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J. Biol. Chem., Vol. 281, Issue 5, 2497-2505, February 3, 2006

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Calcium-permeable Acid-sensing Ion Channel Is a Molecular Target of the Neurotoxic Metal Ion Lead^*

Wei Wang¹, Bo Duan¹, Han Xu, Lin Xu^¶2, and Tian-Le Xu³

From the Institute of Neuroscience and Key Laboratory of Neurobiology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, the School of Life Sciences, University of Science and Technology of China, Hefei 230027, and the ^¶Laboratory of Learning and Memory, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China

Received for publication, June 30, 2005 , and in revised form, November 28, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Acid-sensing ion channels (ASICs) are emerging as fundamental players in the regulation of neural plasticity and in pathological conditions. Here we showed that lead (Pb²⁺), a well known neurotoxic metal ion, reversibly and concentration-dependently inhibited ASIC currents in the acutely dissociated spinal dorsal horn and hippocampal CA1 neurons of rats. In vitro expression of ASIC subunits in combination demonstrated that both ASIC1 and -3 subunits were sensitive to Pb²⁺. Mechanistically, Pb²⁺ reduced the pH sensitivity of ASICs independent of membrane voltage change. Moreover, Pb²⁺ inhibited the ASIC-mediated membrane depolarization and the elevation of intracellular Ca²⁺ concentration. In addition, we compared the effect of Pb²⁺ with that of Ca²⁺ or amiloride to explore the possible interactions of Pb²⁺ and Ca²⁺ in regulating ASICs, and we found that Pb²⁺ inhibited ASIC currents independent of the amiloride/Ca²⁺ blockade. Because ASIC1b and -3 subunits are mainly expressed in peripheral neurons, our data identified ASIC1a-containing Ca²⁺-permeable ASIC as a novel central target of Pb²⁺ action, which may contribute to Pb²⁺ neurotoxicity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Acid-sensing ion channels (ASICs)⁴ belong to the members of voltage-insensitive, amiloride-sensitive epithelial Na⁺ channel/degenerin superfamily (1). To date, four genes encoding six ASIC subunit proteins, including ASIC1a, -1b, -2a, -2b, -3, and -4, have been identified. ASIC1b and -2b are splice variants of ASIC1a and -2a, respectively. Functional ASICs are thought to be tetrameric assemblies of homomeric or heteromeric ASIC subunits (2). Studies of the structure of ASICs indicate that ASICs consist of two transmembrane domains (TM1 and TM2) and a large extracellular loop, and the pre-TM2 region is essential for Ca²⁺ permeability and the gating of this channel (3, 4). Some of the endogenous cations such as the divalent metal ions Ca²⁺, Zn²⁺, and Mg²⁺ (5–15) and the polyvalent cation spermine (5) have been shown to modify the gating and activity of specific ASICs.

Of all ASIC subunits, ASIC1a subunit renders ASICs a high Ca²⁺ permeability (16, 17). The unraveling of ASIC1a in synapse function (18, 19) would be a plausible cellular mechanism responsible for a variety of physiological and pathological processes such as learning and memory (18–20), nociceptive transmission (15, 21), visceral sensation (22), and ischemic cell death (17). Therefore, it is reasonable to speculate that disturbance of ASIC1a function would impair the contribution of ASICs to synaptic function.

Lead (Pb²⁺) is a well known divalent metal ion because of its harm to the human body. It is widely accepted that Pb²⁺-induced impairment of synaptic function is one of the fundamental cellular mechanisms of Pb²⁺ neurotoxicity in the central nervous system (CNS) (23–25). Considering the importance of ASICs in synaptic physiology, we asked whether ASICs are novel molecular targets of Pb²⁺ action. Furthermore, because Ca²⁺ and Pb²⁺ are both divalent cations, we asked whether they share similar mechanisms in modulating ASICs. To answer these questions, we performed whole-cell patch clamp recordings in rat spinal dorsal horn (SDH) and hippocampal CA1 neurons as well as CHO cells transfected with five major ASIC subunits, 1a, 1b, 2a, 2b, and 3 in combination. We found that the ASIC1a, -1b, and -3 subunits were sensitive to Pb²⁺. Because ASIC1b and -3 are mainly expressed in peripheral neurons, our study identifies ASIC1a subunit as a novel molecular target of Pb²⁺ in CNS neurons, which may contribute to Pb²⁺ neurotoxicity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Acute Isolation of Rat CNS Neurons—The use and care of experimental animals were approved by the Anhui Health Department, China. Neurons from rat SDH or hippocampal CA1 region were mechanically dissociated as described previously (11, 15). In brief, 2-week-old Wistar rats were sacrificed by decapitation, and then a segment of lumbosacral (L4–S2) spinal cord or brain was quickly removed and immersed into an ice-cold incubation solution (see below for composition). The spinal segment or the brain was sectioned at 400 µm with a vibratome tissue slicer (LEICA VT1000S, Leica Instruments Ltd., Wetzlar, Germany). Before being sectioned, the attached dorsal rootlets and pia matter of nervous tissue were peeled off. A vibration-isolation system was then used to mechanically dissociate the neurons from the SDH or hippocampus CA1 region (11, 15). Briefly, a fire-polished glass pipette mounted on a vibrator was placed lightly on the surface of the SDH or hippocampal CA1 region of the slice and vibrated horizontally at 5–10 Hz for about 5 min under the control of a pulse generator. The slices were then discarded, and the mechanically isolated neurons were attached to the bottom of the culture dish within 20 min, ready for electrophysiological experiments.

Transfection of CHO Cells—All constructs were expressed in CHO cells. CHO cells were cultured at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air. Cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 2 mM L-glutamine, 10% fetal bovine serum, and 100 units/ml penicillin/streptomycin (all from Invitrogen). Transient transfection of CHO cells was carried out using the conventional calcium phosphate method. Co-transfection with a green fluorescent protein expression vector, pEGFP-N1, was used to enable identification of transfected cells for patch clamping by monitoring green fluorescent protein fluorescence in some experiments. When more than one ASIC subunit was expressed, the multiplasmids were co-transfected in a ratio of 1:1. Electrophysiological recordings were performed 16–48 h after transfection. The GW1-CMV-ASIC1a and GW1-CMV-ASIC2a were generous gifts from Dr. Jun Xia (Hong Kong University of Science and Technology, Hong Kong, China), and the two plasmids were subcloned into pEGFP-C3 that was digested with HindIII and XhoI for subsequent experiments. The pcDNA3-ASIC1b was kindly from Dr. Zhi-Gang Xiong (Legacy Clinical Research Center, Portland, OR). The pN1z-ASIC2b was kindly provided by Dr. Philip K. Ahring (NeuroSearch A/S, Ballerup, Denmark), and PCI-ASIC3 was from Rainer Waldmann (Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France). The pEGFP-N1 was from Dr. Jian-hong Luo (Zhejiang University Faculty of Medicine, Hangzhou, China).

Electrophysiology—The electrophysiological recordings were performed in the conventional whole-cell patch clamp recording configuration under voltage clamp or current clamp conditions. Patch pipettes were pulled from glass capillaries with an outer diameter of 1.5 mm (Narishige, Tokyo, Japan) on a two-stage puller (PP-830, Narishige Co. Ltd., Tokyo, Japan). The resistance between the recording electrode filled with pipette solution and the reference electrode was 4–6 megohms. Membrane currents were measured using a patch clamp amplifier (Axopatch 200B, Axon Instruments, Foster City, CA), filtered at 1 kHz, sampled, and analyzed using a DigiData 1320A interface and a computer with the pCLAMP system (Version 8.0, Axon Instruments, Foster City, CA). The series resistance, estimated from optical cancellation of the capacity transient, was 10–30 megohms, and in most experiments, 70–90% series resistance was compensated. The membrane potential was voltage clamped at –50 mV throughout the experiments under voltage clamp conditions except when the current-voltage (I-V) relationships for ASIC currents were examined. All experiments were carried out at room temperature (22–25 °C).

Primary SDH and Hippocampal Neuronal Cultures—SDH neurons from 15-day-old embryonic Sprague-Dawley rats or hippocampal neurons from 18-day-old embryonic Sprague-Dawley rats were isolated by a standard enzyme treatment protocol. Briefly, SDH or hippocampi of rats were dissociated in Ca²⁺-free saline with sucrose (20 mM) and plated (1–2 x 10⁵ cell/ml) on poly-D-lysine (Sigma)-coated cover glasses. The neurons were grown in Dulbecco's modified Eagle's medium with L-glutamine plus 10% fetal bovine serum and 10% F-12 nutrient mixture (Invitrogen). After 1 day, Neurobasal medium (1.5 ml, Invitrogen) with 2% B27 (Invitrogen) was replaced every 3–4 days. Treatment with 5-fluoro-2'-deoxyuridine (20 µg/ml, Sigma) on the 4th day after plating was used to block cell division of non-neuronal cells, which helped to stabilize the cell population. The cultures were maintained at 37 °C in a 5% CO₂-humidified atmosphere. Neurons were used for Ca²⁺ imaging 9–14 days after plating.

Ca²⁺ Imaging—Cultured SDH or hippocampal neurons grown on 8 x 8-mm glass coverslips were washed three times with phosphate-buffered saline and incubated with 1 µM fura-2-acetoxymethyl ester for 20 min at 37 °C, washed three times, and incubated in standard extracellular solution for 30 min. Coverslips were transferred to a perfusion chamber on an inverted microscope (Nikon TE2000-E, Japan). Experiments were performed by using a 40x UV fluor oil-immersion objective lens, and images were recorded by a cooled CCD camera (Hamamatsu Photonics, Hamamatsu City, Japan). To block potential Ca²⁺ entry through voltage-gated Ca²⁺ channels or glutamate receptors, 10 µM nifedipine, 20 µM D-2-amino-5-phosphonopentanoic acid, and 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione were added to the bath solution. The fluorescence excitation source was a 75-watt xenon arc lamp. Ratio images were acquired by using alternating excitation wavelengths (340/380 nm) with a monochromator (Till Polychrome IV, Munich, Germany), and fura-2 fluorescence was detected at emission wavelength of 510 nm. Digitized images were acquired and analyzed in a personal computer controlled by SimplePCI (Compix Inc.). Ratio images (340/380 nm) were analyzed by averaging pixel ratio values in circumscribed regions of cells in the field of view. The values were exported to Origin 7.0 for further analysis.

Solutions and Chemicals—The ionic composition of the incubation solution was as follows (mM): 124 NaCl, 24 NaHCO₃, 5 KCl, 1.2 KH₂PO₄, 2.4 CaCl₂, 1.3 MgSO₄, 10 glucose, aerated with 95% O₂ and 5% CO₂ to a final pH of 7.4. The standard extracellular solution contained the following (mM): 150 NaCl, 5 KCl, 1 MgCl₂, 2 CaCl₂, 10 glucose, buffered to various pH values with either 10 mM HEPES (pH 6.0–7.4) or 10 mM MES (pH <6.0). The osmolarity of all external solutions was adjusted to 325–330 mosmol liter^–1 with sucrose. The patch pipette solution for whole-cell patch clamp recording was as follows (mM): 120 KCl, 30 NaCl, 0.5 CaCl₂, 1 MgCl₂, 5 EGTA, 2 MgATP, and 10 HEPES; pH was adjusted to 7.2 with Tris base. The osmolarity of the pipette solution was adjusted to 280–300 mosmol liter^–1 with sucrose. The extracellular pH was adjusted to different values by the addition of 1 N NaOH or 1 N HCl and was routinely checked before and during experiments. When the I-V relationships for ASIC currents were examined, 300 nM tetrodotoxin and 100 µM CdCl₂ were added to the extracellular solutions, and KCl was replaced with equimolar CsCl in the pipette solution. Changes in divalent ion content of the bathing solution were kept iso-osmotic.

All chemicals were purchased from Sigma. Lead acetate was dissolved in distilled water at a concentration of 20 mM to make stock. The stock solutions were diluted in the extracellular solutions just before each experiment to avoid precipitation. The solution containing a high concentration of Ca²⁺ was obtained in the same way. Drugs were applied using a rapid application technique termed the "Y-tube" method, which allowed a complete exchange of external solution surrounding a neuron within 20 ms (15).

Data Analysis—Clampfit software was used for data analysis. The continuous theoretical curves for concentration-response relationships for ASIC currents in the presence or absence of Pb²⁺ were obtained according to a modified Michaelis-Menten equation by the method of least squares (the Newton-Raphson method) after normalizing the amplitude of the response shown in Equation 1,

(Eq. 1)

where I is the normalized value of the current; I_max is the maximal response; C is the drug concentration; EC₅₀ is the concentration that induced the half-maximal response; and h is the apparent Hill coefficient. The curve for the effect of Pb²⁺ or amiloride on ASIC currents was fitted to the following Equation 2,

(Eq. 2)

where IC₅₀ represents the concentration that induced the half-maximal inhibitory effect, and the other abbreviations are the same as used in Equation 1. The recovery of the rapidly decaying current was fitted to monoexponential function shown in Equation 3,

(Eq. 3)

where A₁ is the amplitude; is the decay time constant; and y₀ is the offset.

View larger version (24K): [in this window] [in a new window]   FIGURE 1. Pb2+ reversibly and concentration-dependently inhibited the ASIC currents in acutely dissociated SDH (A) and hippocampal CA1 neurons (B) of rats. The neurons were pre-perfused with Pb2+ for about 15 s before simultaneous application with an acidic solution. All responses were normalized to the peak current induced by extracellular pH 6.0 solution. Each point represents the average response of 6–10 neurons. In this and subsequent figures, the horizontal bars indicate the drug application duration, and the vertical bars show the mean ± S.E. The holding potential (VH) was –50 mV.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Inhibition of ASIC-mediated Currents by Pb²⁺ in Acutely Dissociated SDH and Hippocampal CA1 Neurons—The role of ASICs in synaptic function has been characterized in CNS neurons (18, 19). Our previous studies indicate that the acid-induced currents in acutely dissociated SDH (15) and hippocampal CA1 (11) neurons are mediated by ASICs. Therefore, we examined the effects of Pb²⁺ on ASIC-mediated currents in these neurons under whole-cell voltage clamp configuration. After recording a stable control in ASIC current, we pre-perfused the neurons with 10 µM Pb²⁺ for 15 s, and then the ASICs were activated again by acidic solutions in the presence of 10 µM Pb²⁺. Pb²⁺ reversibly reduced the amplitude of the ASIC currents induced by pH 6.0 external solution in both SDH (Fig. 1A) and CA1 (Fig. 1B) neurons at a holding potential (V_H) of –50 mV. Application of Pb²⁺ alone did not evoke any detectable current. The Pb²⁺-induced inhibition of the ASIC currents was concentration-dependent. In SDH neurons, the IC₅₀ and Hill coefficients (Equation 2) of the inhibition-response curves were 8.7 µM and 1.2, respectively (n = 10); and in CA1 neurons, the two values were 7.1 µM and 1.2, respectively (n = 6). However, Pb²⁺ (10 µM) did not exert any apparent effects on the decay time constant (, Equation 3) of the currents in either SDH neurons (without Pb²⁺, = 1.1 ± 0.1 s, and with Pb²⁺, = 1.3 ± 0.1 s, n = 10) or CA1 neurons (without Pb²⁺, = 1.2 ± 0.1 s, and with Pb²⁺, = 1.3 ± 0.1 s, n = 6).

ASIC1 and -3 Subunits Were Sensitive to Pb²⁺—To study the subunit responsible for the Pb²⁺-induced inhibition, we expressed five major ASIC subunits 1a, 1b, 2a, 2b and 3 in CHO cells. ASIC currents were elicited by external acidic solutions in these cells at a holding potential of –50 mV. We employed a moderate acidic solution of pH 6.0 to evoke ASIC currents mediated by homomeric ASIC1a-, 1b-, or heteromeric ASIC1a-containing channels, and an acidic solution of pH 5.0 was applied to activate homomeric ASIC2a- or heteromeric ASIC2a-containing channels or homomeric ASIC3 channels. As in neurons, after control ASIC currents became stable, we pre-perfused the CHO cells with Pb²⁺ for 15 s, and then the ASIC currents were induced in the presence of Pb²⁺ (Fig. 2A). We found that Pb²⁺ significantly reduced the amplitude of the ASIC currents mediated by homomeric 1a and 1b and heteromeric 1a + 2a and 1a + 2b channels in concentration-dependent manners with the IC₅₀ and Hill coefficient of 3.7 µM and 2.2, 1.5 µM and 1.8, 4.9 µM and 2.3, 2.8 µM and 1.5, respectively (n = 5–7) (Fig. 2). Similar to the effect on CNS neurons, Pb²⁺-induced inhibition in CHO cells was reversible after the removal of Pb²⁺. In addition, Pb²⁺ did not affect the kinetics of ASIC currents in CHO cells (data not shown). In contrast, Pb²⁺ exerted no significant inhibitory effects on the ASIC currents mediated by homomeric 2a and heteromeric 2a + 2b channels at the concentrations ranging from 0.1 to 100 µM (n = 4–5). Pb²⁺ exerted no significant inhibition on homomeric ASIC3 channel at concentrations ranging from 0.1 to 10 µM but decreased its current amplitude at a high concentration of 100 µM (36.1 ± 5.3% of the control, n = 5, p < 0.01, Student's paired t test), suggesting the existence of low affinity site(s) for Pb²⁺ on ASIC3 subunit.

Pb²⁺ Prevented Neuronal Ca²⁺ Elevation Mediated by ASIC Activation—The ASIC1a subunit, which renders ASICs a high Ca²⁺ permeability (16), is abundantly expressed in SDH (15) and hippocampal (14, 18, 20, 26) neurons, whereas the splice variants ASIC1b and ASIC3 subunits are expressed in peripheral neurons (27, 28). Thus we hypothesized that Pb²⁺ affects ASIC-mediated Ca²⁺ entry by specifically inhibiting ASIC1a channels in CNS neurons. To test the hypothesis, we performed Ca²⁺ imaging experiments in cultured SDH and hippocampal neurons. We observed an elevation of [Ca²⁺]_i when extracellular acidic solution was applied to these cultured neurons (Fig. 3A). This acid-induced elevation of [Ca²⁺]_i was pH-dependent and was reversibly blocked by 100 µM amiloride (data not shown). Pb²⁺ (10 µM) reversibly reduced the ASIC-mediated elevation of [Ca²⁺]_i in cultured SDH (59.7 ± 6.4% of control, n = 6) or hippocampal neurons (64.3 ± 11.2% of control, n = 6) (Fig. 3). In addition, no significant difference of Pb²⁺ inhibition was observed between hippocampal and SDH neurons (Fig. 3B). Because the blockers of ionic glutamate receptors and voltagegated Ca²⁺ channels were added into the bath solution to prevent potential Ca²⁺ entry through these receptors/channels (see "Experimental Procedures"), we can attribute the Pb²⁺-induced reduction in [Ca²⁺]_i to Pb²⁺-induced inhibition of neuronal ASIC1a-containing channels.

Pb²⁺ Inhibited ASIC Currents Independent of Membrane Voltage—In the subsequent experiments, we investigated further the mechanisms of Pb²⁺-induced inhibition of ASIC currents in acutely dissociated SDH neurons, in which homomeric ASIC1a channel-mediated currents were dominant (15). We first determined whether alternation of membrane potential affects the inhibitory effect of Pb²⁺. We changed the V_H of the tested neurons from –60 to +60 mV in the absence or presence of 10 µM Pb²⁺ (Fig. 4A), and we plotted the current-voltage curve (Fig. 4B). The reversal potential of the ASIC currents was not altered by Pb²⁺ (without Pb²⁺, 33.5 ± 3.7 mV and with Pb²⁺, 37.0 ± 4.9 mV; n = 7), suggesting that Pb²⁺ inhibited ASIC function without changing its ion selectivity. Moreover, the Pb²⁺-induced inhibition was independent of V_H (Fig. 4C), suggesting that the action site(s) of Pb²⁺ are not within the channel pore where Pb²⁺ would experience the membrane electric field.

View larger version (24K): [in this window] [in a new window]   FIGURE 2. Subunit specificity of Pb2+ inhibition on ASICs. A, representative traces showing the effects of 10 µM Pb2+ on acid-induced currents mediated by homomeric ASIC1a, -1b, -2a, and -3 or heteromeric 1a + 2a, 1a + 2b, and 2a + 2b channels expressed in CHO cells. The effects of Pb2+ on ASIC currents were reversible. B, statistical data showing that Pb2+ concentration-dependently inhibited ASIC1-containing channels (both homomeric 1a and 1b channels and heteromeric 1a + 2a, 1a + 2b channels). Note that Pb2+ at a high concentration of 100 µM also significantly inhibited homomeric ASIC3 channels. Each point is the average of 4–7 cells. **, p < 0.01 (n = 5, Student's paired t test), compared with control. The VH was –50 mV.

Previous studies have shown that Pb²⁺ stimulates intracellular protein kinases such as protein kinase C and protein kinase A (23, 29, 30), which may be associated with ASIC activity (31–33). To examine whether intracellular signaling messengers are involved in mediating Pb²⁺ inhibition of ASIC currents, we studied the effects of 10 µM Pb²⁺ at various pretreatment durations. As shown in Fig. 5B, alternation of Pb²⁺ pretreatment duration from 15 to 120 s did not affect its inhibitory potency (n = 8; p > 0.05, ANOVA). In addition, when loaded into neurons via the patch electrode, staurosporine (10 µM), a nonselective protein kinases inhibitor, or BAPTA (10 mM), the Ca²⁺ chelator, did not alter the inhibition of ASIC currents by 10 µM Pb²⁺ (Fig. 5C). These results suggest that intracellular signaling messengers are not involved in the Pb²⁺-induced inhibition of ASIC currents.

Distinctions between Pb²⁺ and Ca²⁺ Modulation of ASICs—To date, a series of studies on the interaction of Ca²⁺, the physiologically relevant divalent metal ion, with ASICs has been made (5, 9–15). Because Pb²⁺ binds to a protein that usually contains Ca²⁺ binding domain(s) (23), we co-applied these two divalent cations to assess their possible interactions in mediating ASIC regulation. We employed three different modes of Ca²⁺ or Pb²⁺ application to differentiate the effects of the two divalent cations. In co-application protocol (protocol a), neurons were co-applied with external Ca²⁺ or Pb²⁺ and pH 6.0 solution; in sequential application protocol (protocol b), neurons were applied with pH 6.0 solution alone immediately after 15 s of perfusion of Ca²⁺ or Pb²⁺; in pretreatment protocol (protocol c), neurons were pretreated with Ca²⁺ or Pb²⁺ for 15 s and then applied with Ca²⁺ or Pb²⁺ and acidic solution together. As shown in Fig. 6, A and C, different sequences of Ca²⁺ (10 mM) application produced different effects on the ASIC currents. Most notably, application of Ca²⁺ immediately before but not during application of pH 6.0 solution resulted in a significant enhancement of ASIC currents. In contrast, when three different protocols of drug application were employed, Pb²⁺ always produced an inhibitory effect on ASIC currents (Fig. 6, B and C). These findings suggest that Pb²⁺ modulates ASICs in a manner different from that of Ca²⁺.

View larger version (19K): [in this window] [in a new window]   FIGURE 3. Pb2+ reduced ASIC-mediated elevation of [Ca2+]i in cultured SDH and hippocampal neurons. A, representative 340/380 nm ratio showing the changes in [Ca2+]i induced by pH 5.0 solution in the absence or presence of 10 µM Pb2+. The reduction of [Ca2+]i elevation was completely recovered after washout of Pb2+. B, statistical data illustrating the inhibition of ASIC-mediated elevation of [Ca2+]i by 10µM Pb2+ in the SDH and hippocampal neurons, respectively. Each column represents the average response of six neurons. n.s. means no significant difference (p > 0.05, ANOVA).

View larger version (15K): [in this window] [in a new window]   FIGURE 4. Effect of altered membrane potential on the modulation of ASIC currents in acutely dissociated SDH neurons by Pb2+ or Ca2+. A, sample recordings of ASIC currents elicited by extracellular pH 6.0 solution in the absence or presence of 10 µM Pb2+ in a representative neuron voltage clamped at various VH values (from –60 to +60 mV). B, the current-voltage (I-V) relationship for the ASIC currents elicited by extracellular pH 6.0 solution in the absence or presence of 10 µM Pb2+. The reversal potentials were both close to the theoretical Na+ equilibrium potential. C, scatter graph showing the relative inhibition of the peak currents recorded at various VH values in the presence of 10 µM Pb2+ or 10 mM Ca2+. Pb2+-induced inhibition of ASIC currents did not depend on VH change (n = 7; p > 0.05, ANOVA). The inhibition of ASIC currents by 10 mM Ca2+ was slightly decreased under more negative VH values.

View larger version (19K): [in this window] [in a new window]   FIGURE 5. Inhibition of ASIC currents by Pb2+ in acutely dissociated SDH neurons was pH-dependent but was independent of intracellular signal messengers. A, left, representative traces from an SDH neuron illustrating the effect of 10 µM Pb2+ on ASIC currents induced by extracellular pH 6.0 and pH 4.0 solutions. The pH 6.0 is the concentration around pH50 and pH 4.0 represents the saturating concentration. Right, Pb2+ right-shifted the concentration-response curve of ASIC currents in a parallel manner without altering the Hill coefficient (with Pb2+ 1.4, without Pb2+ 1.3) and maximal or minimal ASIC currents. Each point represents the average response of 5–7 neurons. All responses were normalized to the peak current induced by extracellular pH 6.0 solution (*). B, the effects of various pre-perfusion times of Pb2+ on ASIC inhibition. The graph indicating that the inhibitory potency of 10 µM Pb2+ was unchanged with the Pb2+ pre-perfusion time varied from 15 to 120 s (n = 8; p > 0.05, ANOVA). C, statistical data show that intracellular application of staurosporine (10 µM) and BAPTA (10 mM) did not significantly affect the inhibition of ASIC currents by 10 µM Pb2+ (n = 5–7, p > 0.05, unpaired Student's t test). The VH was –50 mV.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Previous studies indicate that the accepted blood Pb²⁺ levels that cause neurocognitive deficits in children are 10 µg/dl (0.5 µM) or higher (43), and neurological symptoms can be seen at the blood Pb²⁺ level above 60 µg/dl (2.9 µM) (44). In our experiments, the concentrations of Pb²⁺ that produce direct inhibition on native ASIC channels are above 1 µM, and the IC₅₀ of Pb²⁺ inhibition of ASIC currents in SDH or hippocampal CA1 neurons is within the range of 7–9 µM (Fig. 1). These data suggest that the direct inhibition of ASICs is more likely to play a role in acute rather than chronic intoxication in vivo.

Possible Mechanisms Underlying the Inhibition of ASICs by Pb²⁺—We employed patch clamp recording to study the effects of Pb²⁺ on ASICs, and we demonstrated that Pb²⁺ action site(s) are located on the exterior surface of the ASIC1 subunit based on the following findings. First, the Pb²⁺-induced inhibition of ASIC current, which did not necessitate any pretreatment (Fig. 5B), was immediate and reversible (Fig. 1). In addition, we did not observe any significant differences in Pb²⁺-induced inhibition of ASIC currents between short term and prolonged Pb²⁺ pretreatment (Fig. 6B). Second, Pb²⁺ has been demonstrated to stimulate intracellular protein kinases (23–25, 29, 30) that may be associated with ASIC activity (31–33). However, in our experiments, the intracellularly loaded nonselective protein kinase inhibitor staurosporine or the Ca²⁺ chelator BAPTA did not affect Pb²⁺ inhibition of the ASIC currents, excluding the involvement of intracellular signaling messengers in the modulation of ASIC currents (Fig. 5C). Finally, the Pb²⁺-induced inhibition was independent on membrane potentials, suggesting that Pb²⁺ action site(s) are not within the channel pore where it would experience the membrane electric field (Fig. 4C).

View larger version (23K): [in this window] [in a new window]   FIGURE 6. Distinctions between Pb2+ and Ca2+ in modulating ASIC currents in acutely dissociated SDH neurons. A and B, representative traces illustrating 10 mM Ca2+ (A) or 10 µM Pb2+ (B) modulated ASIC currents in three different modes of drug application (a–c). C, statistical results showing the relative values of ASIC currents in the presence of 10 mM Ca2+ or 10 µM Pb2+ in the three modes indicated in A and B. All responses were normalized to the peak current induced by extracellular pH 6.0 solution (dotted line). Each graph was the average response of eight neurons. ** and *** mean significant difference (**, p < 0.01; ***, p < 0.001, ANOVA). The VH was –50 mV.

View larger version (19K): [in this window] [in a new window]   FIGURE 7. Noncompetitive interaction of Pb2+ and amiloride (17) ASIC currents in the acutely dissociated SDH neurons. A, representative traces from a single SDH neuron illustrating the effects of co-application of 0.1 µM amiloride (AMI) with 6 or 60 µM Pb2+, respectively, on ASIC currents induced by pH 6.0 solution. B, the concentration-response relationship for the inhibition of ASIC currents by AMI in the absence and presence of 6 or 60 µM Pb2+. All responses were normalized to the peak current induced by extracellular pH 6.0 solution. Each point represents the average response of 3–7 neurons. The VH was –50 mV.

View larger version (18K): [in this window] [in a new window]   FIGURE 8. Pb2+ reduced ASIC-mediated membrane depolarization in the acutely dissociated SDH neurons. A, representative traces showing that membrane depolarization induced by pH 6.5 solution. This depolarization was reversibly inhibited by 10 µM Pb2+ and 100 µM AMI. B, statistical data showing acidic solution-induced changes in membrane potential (Vm) in the absence or presence of 10 µM Pb2+. ** means significant difference (p < 0.01, Student's paired t test). The membrane potential was recorded in current clamp mode. Each column was the average of 6–8 neurons. Dashed line indicates the resting potential.

Functional Implications—The mammalian SDH is the first central site for integration, relay, and modulation of nociceptive information from nociceptors. Our previous study suggests that ASICs may participate in central nociception in spinal second-order sensory neurons (15). Thus, Pb²⁺-induced inhibition of ASIC currents in the SDH neurons may disrupt the sensory processing in the SDH, resulting in the abnormality of spinal nociception. In addition, a recent report implicates the involvement of ASIC1 in normal visceral mechanosensation (22). The mice lacking the ASIC1 gene exhibited increased mechano-sensitivity that is correlated to abdominal discomfort, including bloating, cramping, and severe pain. Moreover, clinical case reports have shown that patients acutely or chronically exposed to Pb²⁺ often display gastrointestinal symptoms of abdominal pain (48, 49). If Pb²⁺ modulates peripheral ASIC1a in a similar fashion, the reduction of ASIC1a current in visceral sensory neurons may participate in the abnormality in visceral sensation.

We demonstrate that ASICs of CNS neurons are a novel target of Pb²⁺ action. However, the definite relationship between the effect of Pb²⁺ on ASICs and Pb²⁺-induced neurotoxicity should be established through further investigations. We observed a direct Pb²⁺-induced inhibition of ASIC currents, which may play an important role in acute Pb²⁺ intoxication. As for the chronic Pb²⁺ exposure, the effects of Pb²⁺ on neuronal ASIC expression at the transcriptional or translational levels should be examined. Moreover, to explore the role of ASICs in Pb²⁺ intoxication-related pathological processes, more knowledge in the involvement of ASICs themselves in synaptic physiology and cellular functions is required.

	FOOTNOTES

 
^* This work was supported by the National Natural Science Foundation of China Grants 30125015 and 30321002, the National Basic Research Program of China Grant 2006CB500803, and the Shanghai Municipal Government. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence may be addressed: Laboratory of Learning and Memory, Kunming Institute of Zoology, Chinese Academy of Sciences, 32 Jiaochang Donglu, Kunming 650223, Yunnan, China. Tel.: 86-871-519-5402; Fax: 86-871-519-1823; E-mail: lxu{at}vip.163.com. ³ To whom correspondence may be addressed: Institute of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-yang Road, Shanghai 200031, China. Tel.: 86-21-54921751; Fax: 86-21-54921735; E-mail: tlxu{at}ion.ac.cn.

⁴ The abbreviations used are: ASICs, acid-sensing ion channels; SDH, spinal dorsal horn; TM, transmembrane domain; CNS, central nervous system; NMDAR, N-methyl-D-aspartate receptor; LTP, long term potentiation; CHO, Chinese hamster ovary; MES, 4-morpholineethanesulfonic acid; ANOVA, analysis ov variance; BAPTA, ,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; [Ca²⁺]_i, intracellular Ca²⁺ concentration.

	ACKNOWLEDGMENTS

 
We thank Dr. Z.-G. Xiong for helpful discussions. We thank Drs. L. J. Wu, J. Gao, X. L. Zhao, and X. B. Zhang for technical assistance. We also thank Dr. J. M. Wu for expert assistance with the sequence alignment and analysis.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Waldmann, R., and Lazdunski, M. (1998) Curr. Opin. Neurobiol. 8, 418–424[CrossRef][Medline] [Order article via Infotrieve]
2. Krishtal, O. (2003) Trends Neurosci. 26, 477–483[CrossRef][Medline] [Order article via Infotrieve]
3. Darboux, I., Lingueglia, E., Champigny, G., Coscoy, S., Barbry, P., and Lazdunski, M. (1998) J. Biol. Chem. 273, 9424–9429[Abstract/Free Full Text]
4. Bassler, E. L., Ngo-Anh, T. J., Geisler, H. S., Ruppersberg, J. P., and Grunder, S. (2001) J. Biol. Chem. 276, 33782–33787[Abstract/Free Full Text]
5. Babini, E., Paukert, M., Geisler, H. S., and Grunder, S. (2002) J. Biol. Chem. 277, 41597–41603[Abstract/Free Full Text]
6. Baron, A., Schaefer, L., Lingueglia, E., Champigny, G., and Lazdunski, M. (2001) J. Biol. Chem. 276, 35361–35367[Abstract/Free Full Text]
7. Baron, A., Waldmann, R., and Lazdunski, M. (2002) J. Physiol. (Lond.) 539, 485–494[Abstract/Free Full Text]
8. Chu, X. P., Wemmie, J. A., Wang, W. Z., Zhu, X. M., Saugstad, J. A., Price, M. P., Simon, R. P., and Xiong, Z. G. (2004) J. Neurosci. 24, 8678–8689[Abstract/Free Full Text]
9. Coric, T., Zhang, P., Todorovic, N., and Canessa, C. M. (2003) J. Biol. Chem. 278, 45240–45247[Abstract/Free Full Text]
10. de Weille, J., and Bassilana, F. (2001) Brain Res. 900, 277–281[CrossRef][Medline] [Order article via Infotrieve]
11. Gao, J., Wu, L. J., Xu, L., and Xu, T. L. (2004) Brain Res. 1017, 197–207[CrossRef][Medline] [Order article via Infotrieve]
12. Immke, D. C., and McCleskey, E. W. (2003) Neuron 37, 75–84[CrossRef][Medline] [Order article via Infotrieve]
13. Paukert, M., Babini, E., Pusch, M., and Grunder, S. (2004) J. Gen. Physiol. 124, 383–394[Abstract/Free Full Text]
14. Waldmann, R., Champigny, G., Bassilana, F., Heurteaux, C., and Lazdunski, M. (1997) Nature 386, 173–177[CrossRef][Medline] [Order article via Infotrieve]
15. Wu, L. J., Duan, B., Mei, Y. D., Gao, J., Chen, J. G., Zhuo, M., Xu, L., Wu, M., and Xu, T. L. (2004) J. Biol. Chem. 279, 43716–43724[Abstract/Free Full Text]
16. Yermolaieva, O., Leonard, A. S., Schnizler, M. K., Abboud, F. M., and Welsh, M. J. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 6752–6757[Abstract/Free Full Text]
17. Xiong, Z. G., Zhu, X. M., Chu, X. P., Minami, M., Hey, J., Wei, W. L., MacDonald, J. F., Wemmie, J. A., Price, M. P., Welsh, M. J., and Simon, R. P. (2004) Cell 118, 687–698[CrossRef][Medline] [Order article via Infotrieve]
18. Wemmie, J. A., Askwith, C. C., Lamani, E., Cassell, M. D., Freeman, J. H., Jr., and Welsh, M. J. (2003) J. Neurosci. 23, 5496–5502[Abstract/Free Full Text]
19. Wemmie, J. A., Chen, J., Askwith, C. C., Hruska-Hageman, A. M., Price, M. P., Nolan, B. C., Yoder, P. G., Lamani, E., Hoshi, T., Freeman, J. H., Jr., and Welsh, M. J. (2002) Neuron 34, 463–477[CrossRef][Medline] [Order article via Infotrieve]
20. Wemmie, J. A., Coryell, M. W., Askwith, C. C., Lamani, E., Leonard, A. S., Sigmund, C. D., and Welsh, M. J. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 3621–3626[Abstract/Free Full Text]
21. Voilley, N., de Weille, J., Mamet, J., and Lazdunski, M. (2001) J. Neurosci. 21, 8026–8033[Abstract/Free Full Text]
22. Page, A. J., Brierley, S. M., Martin, C. M., Martinez-Salgado, C., Wemmie, J. A., Brennan, T. J., Symonds, E., Omari, T., Lewin, G. R., Welsh, M. J., and Blackshaw, L. A. (2004) Gastroenterology 127, 1739–1747[CrossRef][Medline] [Order article via Infotrieve]
23. Lidsky, T. I., and Schneider, J. S. (2003) Brain 126, 5–19[Abstract/Free Full Text]
24. Nihei, M. K., and Guilarte, T. R. (2001) Neurotoxicology 22, 635–643[CrossRef][Medline] [Order article via Infotrieve]
25. Suszkiw, J. B. (2004) Neurotoxicology 25, 599–604[CrossRef][Medline] [Order article via Infotrieve]
26. Alvarez de la Rosa, D., Krueger, S. R., Kolar, A., Shao, D., Fitzsimonds, R. M., and Canessa, C. M. (2003) J. Physiol. (Lond.) 546, 77–87[Abstract/Free Full Text]
27. Chen, C. C., England, S., Akopian, A. N., and Wood, J. N. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 10240–10245[Abstract/Free Full Text]
28. Waldmann, R., Bassilana, F., de Weille, J., Champigny, G., Heurteaux, C., and Lazdunski, M. (1997) J. Biol. Chem. 272, 20975–20978[Abstract/Free Full Text]
29. Markovac, J., and Goldstein, G. W. (1988) Nature 334, 71–73[CrossRef][Medline] [Order article via Infotrieve]
30. Kern, M., and Audesirk, G. (1995) Toxicol. Appl. Pharmacol. 134, 111–123[CrossRef][Medline] [Order article via Infotrieve]
31. Baron, A., Deval, E., Salinas, M., Lingueglia, E., Voilley, N., and Lazdunski, M. (2002) J. Biol. Chem. 277, 50463–50468[Abstract/Free Full Text]
32. Deval, E., Salinas, M., Baron, A., Lingueglia, E., and Lazdunski, M. (2004) J. Biol. Chem. 279, 19531–19539[Abstract/Free Full Text]
33. Leonard, A. S., Yermolaieva, O., Hruska-Hageman, A., Askwith, C. C., Price, M. P., Wemmie, J. A., and Welsh, M. J. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2029–2034[Abstract/Free Full Text]
34. Chesler, M. (2003) Physiol. Rev. 83, 1183–1221[Abstract/Free Full Text]
35. Vukicevic, M., and Kellenberger, S. (2004) Am. J. Physiol. 287, C682–C690[CrossRef]
36. Riedel, G., Platt, B., and Micheau, J. (2003) Behav. Brain Res. 140, 1–47[CrossRef][Medline] [Order article via Infotrieve]
37. Lynch, M. A. (2004) Physiol. Rev. 84, 87–136[Abstract/Free Full Text]
38. Lasley, S. M., and Gilbert, M. E. (2000) Neurotoxicology 21, 1057–1068[Medline] [Order article via Infotrieve]
39. Kuhlmann, A. C., McGlothan, J. L., and Guilarte, T. R. (1997) Neurosci. Lett. 233, 101–104[CrossRef][Medline] [Order article via Infotrieve]
40. Nihei, M. K., Desmond, N. L., McGlothan, J. L., Kuhlmann, A. C., and Guilarte, T. R. (2000) Neuroscience 99, 233–242[CrossRef][Medline] [Order article via Infotrieve]
41. Gilbert, M. E., Mack, C. M., and Lasley, S. M. (1996) Brain Res. 736, 118–124[CrossRef][Medline] [Order article via Infotrieve]
42. Jett, D. A., Kuhlmann, A. C., Farmer, S. J., and Guilarte, T. R. (1997) Pharmacol. Biochem. Behav. 57, 271–279[CrossRef][Medline] [Order article via Infotrieve]
43. Centers for Disease Control (1991) Preventing Lead Poisoning in Young Children, Atlanta, GA
44. Needleman, H. (2004) Annu. Rev. Med. 55, 209–222[CrossRef][Medline] [Order article via Infotrieve]
45. Sun, X., Tian, X., Tomsig, J. L., and Suszkiw, J. B. (1999) Toxicol. Appl. Pharmacol. 156, 40–45[CrossRef][Medline] [Order article via Infotrieve]
46. Vyas, M. N., Jacobson, B. L., and Quiocho, F. A. (1989) J. Biol. Chem. 264, 20817–20821[Abstract/Free Full Text]
47. Ghering, A. B., Jenkins, L. M., Schenck, B. L., Deo, S., Mayer, R. A., Pikaart, M. J., Omichinski, J. G., and Godwin, H. A. (2005) J. Am. Chem. Soc. 127, 3751–3759[CrossRef][Medline] [Order article via Infotrieve]
48. Beigel, Y., Ostfeld, I., and Schoenfeld, N. (1998) N. Engl. J. Med. 339, 827–830[Free Full Text]
49. Coyle, P., Kosnett, M. J., and Hipkins, K. (2005) Am. J. Ind. Med. 47, 172–175[CrossRef][Medline] [Order article via Infotrieve]

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