesirk, 1989, 1991; Reuveny and Narahashi, 1991; Oortgiesen et al., 1993) and evidently uses these channels as a means of entry into cells (Simons and Pocock, 1987). Inhibitory effects of acute exposure to Pb²⁺ on native Ca²⁺ channels have been reported for invertebrate neurons (Audesirk and Audesirk, 1989; Büsselberg et al., 1991), rat dorsal root ganglion neurons (Evans et al., 1991; Büsselberg et al., 1994) pheochromocytoma cells (PC12) (Hegg and Miletic, 1996, 1998; Shafer, 1998), mouse and human neuroblastoma (Audesirk and Audesirk, 1991; Reuveny and Narahashi, 1991; Oortgiesen et al., 1993), and bovine chromaffin cells (Tomsig and Suszkiw, 1991; Sun and Suszkiw, 1995). However, although different cell types show apparently differential sensitivity to Pb²⁺, there are very few reports on the effects of Pb²⁺ on specific defined subtypes of Ca²⁺ channels, and there are no comparative studies using recombinant channels to test for differences among distinct Ca²⁺ channel phenotypes. Moreover, despite the clear neurotoxicity of Pb²⁺, there are no reports of effects of the metal on heterologously-expressed cloned neuronal Ca²⁺ channels, although Bernal et al. (1997) reported that Pb²⁺ suppressed the function of stably expressed L-type cardiac Ca²⁺ channels from rabbit. The objective of the present study was to compare and characterize the acute effects of Pb²⁺ on isolated, distinct phenotypes of voltage-activated Ca²⁺ channels typically expressed in neurons. Because of the multitude of intracellular actions that Pb²⁺ has, many of which could influence Ca²⁺ channel function, such as its well-known interaction with protein kinase C (Markovac and Goldstein, 1988), we limited the comparison to effects that are likely to occur solely at the membrane level to eliminate the confounding problem of possible interaction of Pb²⁺ with specific intracellular components of the channel. As such, we used a single, constant  subunit. We discuss how the effects of Pb²⁺ on recombinant channels compare with those presumably similar channel phenotypes (based on pharmacological sensitivities) when expressed in a native setting. Transiently expressed human neuronal voltage-dependent Ca²⁺ channels were used to examine toxic effects of Pb²⁺ as applied acutely in the extracellular solution on one specific subtype of Ca²⁺ channel at a time. Expression cDNA clones of _1C, _1B, or _1E subunits coding for neuronal L-, N-, and R-subtypes, respectively, were combined with a constant ₂ and ₃ Ca²⁺ channel subunits of human neuronal origin to transfect human embryonic kidney (HEK) 293 cells. Jellyfish green fluorescent protein (GFP) was used as a cotransfection reporter. Characteristics of current conducted through each channel subtype as well as the effects of Pb²⁺ on that current were examined after transient transfection using whole-cell, voltage-clamp recording techniques and Ba²⁺ as charge carrier.

	Materials and Methods

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Materials. HEK 293 cells were purchased from the American Type Culture Collection (Manassas, VA). All reagents were pure or ultrapure laboratory grade unless specifically noted. ATP-Mg, cAMP, HEPES, EGTA, and tetrodotoxin were all obtained from Sigma Chemical Co. (St. Louis, MO). Stock solutions (10 mM) of lead acetate (J. T. Baker Chemical Co., Phillipsburg, NJ) were prepared weekly in double-distilled water, from which test solutions were prepared in extracellular solution just before each experiment. Expression cDNA clone plasmids of human neuronal Ca²⁺ channel subunits used in the study were all generously provided by Dr. Kenneth A. Stauderman of SIBIA Neurosciences (San Diego, CA), now Merck Research Laboratories. The human gene source tissues were as follows: _1B-1, neuroblastoma cell line IMR32 (Williams et al., 1992b); _1C-1, hippocampus (M. E. Williams, unpublished observations); _1E-3, hippocampus (Williams et al., 1994); ₂, brainstem and basal ganglia (Williams et al., 1992a); and ₃, hippocampus (M. E. Williams, unpublished observations). GFP sequences were removed from pEGFP-1 (BD Biosciences Clontech, Palo Alto, CA) and subcloned in pCDNA3.1 (Invitrogen, Carlsbad, CA) in our laboratory.

Cell Culture and Transfection. HEK 293 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% (w/v) fetal bovine serum, and penicillin, streptomycin, and antimycotic mixture (final concentrations: 100 U/ml penicillin; 100 µg/ml streptomycin; and 0.25 µg/ml amphotericin B as Fungizone) (Invitrogen) in a 5% CO₂ environment. One day before gene transfer, cells were plated at a density of 5 × 10⁵ on 35-mm culture dishes. Cells were transfected with a mixture of plasmids containing either _1B-1, _1C-1, or _1E-3 Ca²⁺ channel subunits together with _2b, _3a, and the GFP cDNA clone, using Fugene 6 (Roche Molecular Biochemicals, Indianapolis, IN) following the manufacturer's instructions. Reactions contained a total of 3 µl of Fugene 6 and 1 µg of plasmid DNA containing the three channel subunits in 1:1:1 M ratio, with GFP plasmid at 20% of the total DNA. Two days were allowed for optimal, transient expression of proteins, at which time the cells were examined for GFP expression. Cells from dishes with a number of green fluorescent cells were replated at a low density to isolate a population of individual cells and allowed to recover at least 2 h to facilitate recording. Recordings were typically made from cells from a minimum of three independent transfections.

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 bath 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 adjusted to 7.2 at room temperature (23-25°C) using tetraethylammonium hydroxide. Ba²⁺ was used as charge carrier because in control experiments, amplitudes of currents carried by Ca²⁺ varied significantly among cells of the different phenotypes (results not shown). Thus a constant [Ba²⁺]_e was used to allow results to be compared across phenotypes more readily. Patch-clamp pipettes with resistance between 6 and 8 M were prepared from glass capillaries (1.5-mm i.d.; World Precision Instruments, New Haven, CT) using a two-stage microelectrode 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 adjusted to 7.2 at room temperature (23-25°C) 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 Ba²⁺ currents (I_Ba) through transiently-expressed Ca²⁺ channels.

3

Statistical Analysis. Origin (Origin Labs, Northampton, MA) and pClamp (Axon Instruments) software suites were used to perform linear and nonlinear fit of data. Statistical comparisons were analyzed using Student's t test or one-way analysis of variance. Results are expressed as mean ± S.E.M., and p < 0.05 was considered statistically significant.

	Results

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Concentration-Dependence of Effect of Pb²⁺on I_Ba. Ca²⁺ channels transiently expressed in HEK 293 cells using a constant _2b and _3a subunit yielded current characteristic of the ₁ subunit used in the experiment. These correspond to L-type using _1C, N-type using _1B, and R-type using _1E. Current amplitudes for all three subtypes were voltage-dependent. No significant inward current was observed for L-type channels until the depolarizing step reached 30mV; it reached maximum amplitude at +10 mV and reversed sign at approximately +60 mV. The N-type current activated at approximately 10 mV and reached maximum amplitude at +10 mV, reversing sign at approximately +60 mV. R-type current began to activate at about 20 mV, reached maximum amplitude at 0 mV, and reversed sign at +50 mV. Current inactivation of R-type channels was faster based on inspection than for either L- or N-type channels.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Representative current traces showing effects of Pb2+ on A (1C), B (1B), and C (1E) types of voltage-activated Ca2+ channels transiently expressed in HEK 293 cells. One of the three classes of 1 subunits (1C, 1B, and 1E) of human neuronal Ca2+ channels was expressed in HEK 293 cells together with 2b and 3a subunits in each experiment. Whole-cell Ba2+ (20 mM Ba2+) currents were evoked by 150-ms depolarizations from a holding potential of 70 mV (1C) or 90 mV (1B and 1E) to a test potential of 0 mV (1C and 1E) or +20 mV (1B). The effect of 0.1 µM and 1.0 µM Pb2+ on elicited currents is shown. Current responses were filtered at 2 kHz and leak current was not subtracted.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent inhibition of Pb2+ on IBa in HEK 293 cells expressing either 1C, 1B, or 1E subunit of human neuronal Ca2+ channels together with the 2b and 3a subunits. A, time course of block with different concentrations of Pb2+ on normalized peak current. B, amplitude of inward Ba2+ currents (20 mM Ba2+) recorded before and after a 2-min exposure to different concentrations of Pb2+ were fitted using I[Pb2+]/IControl = [1+([Pb2+]e/IC50)n]1 with an IC50 = 0.38, 1.31, and 0.10 µM for 1C, 1B, and 1E, respectively. Values shown are the mean ± SEM of seven to nine different cells. Cells expressing Ca2+ channels containing 1C, 1B, or 1E subunit together with 2b and 3a subunits were depolarized from 70 to 0 mV, 90 to +20 mV, or 90 to 0 mV, respectively, at a stimulation frequency of 0.1 Hz. Current responses were filtered at 2 kHz and leak current was subtracted.

Voltage-Dependence of Reduction of I_Ba by Pb²⁺ . To examine whether the decline of I_Ba caused by Pb²⁺ is voltage-dependent, we compared the current-voltage relationships in the presence or absence of Pb²⁺. After a 2-min exposure to 0.1, 0.5, and 1 µM Pb²⁺ (concentrations approximating the IC₅₀ values) for L-, N-, and R- Ca²⁺ channel subtype-expressing cells, respectively, Pb²⁺ reduced peak current amplitude at all potentials that elicited current but did not alter either the threshold of activation of I_Ba or the reversal potential. The potential at which maximum current was elicited was also not altered by Pb²⁺ for any of the channels. Inhibition of peak current by Pb²⁺ was similar at all voltage steps and there was little suggestion of any voltage-dependent reduction of peak current by Pb²⁺ (Fig. 3A). Conversion of the current-voltage curves to conductance-voltage curves by dividing the observed current by the driving force yielded the plots shown in Fig. 3B. Boltzmann fits to the data were used to calculate the voltage at which half the channels open (V_1/2). V_1/2 was 1.6 mV in control and 0.2 mV after 2-min exposure to 0.5 µM Pb²⁺ in L-type, V_1/2 = 1.5 and 1.4 mV before and after 2-min exposure to 1.0 µM Pb²⁺ in N-type, and V_1/2 = 10.3 and 11.0 mV before and after exposure of 0.1 µM Pb²⁺ in R-type channels. None of these differences were significant (p > 0.05).

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Effect of Pb2+ on current-voltage relationship and conductance of IBa in HEK 293 cells expressing either 1C, 1B, or 1E subunit together with 2b and 3a subunits of human neuronal Ca2+ channels. A, current-voltage relationship of IBa (20 mM Ba2+) recorded before and after 2-min exposure of 0.5, 1.0, and 0.1 µM Pb2+ for 1C, 1B, and 1E, respectively. B, conductance-voltage curves were obtained from the current-voltage relationships in A. The conductance-voltage curves were fitted using the Boltzmann equation: G(V) = Gmax/(1+exp[(V  V1/2)/k]), with control V1/2 = 1.6 mV, k = 6.0 mV and, after 2-min 0.5 µM Pb2+ exposure, V1/2 = 0.2 mV, k = 7.1 mV for 1C; with control V1/2 = 1.5 mV, k = 4.4 mV and, after 2-min 1.0 µM Pb2+ exposure, V1/2 = 1.4 mV, k = 3.5 mV for 1B; and with control V1/2 = 10.3 mV, k = 6.9 mV and, after 2-min 0.1 µM Pb2+ exposure, V1/2 = 11.0 mV, k = 7.7 mV for 1E. Values shown are the mean ± S.E.M. of 4 to 14 different cells. Cells expressing Ca2+ channels containing 1C, 1B, or 1E subunit were depolarized from 70 to 0 mV, 90 to +20 mV, or 90 to 0 mV, respectively. Current responses were filtered at 2 kHz and leak current was subtracted.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Effect of Pb2+ on voltage-dependence of steady-state of inactivation curves of IBa currents (20 mM Ba2+) in HEK 293 cells expressing human neuronal 1C, 1B, or 1E subunit mediated L-, N-, or R-type Ca2+ channels, respectively. A, the normalized peak currents plotted versus the voltage of an 8-s conditioning prepulse during a 480-ms test pulse used to depolarize transfected cells from 70 to 0 mV, 90 to +20 mV, or 90 to 0 mV for 1C, 1B, or 1E subunit-expressing cells, respectively. The peak currents before (control, ) and after 3-min exposure of Pb2+ () were normalized using the largest current from control recorded after conditioning prepulses from 100 to 0 mV. The dashed lines () showing the peak currents after 3-min exposure of Pb2+ were normalized using the largest current from the same group within conditioning prepulses. The smooth curve is a Boltzmann function, I/Imax = [1 + exp{(V  V1/2)/k}]1, with control V1/2 = 44.4 mV, k = 12.4 mV, and after 3-min 0.5 µM Pb2+ exposure, V1/2 = 45.0 mV, k = 12.1 mV for 1C; with control V1/2 = 65.9 mV, k = 9.6 mV, and after 3-min 1.0 µM Pb2+ exposure, V1/2 = 72.2 mV, k = 10.0 mV for 1B (p < 0.05), and with control V1/2 = 69.8 mV, k = 9.5 mV, and after 3-min 0.1 µM Pb2+ exposure, V1/2 = 79.7 mV, k = 8.2 mV for 1E (p < 0.05). Values shown are the mean ± S.E.M. of five to seven different cells. Current responses were filtered at 2 kHz and leak current was subtracted. B, the percentage of block by Pb2+ over the range of voltages calculated from A. Block by Pb2+ was voltage-dependent.

Reversibility of Reduction of I_Ba Caused by Pb²⁺. Previous studies of native channels have shown that Pb²⁺-induced reduction of I_Ba exhibited differential reversibility to wash with Pb²⁺-free solution among different preparations (Audesirk and Audesirk, 1991; Reuveny and Narahashi, 1991; Hegg and Miletic, 1996). Therefore, we compared the reversibility of Pb²⁺-induced reduction of I_Ba in HEK 293 cells expressing the three types of recombinant channels by washing the cells with Pb²⁺-free solution for 3 min after 1-min exposure to 10 µM Pb^2+. In our hands, Pb²⁺-free extracellular solution reversed 90, 92, and 71% of the Pb²⁺-elicited block of current for L-, N-, and R-subtype-expressing cells, respectively (Fig. 5). Thus, the effects of Pb²⁺ on L- and N-type Ca²⁺ channels were almost completely reversible, whereas the Pb²⁺ block of R-type Ca²⁺ channels was incompletely reversible. _1E encoded Ca²⁺ channels of human neuronal origin showed higher sensitivity and greater washout resistance after treatment with Pb²⁺ than did _1B or _1C encoded channels.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Reversible reduction of peak Ba2+ current (20 mM Ba2+) by 10 µM Pb2+ in HEK 293 cells expressing human neuronal 1C, 1B, or 1E subunit mediated L-, N-, or R-type Ca2+ channels, respectively. A, peak current was rapidly blocked by 10 µM Pb2+ during 1-min exposure; washing with Pb2+-free extracellular solution reversed the reduction of current caused by Pb2+ in all three subtypes. B, amplitude of IBa recorded before and after 1-min exposure to Pb2+ and after 3-min washing. Values shown are the mean ± S.E.M. of four to six different cells. *, significantly different from control. The value in the presence of 10.0 µM Pb2+ was significantly different for 1C, 1B, and 1E (p < 0.05) compared with respective subtypes in control; the value after 3-min of wash was not significantly different for 1C (p > 0.05), but was significantly different for 1B (p < 0.05) and 1E (p < 0.05). Cells expressing Ca2+ channels containing 1C, 1B or 1E subunit were depolarized from 70 to 0 mV, 90 to +20 mV, or 90 to 0 mV, respectively, at a stimulation frequency of 0.1 Hz. Current responses were filtered at 2 kHz and leak current was subtracted.

Concentration- and Voltage-Dependent Effects of Pb²⁺ on Inactivation Rate of I_Ba. Despite its strong reduction of peak current, Pb²⁺ seemed to accelerate the inactivation of I_Ba in cells expressing any of the three subtypes of Ca²⁺ channels. To explore this issue in greater detail, the concentration- and voltage-dependence of effects of Pb²⁺ on inactivation rate of I_Ba were examined. Figure 6A shows current through L-type channels in the absence or presence of three different concentrations of Pb²⁺. The normalized current traces show that Pb²⁺ caused faster decay of L-type current in a concentration-dependent manner. To calculate the rate of current decay, we analyzed the portion of current that inactivated at 20 ms of depolarization (measured as [peak I_Ba  I_20ms]/peak I_Ba) and plotted it as a function of concentration of Pb²⁺ in Fig. 6B. The current decay rates varied in an almost linear fashion with increasing concentrations of Pb²⁺ (r = 0.92). A similar concentration-dependent effect was observed with N- and R-type Ca²⁺ channel-expressing cells (Figs. 7 and 8). In N-type Ca²⁺ channel-expressing cells, the current decay at 20 ms of depolarization was enhanced significantly from 25.0 ± 2.6% in control to 29.5 ± 2.7% with 0.1 µM Pb²⁺ (p < 0.05) and 45.8 ± 3.3% with 1.0 µM Pb²⁺ (p < 0.05) (Fig. 7B). In R-type Ca²⁺ channel-expressing cells, the current decay at 20 ms of depolarization was also increased from 48.5 ± 4.3% in control to 54.7 ± 4.6, 57.2 ± 4.5, and 55.9 ± 5.7% for 0.01, 0.1, and 1.0 µM Pb²⁺, respectively.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Concentration-dependent effect of Pb2+ on decay rate of Ba2+ current (20 mM Ba2+) in HEK 293 cells expressing human neuronal 1C subunit mediated L-type Ca2+ channels. A, representative traces on the left showing original raw currents through 1C channels elicited by depolarization from a holding potential of 70 to 0 mV in control and during exposure to three different concentrations of Pb2+. The current traces normalized to peak current amplitudes are shown in the traces on the right. The rate of current decay during exposure to Pb2+ exhibited concentration-dependence. B, the percentage current decay at 20 ms of depolarization was plotted as a function of concentrations of Pb2+. Values shown are the mean ± S.E.M. of nine different cells. Pulse steps (150 ms) from 70 to 0 mV were used to examine the decay phase and current responses were filtered at 2 kHz. As shown by the asterisks (*), the current decay in presence of Pb2+ was significantly different from the control value at 0.01, 0.1 Pb2+, and 1.0 µM Pb2+ (p < 0.05).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Concentration-dependent effect of Pb2+ on decay rate of IBa (20 mM Ba2+) in HEK 293 cells expressing human neuronal 1B subunit-mediated N-type Ca2+ channels. A, representative traces on the left showing original currents through 1B channels elicited by depolarization from a holding potential of 90 to +20 mV in control and in the presence of 0.1 and 1.0 µM Pb2+. The current traces normalized to peak current amplitudes are shown on the right. The rate of current decay during exposure to Pb2+ exhibited concentration-dependence. B, the percentage decay at 20 ms of depolarization was plotted as a function of concentrations of Pb2+. Values of the mean ± S.E.M. are from six different cells in which 150-ms pulse steps were used to examine the decay phase and current responses were filtered at 2 kHz. *, current decay in the presence of Pb2+ was significantly different from the control value at both concentrations of 0.1 and 1.0 µM Pb2+ (p < 0.05).

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Concentration-dependent effect of Pb2+ on decay rate of Ba2+ current (20 mM Ba2+) in HEK 293 cells expressing human neuronal 1E subunit mediated R-type Ca2+ channels. A, representative traces on the left showing original currents through 1E channels elicited by depolarization from a holding potential of 90 to 0 mV in control and in presence of different concentrations of Pb2+. The currents normalized to peak current amplitudes are shown on the right. The rate of current decay during exposure to Pb2+ exhibits concentration-dependence. B, the percentage decay at 20 ms of depolarization was plotted as a function of Pb2+ concentration. Values of the mean ± S.E.M. are from five different cells in which 150-ms pulse steps were used to examine the decay phase; current responses were filtered at 2 kHz. *, current decay in the presence of Pb2+ were significantly different from the control value at 0.01, 0.1, and 1.0 µM Pb2+ (p < 0.05).

View larger version (23K): [in this window] [in a new window]   Fig. 9.   Voltage-dependent effect of Pb2+ on decay rate of Ba2+ current (20 mM Ba2+) in HEK 293 cells expressing human neuronal 1C subunit-mediated L-type Ca2+ channels. A, representative traces showing effect of 0.5 µM Pb2+ on Ba2+ current at different membrane potentials. Currents, shown normalized to maximum current amplitude, were elicited by depolarization from a holding potential of 70 mV to different membrane potentials in the absence (control) and presence of 0.5 µM Pb2+. The rate of current decay by Pb2+ at different membrane potentials exhibited voltage-dependence. B, the current decay at 20 ms of depolarization as percentages was plotted as a function of membrane potentials. Values shown are the mean ± S.E.M. of five to eight different cells. Pulse steps (150 ms) were used to examine the decay phase and current responses were filtered at 2 kHz. *, current decay in the presence of 0.5 µM Pb2+ was significantly different at 10, 20, 30, and 40 mV (p < 0.05) of membrane potential compared with the respective membrane potential in control.

View larger version (21K): [in this window] [in a new window]   Fig. 10.   Voltage-dependent effect of Pb2+ on decay rate of Ba2+ current (20 mM Ba2+) in HEK 293 cells expressing human neuronal 1B subunit mediated N-type Ca2+ channels. A, representative traces showing effect of 1.0 µM Pb2+ on Ba2+ current at different membrane potentials. Currents, shown normalized to maximum current amplitude, were elicited by depolarization from a holding potential of 90 mV to different membrane potentials in the absence and presence of 1.0 µM Pb2+. The rate of current decay by Pb2+ at different membrane potentials exhibited voltage-dependence. B, the percentage rate of current decay at 20 ms of depolarization as percentages was plotted as a function of membrane potential. Values shown are the mean ± S.E.M. of six different cells. Pulse steps (150 ms) were used to examine the decay phase and current responses were filtered at 2 kHz. *, current decay in the presence of 1.0 µM Pb2+ was significantly different at 0 to 40 mV of membrane potential compared with the respective membrane potential in control Pb2+-free (p < 0.05).

View larger version (22K): [in this window] [in a new window]   Fig. 11.   Voltage-dependent effect of Pb2+ on decay rate of Ba2+ current (20 mM Ba2+) in HEK 293 cells expressing human neuronal 1E subunit mediated R-type Ca2+ channels. A, representative traces showing effect of 0.1 µM Pb2+ on Ba2+ current at different membrane potentials. Currents, shown normalized to maximum current amplitude, were elicited by depolarization from a holding potential of 90 mV to different membrane potentials in the absence and presence of 0.1 µM Pb2+. The current decay rates by Pb2+ at different membrane potentials exhibited voltage-dependence. B, the rate of current decay at 20 ms of depolarization as percentages was plotted as a function of membrane potentials. Values shown are the mean ± S.E.M. of six different cells. Pulse steps (150 ms) were used to examine the decay phase and current responses were filtered at 2 kHz. *, decay value in the presence of 0.1 µM Pb2+ was significantly different at 10, 0, 10, 20, 30, and 40 mV (p < 0.05) of membrane potential compared with the respective membrane potential in control.

Kinetics of Pb²⁺-Induced Inactivation of I_Ba. The inactivation time constants were estimated by fitting the I_Ba decay to a biexponential function. In controls, the I_Ba kinetics at 10 mV in L-type channels exhibited a biexponential distribution; however, the N- and R-type currents showed single exponential distribution. In the presence of Pb²⁺, the fast and slow components in I_Ba of L-type current were accelerated slightly but nonsignificantly from _{fast, control} = 16.2 ± 2.0 ms to _{fast, Pb} = 12.2 ± 2.2 ms, and from _{slow, control} = 72.5 ± 14.3 ms to _{slow, Pb} = 53.6 ± 6.9 ms with 0.5 µM Pb²⁺ (p > 0.05; Fig. 12A). At 1.0 µM, Pb²⁺ modulated the N-type current with a biexponential distribution; the transient fast component in the current decay was induced with _{fast, Pb} = 13.0 ± 2.9 ms and the slower inactivation time constant of the I_Ba was not significantly affected by Pb²⁺ (Fig. 12B). In R-type channels, I_Ba inactivation ensued with a fast time constant of 28.6 ± 2.2 ms and absence of intrinsic slow inactivation. Pb²⁺ significantly accelerated the decay time constant to 21.9 ± 1.6 ms (p < 0.05, Fig. 12C).

View larger version (25K): [in this window] [in a new window]   Fig. 12.   Pb2+-induced modulation of current Ba2+ current (20 mM Ba2+) decay in HEK 293 cells expressing human neuronal 1C (A), 1B (B), or 1E (C) subunit mediated L-, N-, or R-type Ca2+ channels, respectively. Representative traces showing inward currents through the indicated channels during 150-ms depolarization from holding potential of 70 mV (1C) or 90 mV (1B and 1E) to 0 mV (1C and 1E) or +20 mV (1B) in control and in the presence of Pb2+ (0.5 µM for 1C, 1.0 µM for 1B, and 0.1 µM for 1E) (left). The time constants of fast (fast, middle) and slow (slow, right) in control and in the presence of Pb2+ were estimated by fitting biexponential functions to the current traces. Values of the mean ± S.E.M. are from five to six different cells. The current responses were filtered at 2 kHz and leak current was subtracted. Relatively speaking, no fast component in inactivation for 1B and no slow component in inactivation for 1E was observed. *, decay time constant in the presence of Pb2+ was significantly different from the control value for 1E (p < 0.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The present study using transient expression from cDNA clones of specific subtypes of human neuronal Ca²⁺ channels was designed to characterize and compare the effects of Pb²⁺ on distinct subtypes of Ca²⁺ channels in isolation. Currently, there is only one previous report on the effects of Pb²⁺ on Ca²⁺ channels in isolation, addressing rabbit cardiac L-type Ca²⁺ channels stably expressed in HEK 293 cells (Bernal et al., 1997). Our results support several aspects of previous studies done on corresponding native channels and extends them using a system transiently expressing only one kind of channel in cells in which they are not normally expressed and in which only the pore-forming element of the channel varied. We demonstrate here that: 1) Pb²⁺ is a reversible and potent inhibitor of currents expressed by cloned human neuronal _1C, _1B, and _1E subunit-containing L-, N-, or R-type Ca²⁺ channels, respectively, expressed in HEK 293 cells. This Pb²⁺-induced inhibition is effective at micromolar or submicromolar concentrations in a concentration-dependent manner, but the potency of Pb²⁺ for various Ca²⁺ channel phenotypes varies considerably. 2) The inactivation kinetics of the three types of channels studied is affected differently by Pb²⁺. 3) Block by Pb²⁺ of _1B- and _1C- containing channels is more easily reversed than those of _1E -containing channels.

In our hands, cloned human L-, N-, and R-type channels showed current characteristics and pharmacology essentially similar to native channels of the corresponding types in mammalian cells. The reduction of current amplitudes and reversibility of block by Pb²⁺ in our studies illustrate some differences among these three channel subtypes. These differences are summarized in tabular form in Table 1. The current from _1E subunit-containing channel (R-type) is more sensitive to Pb²⁺ than are those from _1C (L-type) or _1B (N-type) channel-expressing cells. The peak current inhibition caused by Pb²⁺ was almost completely reversed for L- and N-type current (90 and 92%) but only incompletely for R-type current (71%) by washing with Pb²⁺-free solution. The potency of Pb²⁺ as a blocker is quite high; in the presence of 20 mM Ba²⁺ as charge carrier, for both R- and L-type currents, the apparent IC₅₀ values were less than 1 µM total added Pb²⁺. Furthermore, inhibition occurs very rapidly and at least for _1B and _1C readily reaches a plateau. For _1E current amplitude also declines rapidly in the presence of Pb²⁺ but does not reach an apparent plateau as readily, suggesting that perhaps a slower continuing inhibitory action is occurring. This effect is reminiscent of the actions of neurotoxic mercurials on cloned, heterologously expressed Ca²⁺ channels (Peng et al., 2001, 2002). The current-voltage relationships for all three of these types of Ca²⁺ channels were unaffected by exposure to Pb²⁺. However, the steady-state inactivation relationships were shifted to more negative potentials after exposure to Pb²⁺ for N- and R-type, but not L-type currents. Pb²⁺ accelerated the inactivation rate of current in all three subtypes of Ca²⁺ channels in a concentration- and voltage-dependent manner. Therefore, it seems that Pb²⁺ has high affinity for Ca²⁺ channels in the closed state.

View this table: [in this window] [in a new window]   TABLE 1 Comparative effects of Pb2+ on various parameters in different recombinant Ca2+ channel subtypes 2 and  subunits were used for all studies. 20 mM Ba2+ was the charge carrier. Pb2+ was applied extracellularly by continuous bath superfusion. Reversibility was tested using a 3-min wash with Pb2+-free physiological saline after 1-min exposure to a concentration that approximated the relative IC50 for that channel subtype.

In the present study, we found both similarities and differences in the blocking ability of Pb²⁺ compared with native currents from previous studies. In rat dorsal root ganglion neurons, the component of whole-cell current ascribed to N-type channels, based on its sensitivity to -conotoxin GVIA (IC₅₀ = 1.0 µM), is slightly more sensitive than is L-type current (IC₅₀ = 6.0 µM) (Evans et al., 1991). In N1E-115 neuroblastoma cells, which possess both L- and T-type Ca²⁺ channels, Pb²⁺ inhibited L-type Ca²⁺ channels with an IC₅₀ of 0.7 µM and T-type with an IC₅₀ of 1.3 µM (Audesirk and Audesirk, 1991). In rat hippocampal neurons, Pb²⁺ is somewhat more selective against presumptive L-type channels than N-type channels (Audesirk and Audesirk, 1993). On the basis of IC₅₀ values, our results demonstrate that _1E current (R-type) was most sensitive to Pb²⁺, followed by _1C current (L-type) and then _1B current (N-type). This suggests that differential susceptibility to Pb²⁺ by different types of Ca²⁺ channels occurs even when the various channels are expressed in the same cell type. The differences between our results and previously reported native channel studies could also be due in part to our using the same ₂ and  subunit with all three ₁ subunits.

Washing with Pb²⁺-free solution reversed the block of I_Ba of all three types of Ca²⁺ currents. However, the extent of reversibility of I_Ba in the three subtypes varied. Pb²⁺-induced block of L- and N-type Ca²⁺ channels was more easily reversed than for R-type Ca²⁺ channels. Previous studies have reported both reversible and irreversible Ca²⁺ channel current inhibition by Pb²⁺ in different preparations. In both rat hippocampal neurons (Audesirk and Audesirk, 1993) and N1E-115 cells (Audesirk and Audesirk, 1991; Oortgiesen et al., 1993), inhibition of current flow through Ca²⁺ channels by Pb²⁺ was generally completely reversible. In rat dorsal root ganglion neurons, on the other hand, the effect of Pb²⁺ was only partially reversible (Büsselberg et al., 1994). However, the block of Ca²⁺ current by Pb²⁺ in PC12 cells was irreversible (Hegg and Miletic, 1996). The concentration-independent and washout-resistant block in rat dorsal root ganglion and snail neurons was termed `irreversible inhibition' by Audesirk (1993). In another study, full recovery from Pb²⁺ block of rabbit cloned cardiac L-type Ca²⁺ channel currents expressed in HEK 293 cells required treatment with heavy metal chelators such as meso-2,3-dimercaptosuccinic acid, 2,3-dimercapto-1-propanesulfonic acid, and EDTA (Bernal et al., 1997). In our hands, simple washout almost completely reversed Pb²⁺ block of L- and N-type currents (more than 90% of control); however, R-type current showed some resistance to simple washout. Examining the IC₅₀ values, it seems that _1E subtype-expressing channels may have higher affinity for Pb²⁺ than do _1C and _1B subunit-expressing channels. Two sets of divalent cation binding sites created by four negatively charged glutamate residues, one between each SS1-SS2 pore-lining segment of the four repeated domains, are believed to be present in the pore of Ca²⁺ channels (Parent and Gopalakrishnan, 1995). Thus, the more reversible component of block may represent Pb²⁺ binding to the more externally located lower affinity sites; the washout-resistant block may be attributable to Pb²⁺ binding to the second, more internally located site, from which it dissociates only slowly (Bernal et al., 1997).

The mechanism by which Pb²⁺ blocks voltage-activated Ca²⁺ channel is poorly understood. If Pb²⁺ binds to a site within the channel, the blocking effect should be voltage-dependent as predicted by a simple model of voltage-dependent channel blockade (Woodhull, 1973). In our experiments, Pb²⁺ did not change the voltage at which the maximal current is elicited, which would indicate that there is no change in kinetics of channel activation. Therefore, Pb²⁺ binding may reduce the number of available functional channels, causing reduction in current amplitude rather than changes in the properties of channels through which current is carried. Our results are consistent with those for native channels in rat dorsal root ganglion cells (Büsselberg et al., 1994), N1E-115 neuroblastoma cells (Audesirk and Audesirk, 1991) and rat hippocampal neurons (Audesirk and Audesirk, 1993). However, they are at divergence with another report, in which Pb²⁺ block caused the voltage at which peak current is generated to shift in the hyperpolarizing direction (Büsselberg et al., 1991). Our studies suggest that Pb²⁺-induced block of Ca²⁺ currents may be independent of channel opening and may not necessarily require open channels.

We can also discount Pb²⁺ interacting with membrane surface charges or with other specific high-affinity sites to alter charge screening because actions at these sites have been reported to shift the current-voltage curve and activation curve to more depolarizing potentials (Byerly et al., 1985). Absence of a shift in the current-voltage relationship curves in our studies rules this out as a likely mechanism of Pb²⁺ block of Ca²⁺ channels. Unlike the above, Pb²⁺ induced the steady-state inactivation curves to shift to more negative potentials in N- and R-type channels but not L-type channels. The potency of block was enhanced greatly at hyperpolarizing potentials that promote the channel being in the resting state. At more negative potentials, Pb²⁺ block for the three subtypes of Ca²⁺ channels in our study shows greater potency than that at positive potentials (Fig. 4). These observations suggest that Pb²⁺ has greater affinity for closed channels than for the inactivated state. This is consistent with previous reports in PC12 cells, in which block of I_Ca by Pb²⁺ has been reported to be associated with the closed state of channels (Shafer, 1998).

Inactivation is an important aspect of Ca²⁺ channel gating, which controls the amount of Ca²⁺ entry during an action potential, and plays an important role in tissue-specific Ca²⁺ signaling. Inactivation kinetics of Ca²⁺ channels are determined by the intrinsic properties of their pore-forming ₁-subunits and by interactions with other channel subunits (Hering et al., 2000). The inactivation of Ca²⁺ channels may involve internal or external conformational changes as well as responses to elevation of intracellular [Ca²⁺]. In our study, Pb²⁺ accelerated the inactivation rate of current in all three subtypes of Ca²⁺ channels in a concentration- and voltage-dependent manner, suggesting that Pb²⁺ might modulate the binding site associated with fast inactivation and increase the rate of entry into inactivated states or slow the recovery to resting state. External Pb²⁺ affected the inactivation rate over a range of concentrations that produced substantial block of peak current. Therefore, Pb²⁺ might speed the rate of entry of channels into the inactivated state. This inactivated state can be reached from the open state by inactivation followed by binding, or binding followed by inactivation. Because Pb²⁺ had higher potency at hyperpolarizing potentials than at depolarizing potentials, Pb²⁺ binding with high affinity apparently precedes inactivation and prevents recovery.

Conformational change has been suggested in C-type inactivation of K⁺ channels with extracellular Cd²⁺, tetraethylammonium, and sulfhydryl modifiers (Hoshi et al., 1990; Choi et al., 1991; Lopez-Barneo et al., 1993; Yellen et al., 1994; Baukrowitz and Yellen, 1995; Liu et al., 1996). The kinetics of block by Pb²⁺ of voltage-dependent Ca²⁺ channels in our study supports the possibility that Pb²⁺ may be causing a conformational change in channels resulting in fast inactivation. The Pb²⁺-induced shift of steady-state inactivation is consistent with the inactivation rate of current as demonstrated in Fig. 12. At 20 mV, Pb²⁺ accelerated the fast-inactivation of all three subtypes of channels. This Pb²⁺-induced inactivation state at least partially reflects Pb²⁺-induced transitions of open channels to an inactivated state. Pb²⁺-induced conformational change near the external mouth of the Ca²⁺ channel pore would rapidly facilitate the inactivation time course of currents in all three subtypes of Ca²⁺ channels.

In summary, Pb²⁺ is a potent and generally reversible inhibitor of human neuronal L-, N-, and R-type Ca²⁺ channels expressed in HEK 293 cells. It seems likely that Pb²⁺ blocks Ca²⁺ current by acting at a site external to the channel, where it competes with Ca²⁺, impeding its entry, but the binding to this is not voltage-dependent. Such a site may undergo a conformational change associated with inactivation. Pb²⁺ most likely binds to Ca²⁺ channels in the closed state and speeds the rate of inactivation.