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

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.

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 Ca 2ϩ 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 (Pb 2ϩ ) is a well known divalent metal ion because of its harm to the human body. It is widely accepted that Pb 2ϩ -induced impairment of synaptic function is one of the fundamental cellular mechanisms of Pb 2ϩ neurotoxicity in the central nervous system (CNS) (23)(24)(25). Considering the importance of ASICs in synaptic physiology, we asked whether ASICs are novel molecular targets of Pb 2ϩ action. Furthermore, because Ca 2ϩ and Pb 2ϩ 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 Pb 2ϩ . Because ASIC1b and -3 are mainly expressed in peripheral neurons, our study identifies ASIC1a subunit as a novel molecular target of Pb 2ϩ in CNS neurons, which may contribute to Pb 2ϩ neurotoxicity.

EXPERIMENTAL PROCEDURES
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 m 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. cellular 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 40ϫ UV fluor oil-immersion objective lens, and images were recorded by a cooled CCD camera (Hamamatsu Photonics, Hamamatsu City, Japan). To block potential Ca 2ϩ entry through voltage-gated Ca 2ϩ channels or glutamate receptors, 10 M nifedipine, 20 M D-2-amino-5-phosphonopentanoic acid, and 10 M 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 NaHCO 3 , 5 KCl, 1.2 KH 2 PO 4 , 2.4 CaCl 2 , 1.3 MgSO 4 , 10 glucose, aerated with 95% O 2 and 5% CO 2 to a final pH of 7.4. The standard extracellular solution contained the following (mM): 150 NaCl, 5 KCl, 1 MgCl 2 , 2 CaCl 2 , 10 glucose, buffered to various pH values with either 10 mM HEPES (pH 6.0 -7.4) or 10 mM MES (pH Ͻ6.0). The osmolarity of all external solutions was adjusted to 325-330 mosmol liter Ϫ1 with sucrose. The patch pipette solution for whole-cell patch clamp recording was as follows (mM): 120 KCl, 30 NaCl, 0.5 CaCl 2 , 1 MgCl 2 , 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 Ϫ1 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 M CdCl 2 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 Ca 2ϩ 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 Pb 2ϩ 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, where I is the normalized value of the current; I max is the maximal response; C is the drug concentration; EC 50 is the concentration that induced the half-maximal response; and h is the apparent Hill coefficient. The curve for the effect of Pb 2ϩ or amiloride on ASIC currents was fitted to the following Equation 2, where IC 50 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, where A 1 is the amplitude; is the decay time constant; and y 0 is the offset. All data are shown as the mean Ϯ S.E. Statistical comparison was carried out using Student's t test for comparison of two groups and analysis of variance for multiple comparisons. Statistically significant differences were assessed as p Ͻ 0.05 or p Ͻ 0.01. p and n represent the value of significance and the number of neurons, respectively.

Inhibition of ASIC-mediated Currents by Pb 2ϩ 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 Pb 2ϩ on ASICmediated 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 M Pb 2ϩ for 15 s, and then the ASICs were activated again by acidic solutions in the presence of 10 M Pb 2ϩ . Pb 2ϩ 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 Ϫ50 mV. Application of Pb 2ϩ alone did not evoke any detectable current. The Pb 2ϩ -induced inhibition of the ASIC currents was concentration-dependent. In SDH neurons, the IC 50 and Hill coefficients (Equation 2) of the inhibition-response curves were 8.7 M and 1.2, respectively (n ϭ 10); and in CA1 neurons, the two values were 7.1 M and 1.2, respectively (n ϭ 6). However, Pb 2ϩ (10 M) did not exert any apparent effects on the decay time constant (, Equation 3) of the currents in either SDH neurons (without Pb 2ϩ , ϭ 1.1 Ϯ 0.1 s, and with Pb 2ϩ , ϭ 1.3 Ϯ 0.1 s, n ϭ 10) or CA1 neurons (without Pb 2ϩ , ϭ 1.2 Ϯ 0.1 s, and with Pb 2ϩ , ϭ 1.3 Ϯ 0.1 s, n ϭ 6).
ASIC1 and -3 Subunits Were Sensitive to Pb 2ϩ -To study the subunit responsible for the Pb 2ϩ -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 Ϫ50 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 Pb 2ϩ for 15 s, and then the ASIC currents were induced in the presence of Pb 2ϩ ( Fig. 2A). We found that Pb 2ϩ significantly reduced the amplitude of the ASIC currents mediated by homomeric 1a and 1b and heteromeric 1a ϩ 2a and 1a ϩ 2b channels in concentration-dependent manners with the IC 50 and Hill coefficient of 3.7 M and 2.2, 1.5 M and 1.8, 4.9 M and 2.3, 2.8 M and 1.5, respectively (n ϭ 5-7) (Fig.  2). Similar to the effect on CNS neurons, Pb 2ϩ -induced inhibition in CHO cells was reversible after the removal of Pb 2ϩ . In addition, Pb 2ϩ did not affect the kinetics of ASIC currents in CHO cells (data not shown). In contrast, Pb 2ϩ exerted no significant inhibitory effects on the ASIC currents mediated by homomeric 2a and heteromeric 2a ϩ 2b channels at the concentrations ranging from 0.1 to 100 M (n ϭ 4 -5). Pb 2ϩ exerted no significant inhibition on homomeric ASIC3 channel at concentrations ranging from 0.1 to 10 M but decreased its current amplitude at a high concentration of 100 M (36.1 Ϯ 5.3% of the control, n ϭ 5, p Ͻ 0.01, Student's paired t test), suggesting the existence of low affinity site(s) for Pb 2ϩ on ASIC3 subunit.
Pb 2ϩ Prevented Neuronal Ca 2ϩ Elevation Mediated by ASIC Activation-The ASIC1a subunit, which renders ASICs a high Ca 2ϩ 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 Pb 2ϩ affects ASIC-mediated Ca 2ϩ entry by specifically inhibiting ASIC1a channels in CNS neurons. To test the hypothesis, we performed Ca 2ϩ imaging experiments in cultured SDH and hippocampal neurons. We observed an elevation of [Ca 2ϩ ] i when extracellular acidic solution was applied to these cultured neurons (Fig. 3A). This acid-induced elevation of [Ca 2ϩ ] i was pH-dependent and was reversibly blocked by 100 M amiloride (data not shown). Pb 2ϩ (10 M) reversibly reduced the ASIC-mediated elevation of [Ca 2ϩ ] i in cultured SDH (59.7 Ϯ 6.4% of control, n ϭ 6) or hippocampal neurons (64.3 Ϯ 11.2% of control, n ϭ 6) (Fig. 3). In addition, no significant difference of Pb 2ϩ inhibition was observed between hippocampal and SDH neurons (Fig. 3B). Because the blockers of ionic glutamate receptors and voltagegated Ca 2ϩ channels were added into the bath solution to prevent potential Ca 2ϩ entry through these receptors/channels (see "Experimental Procedures"), we can attribute the Pb 2ϩ -induced reduction in [Ca 2ϩ ] i to Pb 2ϩ -induced inhibition of neuronal ASIC1a-containing channels.
Pb 2ϩ Inhibited ASIC Currents Independent of Membrane Voltage-In the subsequent experiments, we investigated further the mechanisms of

Inhibition of ASIC1a Channels by Pb 2؉
FEBRUARY 3, 2006 • VOLUME 281 • NUMBER 5 Pb 2ϩ -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 Pb 2ϩ . We changed the V H of the tested neurons from Ϫ60 to ϩ60 mV in the absence or presence of 10 M Pb 2ϩ (Fig. 4A), and we plotted the current-voltage curve (Fig. 4B). The reversal potential of the ASIC currents was not altered by Pb 2ϩ (without Pb 2ϩ , 33.5 Ϯ 3.7 mV and with Pb 2ϩ , 37.0 Ϯ 4.9 mV; n ϭ 7), suggesting that Pb 2ϩ inhibited ASIC function without changing its ion selectivity. Moreover, the Pb 2ϩ -induced inhibition was independent of V H (Fig. 4C), suggesting that the action site(s) of Pb 2ϩ are not within the channel pore where Pb 2ϩ would experience the membrane electric field.
Pb 2ϩ Reduced pH Sensitivity of ASICs through a Competitive Mechanism-Next, we examined whether the Pb 2ϩ -induced inhibition of ASICs was modified by extracellular H ϩ . As illustrated in Fig. 5A, Pb 2ϩ (10 M) shifted the activation curve of ASIC currents rightward and reduced the pH value for the half-maximal activation (pH 50 ) from pH 6.4 to pH 5.8, suggesting a reduced pH sensitivity of ASICs in the presence of Pb 2ϩ . Furthermore, Pb 2ϩ did not significantly alter the Hill coefficient (1.1 without Pb 2ϩ ; 1.3 with Pb 2ϩ , n ϭ 5-7, Equation 1) or the maximal or minimal pH values of the activation curve, indicating a competitive mechanism.
Previous studies have shown that Pb 2ϩ stimulates intracellular protein kinases such as protein kinase C and protein kinase A (23, 29, 30), which may be associated with ASIC activity (31)(32)(33). To examine whether intracellular signaling messengers are involved in mediating Pb 2ϩ inhibition of ASIC currents, we studied the effects of 10 M Pb 2ϩ at various pretreatment durations. As shown in Fig. 5B, alternation of Pb 2ϩ pretreatment duration from 15 to 120 s did not affect its inhibitory potency (n ϭ 8; p Ͼ 0.05, ANOVA). In addition, when loaded into neurons via the patch electrode, staurosporine (10 M), a nonselective protein kinases inhibitor, or BAPTA (10 mM), the Ca 2ϩ chelator, did not alter the inhibition of ASIC currents by 10 M Pb 2ϩ (Fig. 5C). These results suggest that intracellular signaling messengers are not involved in the Pb 2ϩ -induced inhibition of ASIC currents.
Distinctions between Pb 2ϩ and Ca 2ϩ Modulation of ASICs-To date, a series of studies on the interaction of Ca 2ϩ , the physiologically relevant divalent metal ion, with ASICs has been made (5, 9 -15). Because Pb 2ϩ binds to a protein that usually contains Ca 2ϩ 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 Ca 2ϩ or Pb 2ϩ application to differentiate the effects of the two divalent cations. In co-application protocol (protocol a), neurons were coapplied with external Ca 2ϩ or Pb 2ϩ 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 Ca 2ϩ or Pb 2ϩ ; in pretreatment protocol (protocol c), neurons were pretreated with Ca 2ϩ or Pb 2ϩ for 15 s and then applied with Ca 2ϩ or Pb 2ϩ and acidic solution together. As shown in Fig. 6, A and C, different sequences of Ca 2ϩ (10 mM) application produced different effects on the ASIC currents. Most notably, application of Ca 2ϩ 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, Pb 2ϩ always produced an inhibitory effect on ASIC currents (Fig. 6, B and C). These findings suggest that Pb 2ϩ modulates ASICs in a manner different from that of Ca 2ϩ .
Noncompetitive Interaction of Pb 2ϩ and Amiloride with ASICs-Recently, it has been shown that Ca 2ϩ competes with amiloride, a known open channel blocker of ASICs, suggesting a Ca 2ϩ -blocking site on ASIC1a at the entrance of the ion pore (13). Because Pb 2ϩ inhibited ASICs at an open state (Fig. 6B-a), co-application of Pb 2ϩ and amiloride during the activation of ASICs was employed to assess whether Pb 2ϩ competes with amiloride in modulating ASIC currents (Fig. 7A). In the absence of Pb 2ϩ , amiloride concentration-dependently blocked the ASIC currents in SDH neurons with an IC 50 and Hill coefficient of 18.0 M and 0.7, respectively (Fig. 7B). In the presence of 6 M Pb 2ϩ , the IC 50 and Hill coefficients of inhibition-response curves for the ASIC currents by amiloride were 11.6 M and 0.8, respectively; and in the presence of 60 M Pb 2ϩ , the IC 50 and Hill coefficients for the inhibition-response curves were 8.3 M and 0.8, respectively (n ϭ 3-7). Compared with the maximal value of the amiloride inhibition-response curve, the maximal responses in the presence of 60 M and 6 M Pb 2ϩ were decreased approximately by 30 and 10%, respectively (n ϭ 5). These data suggest that Pb 2ϩ produces inhibition independent of the amiloride blockade of ASIC1a.
Pb 2ϩ Decreased Acidosis-induced Membrane Depolarization-Our previous study demonstrated that activation of ASICs in SDH neurons induces membrane depolarization, which may contribute to SDH synaptic transmission (15). Because Pb 2ϩ exerts inhibitory effects on ASICs, we examined whether Pb 2ϩ has an impact on acidosis-induced membrane excitation. Membrane potential was recorded in current clamp mode with no current injection. The acutely dissociated SDH neurons had a resting potential of Ϫ51.3 Ϯ 1.8 mV (n ϭ 8). A slight decrease of extracellular pH, which is in the range locally reached by pH fluctuations due to normal neuronal activity (34,35), was applied to induce membrane depolarization. As shown in Fig. 8, extracellular acidic solution of pH 6.5 induced a change in membrane potential of 37.0 Ϯ 2.9 mV (n ϭ 6). This acidosis-induced depolarization was reversibly inhibited by 100 M amiloride (change in membrane potential of 14.2 Ϯ 3.2 mV). In the same neurons, 10 M Pb 2ϩ reversibly reduced the change in membrane potential to 20.8 Ϯ 4.1 mV. Moderate acidic solu-

DISCUSSION
The ASIC1a Subunit Is a Novel Molecular Target of Pb 2ϩ Action in the CNS-Among various molecular targets of Pb 2ϩ neurotoxicity, the N-methyl-D-aspartate receptor (NMDAR) has received much attention because of its pivotal role in activity-dependent synaptic plasticity in the hippocampus, which is considered as a potential neural substrate for learning and memory, and elsewhere during both the developing and mature brain (36,37). Consequently, Pb 2ϩ -induced disruption of NMDARs is considered to be one of the molecular mechanisms underlying Pb 2ϩ neurotoxicity in the CNS (24,38). Thus, Pb 2ϩ -induced impairment of NMDAR-dependent long term potentiation (LTP) in hippocampus is correlated with deficits in spatial memory (39 -42). Although activation of NMDARs leading to influx of external Ca 2ϩ has been demonstrated to be critical for induction of many forms of LTP, it is suggested that elevation of the postsynaptic Ca 2ϩ level through pathways beyond NMDARs may also play important roles for the induction of hippocampal LTP (37). In particular, recent data identified ASIC1a channels as a novel pathway for [Ca 2ϩ ] i elevation in CNS neurons (8,16,17). ASIC1 knock-out mice displaying a mild deficit in spatial memory and a severe deficit in a classical eyeblink conditioning are accompanied by a deficit in hippocampal LTP, which indicates that ASIC1 participates in synaptic plasticity, learning, and memory (19). In this study, we show that ASIC1a-containing channel in SDH and hippocampal CA1 neurons is a novel molecular target of Pb 2ϩ action. Moreover, Pb 2ϩ reduces ASIC-mediated elevation of [Ca 2ϩ ] i and attenuates acid-induced membrane depolarization in these neurons. Considering the wide distribution of ASIC1a subunit in the central and periphery nervous system (14, 15, 18 -20, 26), it is conceivable that Pb 2ϩ -induced inhibition of ASIC signaling could represent a novel molecular mechanism underlying Pb 2ϩ neurotoxicity. On the other hand, it was recently demonstrated that a blockade of ASIC channels protects from postischemic neuronal death (17), suggesting at least some beneficial effects of ASIC1 inhibition. Therefore, although Pb 2ϩ itself is neurotoxic, the structural information based on future studies of Pb 2ϩ -ASIC interaction would provide insights into the design of therapeutic agents against excitotoxic and acidotoxic neuronal damage. Furthermore, the subtype specificity of Pb 2ϩ action suggests a role of Pb 2ϩ in identifying ASIC1acontaining channels in CNS neurons.
Previous studies indicate that the accepted blood Pb 2ϩ levels that cause neurocognitive deficits in children are 10 g/dl (ϳ0.5 M) or higher (43), and neurological symptoms can be seen at the blood Pb 2ϩ level above 60 g/dl (ϳ2.9 M) (44). In our experiments, the concentrations of Pb 2ϩ that produce direct inhibition on native ASIC channels are above 1 M, and the IC 50 of Pb 2ϩ inhibition of ASIC currents in SDH or hippocampal CA1 neurons is within the range of 7-9 M (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 Pb 2ϩ -We employed patch clamp recording to study the effects of Pb 2ϩ on ASICs, and we demonstrated that Pb 2ϩ action site(s) are located on the exterior surface of the ASIC1 subunit based on the following findings. First, the Pb 2ϩ -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 Pb 2ϩ -induced inhibition of ASIC currents between short term and prolonged Pb 2ϩ pretreatment (Fig. 6B). Second, Pb 2ϩ has been demon- strated to stimulate intracellular protein kinases (23-25, 29, 30) that may be associated with ASIC activity (31)(32)(33). However, in our experiments, the intracellularly loaded nonselective protein kinase inhibitor staurosporine or the Ca 2ϩ chelator BAPTA did not affect Pb 2ϩ inhibition of the ASIC currents, excluding the involvement of intracellular signaling messengers in the modulation of ASIC currents (Fig. 5C). Finally, the Pb 2ϩ -induced inhibition was independent on membrane potentials, suggesting that Pb 2ϩ action site(s) are not within the channel pore where it would experience the membrane electric field (Fig. 4C).
The finding that Pb 2ϩ affects ASICs via the extracellular domain of this channel suggests the presence of structural determinants of Pb 2ϩbinding site(s) in the extracellular loop. Because the negatively charged amino acids, glutamate and aspartate (45,46) as well as cysteine (47), have been shown to be associated with Pb 2ϩ coordination in some other proteins, and our data from the heterologous expression experiments indicate that only ASIC1 and -3 subunits are sensitive to Pb 2ϩ inhibition, we speculate that glutamate, aspartate, and cysteine, which are conserved in the extracellular portions of ASIC1 and -3, may be the site(s) of Pb 2ϩ inhibition. Therefore, Asp-303, Glu-359, and Glu-364 of ASIC1a and Asp-290, Glu-344, and Glu-349 of ASIC1b, which are conserved only in ASIC1a and -1b but not present in ASIC2a and -2b, and -3 subunits might represent the high affinity Pb 2ϩ inhibition site(s). In addition, similar residues conserved in ASIC1 and -3 or present only in ASIC3 may constitute the low affinity site(s) for Pb 2ϩ inhibition. Apparently, further investigations are required for the establishment of the action site(s) of Pb 2ϩ on ASICs.
Another finding in our experiments is that Pb 2ϩ rightward-shifted the concentration-response curve for ASIC currents in a parallel manner without significantly altering the maximal value and Hill coefficient (Fig. 5A), which suggests that Pb 2ϩ reduced pH sensitivity of ASICs through a competitive mechanism. Comparatively, Ca 2ϩ has also been shown to modulate ASIC currents in a competitive manner (5,10,13). Therefore, we hypothesized that the two divalent cations may interact with ASIC1a in a similar fashion. However, in our experiments, enhancement or inhibition of ASIC currents by Ca 2ϩ depended on the open or closed state of ASICs, whereas Pb 2ϩ always exerted inhibitory effects on ASIC currents. When Ca 2ϩ was applied at the resting state of ASICs, a significant enhancement of ASIC currents was observed. How-  ever, Ca 2ϩ inhibited ASIC currents when it was applied at the open state of the channels (Fig. 6, A and C).
In a recent study by Paukert et al. (13), the authors proposed a "two sites" model to explain the Ca 2ϩ -induced modulation of ASICs: a blocking site near the entrance of channel pore and a putative modulating site located on the external part of the channels. This model could account for the Ca 2ϩ -induced opposite modulation of ASIC currents observed in the present study. The two sites model, however, cannot explain the mechanism of Pb 2ϩ inhibition on ASIC1a. As mentioned above, whatever sequence of drug application was employed, Pb 2ϩ always exerted inhibitory effects (Fig. 6, B and C). We hypothesized that Pb 2ϩ inhibited ASIC currents by competing H ϩ binding based on the observation that Pb 2ϩ reduced pH sensitivity of ASICs (Fig. 5A). Moreover, this inhibitory site of Pb 2ϩ is distinct from the blocking site of Ca 2ϩ on ASIC1a because Pb 2ϩ inhibited ASIC currents independent of amiloride/Ca 2ϩ blockade (Fig. 7), and the effect of Pb 2ϩ was voltage-independent (Fig.  4C). Taken together, we conclude that Pb 2ϩ inhibits ASICs via a competitive mechanism, which is different from that of Ca 2ϩ and amiloride.
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, Pb 2ϩ -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 mechanosensitivity 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 Pb 2ϩ often display gastrointestinal symptoms of abdominal pain (48,49). If Pb 2ϩ 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 Pb 2ϩ action. However, the definite relationship between the effect of Pb 2ϩ on ASICs and Pb 2ϩ -induced neurotoxicity should be established through further investigations. We observed a direct Pb 2ϩ -induced inhibition of ASIC currents, which may play an important role in acute Pb 2ϩ intoxication. As for the chronic Pb 2ϩ exposure, the effects of Pb 2ϩ on neuronal ASIC expression at the transcriptional or translational levels should be examined. Moreover, to explore the role of ASICs in Pb 2ϩ intoxication-related pathological processes, more knowledge in the involvement of ASICs themselves in synaptic physiology and cellular functions is required.