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Am J Physiol Renal Physiol 293: F236-F244, 2007. First published April 11, 2007; doi:10.1152/ajprenal.00002.2007
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Effect of divalent heavy metals on epithelial Na+ channels in A6 cells

Ling Yu,1,3 Douglas C. Eaton,1,2,3 and My N. Helms1,3

Departments of 1Physiology and 2Pediatrics and 3The Center for Cell and Molecular Signaling, Emory University School of Medicine, Atlanta, Georgia

Submitted 2 January 2007 ; accepted in final form 6 April 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To better understand how renal Na+ reabsorption is altered by heavy metal poisoning, we examined the effects of several divalent heavy metal ions (Zn2+, Ni2+, Cu2+, Pb2+, Cd2+, and Hg2+) on the activity of single epithelial Na+ channels (ENaC) in a renal epithelial cell line (A6). None of the cations changed the single-channel conductance. However, ENaC activity [measured as the number of channels (N) x open probability (Po)] was decreased by Cd2+ and Hg2+ and increased by Cu2+, Zn2+, and Ni2+ but was not changed by Pb2+. Of the cations that induced an increase in Na+ channel function, Zn2+ increased N, Ni2+ increased Po, and Cu2+ increased both. The cysteine modification reagent [2-(trimethylammonium)ethyl]methanethiosulfonate bromide also increased N, whereas diethylpyrocarbonate, which covalently modifies histidine residues, affected neither Po nor N. Cu2+ increased N and stimulated Po by reducing Na+ self-inhibition. Furthermore, we observed that ENaC activity is slightly voltage dependent and that the voltage dependence of ENaC is insensitive to extracellular Na+ concentration; however, apical application of Ni2+ or diethylpyrocarbonate reduced the channel voltage dependence. Thus the voltage sensor of Xenopus ENaC is different from that of typical voltage-gated channels, since voltage appears to be sensed by histidine residues in the extracellular loops of ENaC, rather than by charged amino acids in a transmembrane domain.

divalent cations; single-channel recording; sodium self-inhibition

HEAVY METALS ARE MAJOR ENVIRONMENTAL pollutants that result in globally important health problems. Heavy metals tend to accumulate in specific tissues in the human body, such as liver, bones, and kidneys. Heavy metal toxicity is most commonly associated with cognitive deficits and renal pathology. The renal pathology is not surprising, since, except for some excretion via the gastrointestinal tract, most heavy metals are concentrated and excreted by the kidneys (11). In the central nervous system, heavy metals often target ion channels (18, 32), but whether the renal effects of heavy metals also involve ion channels is less clear. In particular, epithelial Na+ channels (ENaC) in the distal nephron are exposed to high levels of heavy metal because of tubular water reabsorption in earlier parts of the nephron.

ENaC are responsible for the kidney's ability to regulate total body Na+ balance and, thereby, mean blood pressure. ENaC consists of three conserved subunits, {alpha}, beta, and {gamma}. Each subunit has two transmembrane-spanning domains (TM1 and TM2), a large extracellular domain between TM1 and TM2, and NH2 and COOH termini within the cytoplasm (3). Inappropriate alteration of ENaC activity is linked to several human genetic diseases: a gain-of-function mutation is associated with Liddle's syndrome (31), and a loss-of-function mutation leads to pseudohypoaldosteronism type 1 (23, 34). ENaC is also well conserved among different species: identity between Xenopus and human {alpha}-, beta-, and {gamma}-subunit ENaC proteins is 54%, 59%, and 55%, respectively.

Because A6 cells are derived from distal tubules of Xenopus laevis and express ENaC protein, they are a useful model for characterization of ENaC regulation and function. Examination of the effect of heavy metals on ENaC activity in A6 cells should provide useful insight into the mechanisms of kidney cell injury caused by divalent metal cations and, thus, the role of heavy metals in renal pathology.

From previous research on transport in frog skin, it is known that heavy metals affect epithelial Na+ transport (10). The effects of heavy metals were reported as changes of transepithelial short-circuit current (SCC), but the precise characteristics of the channels in the frog skin carrying this current were not clearly defined. More recently, the effects of heavy metals on ENaC have been examined in cultured epithelial cells (6) and ENaC expressed in Xenopus oocytes (1, 29, 30). In these experiments, ENaC were defined as amiloride-sensitive channels, but the currents were measured as transepithelial SSC or as whole cell current. The properties of amiloride-sensitive channels can vary widely (7), but only the channel consisting of {alpha}-, beta-, and {gamma}-subunits with a single-channel conductance of ~5 pS is characteristic of ENaC in principal cells of the distal nephron (7). Single-channel recording unequivocally determines channel characteristics, including unit conductance and channel activity [measured as NPo, i.e., the number of channels (N) x open probability (Po)]. Single-channel measurements can also provide information about N and Po.

In this study, we have examined the effects of apical application (within the patch pipette) of Zn2+, Ni2+, Cu2+, Pb2+, Cd2+, and Hg2+ on ENaC activity. Zn2+, Ni2+, and Cu2+ increased channel activity, Cd2+ and Hg2+ strongly inhibited activity, and Pb2+ had a marginal effect on ENaC function. Our experimental evidence suggests that each metal has its own coordination site in the extracellular loops (ECLs) of Xenopus ENaC. We also found that ENaC in A6 cells is voltage dependent and that the voltage is likely sensed by His residues in the ECLs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
A6 cell culture preparation. A6 cells (subclone 2F3; obtained from Drs. Krahenbul and Rossier) were maintained in plastic tissue culture flasks, as described previously (2). For single-channel patch-clamp experiments, A6 cells were seeded onto collagen-coated permeable support inserts until they reached confluency. A6 cell culture medium, which consists of three parts Coon's medium F-12 and seven parts Leibovitz's medium L-15 modified for amphibian cells with 104 mM NaCl, 25 mM NaHCO3, 10% fetal bovine serum (GIBCO, Grand Island, NY), 1.0% streptomycin, and 0.6% penicillin (Irvine Scientific, Santa Ana, CA) with pH 7.4, was replaced three times per week. Patch-clamp experiments were carried out in A6 cells between passages 97 and 104.

Single-channel recordings. The cell-attached configuration was used in all patch-clamp studies. Micropipettes were pulled from filamented borosilicate glass capillaries (TW-150, World Precision Instruments) with a two-stage vertical puller (Narishige, Tokyo, Japan). Resistance of the pipettes was 7–10 M{Omega} when they were filled with and immersed in patch solution containing (in mM) 96 NaCl, 3.4 KCl, 0.8 CaCl2, 0.8 MgCl2, and 10 HEPES, with pH adjusted to 7.4 with NaOH. Pipettes were backfilled with patch solution or with patch solution containing one of the following compounds: 100 µM ZnCl2, 2 mM NiCl2, 100 µM Pb(acetate)2, 100 µM CuCl2, 100 µM CdCl2, 2–20 µM HgCl2, 2 mM [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET), or 2 mM diethylpyrocarbonate (DEPC). MTSET, DEPC, and Pb(acetate)2 solutions were made fresh daily. All chemical compounds were purchased from Sigma Chemical (St. Louis, MO).

Data analysis. After formation of a high-resistance (~5-G{Omega}) seal, the channel currents were recorded at 1 kHz with an Axopatch 1-D amplifier (Molecular Devices) with a low-pass 100-Hz Bessel filter. Channel activity (NPo) was calculated from pClampfit 9.2 data analysis software (Molecular Devices), N was determined from the maximal number of transitions during 20–25 min of recording, and Po was calculated as the ratio of NPo to N. Values are means ± SE. Differences between groups were evaluated with one-way ANOVA, and differences of properties of the same cell at different membrane potentials were tested by paired t-test. Parameter estimation from curve fitting was done with Sigmaplot and Sigmastat (San Rafael, CA).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The conductance and selectivity of ENaC in cultured renal cells are affected by culture conditions (9). In our work, A6 cells were grown on permeable supports in the presence of aldosterone; under this condition, the only cation channel that was observed was a channel with 4- to 5-pS conductance and slow gating kinetics, which we previously identified as ENaC. These properties are characteristic of Na+ channels found in many tight epithelia. All the results reported here are from this type of channel.

Different heavy metals affect ENaC activity differently. We used single-channel, cell-attached patch-clamp analysis to test the effects of six divalent metals on ENaC activity. These metals were individually applied to the apical surface via the pipette solution. To eliminate variability, which might arise between cells from different passages and even different wells from the same passage, we formed patches and recorded separately from nearby cells in the same culture well, first with a pipette without heavy metal and then with a pipette containing one of the heavy metals. None of the metals produced a statistically significant change in single-channel conductance (Table 1), but all the metals, except Pb2+, produced an easily discernible change in ENaC activity (NPo; Fig. 1). NPo from cells exposed to heavy metals, as well as their respective controls, are summarized in Fig. 2. Figure 2A shows a significant increase of NPo in cells treated with 2 mM Ni2+ compared with control cells (0.95 ± 0.16 vs. 0.40 ± 0.12, P = 0.029). [We also tested the effect of 200 µM Ni2+ on ENaC activity and found no significant effect at this lower concentration (data not shown).] Figure 2A also shows that 100 µM Zn2+ (0.87 ± 0.39) failed to significantly alter ENaC activity when the pipette holding potential was 0 mV. (Zn2+ and Ni2+ treatments shared a common group of control recordings.) In a separate study (Fig. 2B), 100 µM Cu2+ significantly increased NPo almost fivefold compared with control values (from 0.16 ± 0.06 to 0.83 ± 0.22, P = 0.01). Interestingly, the effect of 100 µM Cd2+ and 20 µM Hg2+ (with the same outer shell electronic structure as Zn2+) differed from the effects of Zn2+. In cells exposed to Cd2+, NPo decreased to <15% of its control value (from 2.85 ± 0.55 to 0.34 ± 0.19, P < 0.01). The inhibitory effect of Hg2+ can be seen at concentrations as low as 2 µM (Fig. 2D), and 20 µM Hg2+ decreased the channel activity from the control value of 0.79 ± 0.34 to 0.02 ± 0.004 (P < 0.01). The results presented in Fig. 2D were recorded from cells with a holding potential of –60 mV to increase overall channel activity (see below). The effect of 100 µM Pb2+ was also tested; compared with its control, there was no significant effect (see NPo during initial recording period at 0 mV in Fig. 3D). All results and statistical evaluation are calculated on the basis of patches with active channels. However, the statistical significance would not change if silent patches were included.


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  		Table 1. Unit conductance of ENaC channels in metal-treated and untreated patches

 

Figure 1
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  		Fig. 1. Typical single-channel recordings from epithelial Na+ channels (ENaC) in A6 cells exposed to heavy metals. A6 cells were grown on Nunc filters for 8–12 days to form tight confluent monolayers; then the patch-clamp technique was used to record single-channel activity in the cell-attached configuration. Inward currents are downward deflections. Downward deflections from the closed state are individual channel openings, O. C, closed level. Controls (Ctrl) and treatments were always recorded in pairs from cells growing in the same dish. All traces were recorded at a holding potential of 0 mV.

 

Figure 2
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  		Fig. 2. Effect of divalent heavy metals on ENaC activity. Channel activity is presented as channel density [i.e., number of channels per patch (N)] x open probability (Po). Numbers above columns represent number of patches with active channels; numbers in parentheses represent number of cells on which seals were formed but in which the patches had no active channels ("silent" patches). Average NPo was recorded from cells with active channels. Values are means ± SE. **P < 0.02. A: 100 µM Zn2+ or 2 mM Ni2+ in pipette solution. Zn2+ and Ni2+ share a common control group. NPo was recorded at pipette holding potential of 0 mV. B and C: 100 µM Cu2+ or Cd2+ in pipette solution. Data were compared with their own control groups at 0 mV. D: 20 µM Hg2+ and 2 µM Hg2+ in pipette solution. Data were recorded at –60 mV and compared with control cells.

 

Figure 3
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  		Fig. 3. Activity of ENaC is enhanced by hyperpolarizing membrane potentials. NPo was recorded at pipette holding potential (–Vpip) of 0 to –100 mV in 20-mV steps and again at 0 mV. Duration of the first 0-mV recording period was 7–10 min; remaining potentials were recorded for 2–3 min. NPo for control and treated cells are plotted in pairs vs. pipette holding potential in A, B, C, and D. Results in A, B, and C are recorded from cells in Fig. 2, A, B, and C, which had active channels. In D, there were 13 patches on Pb2+-treated cells and 12 patches on control cells.

 
ENaC voltage dependence is altered by heavy metals. ENaC is typically regarded as a constitutively active channel, the activity of which is modulated by hormonal agents. However, when all three subunits are expressed in Xenopus oocytes, Xenopus ENaC is slightly voltage dependent (5), and native ENaC in rat cortical collecting tubule principal cells is also voltage dependent (21). To test the voltage dependence of Xenopus ENaC in A6 cells, the channel activity was first recorded at a holding potential of 0 mV immediately after formation of a tight seal. Then activity from the same patch was recorded at a more hyperpolarizing membrane potential, from 0 to –100 mV in –20-mV steps. To check whether the channel activity remained stable after the voltage steps, we returned each patch to a holding potential of 0 mV for a final recording period. Because NPo from most of the patch recordings remained stable for ~30 min, for completion of all the recordings at different membrane potentials before loss of channel activity, the duration of the first 0-mV recording period was ~7–10 min and the duration of all subsequent recordings at different holding potentials was 2–3 min. From the results of Fig. 3 (as well as GoGoGoFig. 7A), where NPo is plotted vs. the different pipette holding potentials (–Vpip), it is clear that ENaC activity in A6 cells is enhanced at hyperpolarized membrane potentials. In Fig. 3D, however, the channel activity did not respond strongly to voltage changes. This is likely due to rundown of channel activity. Nevertheless, the increase in NPo at hyperpolarized membrane potentials is clear. The reduced voltage sensitivity was also observed in cells from the same passage recorded in pairs exposed to 100 µM Pb2+ (Fig. 3D).


Figure 4
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  		Fig. 4. Effects of heavy metal on ENaC voltage dependence. Mean NPo values were calculated for individual cells, log NPo vs. voltage was plotted (means ± SE), and values were fitted to the following equation: NPo(V) = NPo(0) exp (–{alpha}VF/RT), where NPo(V) is NPo at voltage V, NPo(0) is NPo at 0 mV, {alpha} is voltage dependence, and R, T, and F are constants. Solid lines are best-fit values for NPo based on mean {alpha}. Determination of {alpha} has the advantage that it is independent of N. A value of {alpha} of 0.30 implies that the voltage-sensing residues in the channel sense ~30% of the membrane field and that there is an e-fold change in Po for an 83-mV change in membrane potential. For concentrations of heavy metals, see Fig. 2 legend.

 

Figure 5
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  		Fig. 5. Effects of heavy metals on voltage dependence of ENaC. Zn2+ and Cu2+ increase voltage dependence, Ni2+ decreases voltage dependence, and Pb2+ does not affect voltage dependence. For concentrations of heavy metals, see Fig. 2 legend. Values are means ± SE. *P < 0.001 vs. control.

 

Figure 6
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  		Fig. 6. Stimulatory effects of Zn2+, Ni2+, and Cu2+ on ENaC activity. A and C: N after treatment vs. control. B and D: Po after treatment vs. control. For concentrations of heavy metals, see Fig. 2 legend. Values are means ± SE. *P < 0.05; **P < 0.02; ***P < 0.01.

 

Figure 7
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  		Fig. 7. Effects of [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) and diethylpyrocarbonate (DEPC) on ENaC activity. A: ENaC activity recorded at different membrane potentials from cells exposed to 2 mM MTSET (n = 24), 2 mM DEPC (n = 19), and their control (n = 19). B: verification of voltage dependence of channel. First recording period shows channel activity as NPo at 0 mV; hyperpolarized value is average NPo of channel activity at –60, –80, and –100 mV. C and D: averaged NPo differentiated into N and average Po. Numbers above columns represent number of cells with active channels. Values are means ± SE. **P < 0.02; ***P < 0.01.

 
The comparison of channel activity at –100 and 0 mV recorded immediately thereafter would be the strongest measure of the channel voltage dependence, but the recording duration at these two voltage steps is too short to provide consistently statistically different values for NPo. Therefore, NPo of each cell at –60 to –100 mV (6–9 min total duation) were averaged and compared with NPo of the same cell during the initial recording period at 0 mV (~7–10 min total duration). The mean value of NPo was derived from 57 control cells at 0 mV (0.252 ± 0.0772) and was significantly different from the mean value for hyperpolarized potentials (0.735 ± 0.177, P = 0.014).

The gating of a voltage-dependent channel is sensed by a cluster of charged amino acids spaced within three to four residues of each other in the transmembrane region; however, there is no such cluster in the transmembrane domains of any of the three ENaC subunits. To explore whether the ECLs of ENaC are involved in sensing voltage and also to test whether heavy metals affect the ability of the channel to sense voltage, we recorded the channel activity at different membrane potentials with pipettes filled with 100 µM Zn2+, 2 mM Ni2+, 100 µM Cu2+, or 100 µM Pb2+. NPo values were plotted vs. pipette holding potential and compared with their respective control measurements (Fig. 3). For evaluation of the effects of heavy metals on channel voltage dependence, the calculated ratio of NPo for each cell at 0 mV was compared with that at hyperpolarized membrane potential (averaged as described above) for each heavy metal and for their respective controls. The ratio was 1.46 ± 0.24 for Ni2+-exposed cells (n = 12) and 2.87 ± 0.59 for control cells (n = 12, P = 0.003); therefore, Ni2+ significantly impaired ENaC voltage dependency. In contrast, ratios for Zn2+-, Cu2+-, and Pb2+-treated cells were not significantly different from controls; however, because the power of the statistical tests is low, the negative results do not exclude a true difference.

Therefore, to obtain a better idea of the channel voltage dependence, we took advantage of the fact that Po changes according to the following expression

Formula 1(1)
where Po(V) is Po at a voltage V, Po(0) is Po at 0 mV, {alpha} is voltage dependence of the channel, and R, T, and F are constants. One way to interpret {alpha}{alpha} is as the fraction of the membrane field detected by charged sites on the channel. The value for {alpha} can be obtained by determining the slope of the relationship between log Po and voltage (or log NPo and voltage, since N only represents a scaling factor). Figure 4 shows just such plots for Ni2+, Cu2+, Zn2+, and Pb2+: Ni2+ decreases, Cu2+ and Zn2+ increase, and Pb2+ does not change the voltage dependence of the channel (measured as slopes of the lines). Figure 5 shows the actual values of {alpha} determined from the slopes for different metals and their respective controls.

Effects of Zn2+, Ni2+, and Cu2+ suggest different binding sites. The channel activity we have presented has been reported as NPo, which combines N and Po. We have shown that N can be determined with a high degree of confidence if the duration of recording is long enough and Po is close enough to 0.5, when Po can be calculated by dividing NPo by N (17). All results summarized in Fig. 3 are from cell recordings of ~20- to 25-min duration, which is sufficient to allow a good estimate of N for each patch. This estimate of N is more accurate when it is determined from recordings held at hyperpolarizing potentials (since Po is closer to 0.5 than at more depolarized potentials). The average N from patches exposed to Zn2+ and Ni2+ were 5.17 ± 0.64 (n = 12) and 3.67 ± 0.90 (n = 12), respectively, compared with the control value of 3.27 ± 0.45 (n = 12; Fig. 6A). In patches exposed to Cu2+, average N was 4.69 ± 0.63 (n = 13), and average N of its paired control cell recordings was 2.85 ± 0.37 (n = 13; Fig. 6C). Since NPo is voltage dependent, Po was calculated from NPo at hyperpolarizing membrane potentials. The average Po from patches exposed to Zn2+ or Ni2+ was 0.306 ± 0.032 (n = 12) or 0.475 ± 0.049 (n = 12), respectively, and average Po of their control recordings was 0.222 ± 0.027 (n = 12; Fig. 6B). For patches exposed to Cu2+, average Po was 0.406 ± 0.033 (n = 13) compared with average control Po of 0.140 ± 0.022 (n = 13; Fig. 6D). When the same calculation was performed for Pb2+-exposed and paired control cells, average N and Po were 3.57 ± 0.77 and 0.19 ± 0.03 (n = 14), respectively, compared with control values of 1.92 ± 0.42 and 0.15 ± 0.3 (n = 12), respectively. In summary, we found that Zn2+ significantly increased N (P = 0.026), Ni2+ significantly increased Po at hyperpolarizing potentials (P = 0.01), and Cu2+ significantly increased N and Po (P = 0.019 and P = 8.4 x 10–5); however, Pb2+ had no significant effect on N or Po. All four metals affect channel activity, albeit in different ways, suggesting that each metal may have different specific binding sites in the ECLs.

MTSET mimics effects of Zn2+ and Cu2+, and DEPC mimics the effect of Ni2+. Divalent metals preferentially bind His and Cys residues (8); indeed, it has been shown that His and Cys residues in ECLs of mouse ENaC are important ligand binding sites for Ni2+ and Zn2+ (29). The effect of heavy metals on ENaC NPo has not been specifically studied. Our present study provides information regarding ligand specificity of the metals. To distinguish the metals' binding sites on ENaC, we covalently modified extracellular Cys or His residues, respectively, by adding MTSET or DEPC to the pipette solution before recording the channel activity at different membrane potentials. The protocol for data collection was the same as that described above, with a common control from cells on the same plates treated with MTSET or DEPC. In Fig. 7A, it is clear that the channel activity is higher at hyperpolarized membrane potentials in control and MTSET-treated, but not DEPC-treated, cells. NPo at hyperpolarized membrane potential was averaged and presented together with NPo at 0 mV in Fig. 7B. Specifically, at 0 mV, NPo of cells exposed to MTSET, DEPC, and the untreated controls were 0.60 ± 0.14 (n = 24), 0.49 ± 0.13 (n = 19), and 0.32 ± 0.08 (n = 19), respectively. Compared with control values, neither MTSET nor DEPC significantly stimulated ENaC activity at 0-mV membrane potential. On the other hand, the average NPo at hyperpolarizing membrane potentials was 1.33 ± 0.24 for cells exposed to MTSET (n = 24) and 0.74 ± 0.21 for cells exposed to DEPC (n = 19), and their control value was 0.923 ± 0.220 (n = 19). Thus the Na+ channels in control and MTSET-treated cells are voltage dependent (P = 0.005 and P = 8.8 x 10–4), whereas the Na+ channels in DEPC-treated cells resemble the ENaC of Ni2+-exposed cells. DEPC (and Ni2+) eliminated the channel voltage dependence measured as the slope of log NPo vs. voltage curves (Fig. 8). No significant difference in NPo at hyperpolarizing membrane potentials was apparent among control and the two reagent treatments in Fig. 7B.


Figure 8
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  		Fig. 8. Effects of 2 mM MTSET and 2 mM DEPC on voltage dependence of ENaC. Voltage dependence was increased by MTSET and decreased by DEPC.

 
However, because the power of the test was low, it is possible that we failed to detect a difference that was actually present. Since NPo consists of two independent variables, N and Po, both of which contribute to the overall variability, we separated the variables on the basis of our best estimates of N. When we examined the effect of MTSET and DEPC on N and Po separately, there was a difference. The average N of control cells was 3.26 ± 0.26 (n = 19). In cells exposed to MTSET, average N was 4.19 ± 0.24 (n = 24), which was significantly different from control (P = 0.038); in cells exposed to DEPC, average N was 2.81 ± 0.19, which was not significantly different from control (n = 19; Fig. 7C). These effects of Cys modification by MTSET are not unlike those observed in Zn2+- and Cu2+-treated cells. Cys modification significantly increased average N, whereas His modification had no effect on N. However, neither MTSET nor DEPC altered ENaC Po at hyperpolarizing membrane potentials (Fig. 7D).

Heavy metals apparently do not alter Po by interaction with His residues. Our results show that Cu2+ and Ni2+ increased Po, but DEPC had no effect on Po, despite the fact that His residues are known to bind Ni2+. To further demonstrate that extracellular His is not involved in the heavy metal effects on Po, we performed another set of experiments in which cells were challenged with DEPC and Cu2+ simultaneously. We used Cu2+, instead of Ni2+, because Cu2+ produced a stronger stimulatory effect on ENaC than Ni2+. Figure 9A shows that channels maintained their voltage dependence after exposure to DEPC + Cu2+. ENaC in cells treated only with DEPC lost their voltage dependence (Figs. 7A and 8). NPo was significantly higher in cells treated with DEPC + Cu2+ than in cells treated with DEPC only (P < 0.05) at all holding potentials except –40 mV. Furthermore, in cells treated with DEPC + Cu2+, hyperpolarizing membrane potentials substantially increased NPo. Figure 9B summarizes NPo at 0 mV and at hyperpolarized potentials. NPo for hyperpolarized cells treated with DEPC + Cu2+ was 1.801 ± 0.257, which is significantly higher than the initial NPo of 1.02 ± 0.23 (n = 14, P = 0.025) at 0 mV in the same cells. The average NPo of hyperpolarized DEPC-treated cells was 0.667 ± 0.181, which is not significantly different from the initial NPo of 0.34 ± 0.12 (n = 11) at 0 mV. As shown in Fig. 9, C and D, cells treated with DEPC + Cu2+ showed an increase in N and Po compared with cells treated with DEPC only: N was 2.06 ± 0.53 for DEPC-treated cells and 4.00 ± 0.73 for cells treated with DEPC + Cu2+ (P = 0.039), and Po was 0.17 ± 0.02 for DEPC-treated cells, which was significantly lower than that for cells treated with DEPC + Cu2+ (0.30 ± 0.03, P = 0.045). Together, these results suggest that DEPC-accessible His residues in the ECLs of ENaC are not the binding site for metal-induced effects on Po.


Figure 9
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  		Fig. 9. Effects of DEPC and DEPC + Cu2+ on ENaC activity. A: NPo of cells exposed to DEPC + Cu2+ and DEPC at different membrane potentials. B: NPo at 0 mV and average NPo at hyperpolarized potentials with DEPC and DEPC + Cu2+. C and D: stimulatory effect of DEPC and Cu2+ + DEPC separated into N and Po. Numbers above columns represent number of cells with active channels. Values are means ± SE (n = 19). *P < 0.05; ***P < 0.01.

 
Stimulatory effects of heavy metals on ENaC activities are due to a reduction of Na+ self-inhibition. Our data show that Zn2+, Ni2+, and Cu2+ stimulate Na+ channel activity. Interestingly, Zn2+ has been reported to stimulate mouse ENaC activity by reducing Na+ self-inhibition (30). Na+ self-inhibition has been observed when external Na+ concentration was >30 mM on oocytes (26). Unfortunately, single-channel activity cannot be resolved with a pipette solution containing only 30 mM Na+.

Therefore, to test metal-induced reduction of Na+ self-inhibition, we recorded single-channel activity with pipette solution containing 40 mM Na+. At this external Na+ concentration, Na+ channel self-inhibition was negligible, whereas the single-channel activity could be resolved clearly (Fig. 10A). We recorded channel activity at holding potentials (–Vpip) from –20 to –100 mV in 20-mV steps with or without 100 µM Cu2+ in the pipette solution. The duration was 8–10 min for the first recording period at –20 mV, 4–5 min at –80 and –100 mV, and 2–3 min at the other potentials (Fig. 10B). At lower extracellular Na+ concentration, Cu2+ no longer stimulated N [4.49 ± 0.29 (n = 12) vs. 4.63 ± 0.56 (n = 11) for treatment vs. control] or Po (0.34 ± 0.03 vs. 0.34 ± 0.04 for treatment vs. control). ENaC remained voltage dependent in control and treated cells. NPo at –20 mV was 0.65 ± 0.14 (control); 0.77 ± 0.16 (treated) and average NPo at –80 and –100 mV was 1.71 ± 0.30 and 1.77 ± 0.23, respectively. Hence, the mechanism of ENaC voltage dependence is independent of Na+ self-inhibition.


Figure 10
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  		Fig. 10. ENaC activity at low external Na+ concentration. A: representative single-channel trace recorded with 40 mM extracellular Na+. B: channel activity recorded at pipette holding potentials from –20 to –100 mV in 20-mV steps and again at –20 mV. NPo with 40 mM extracellular Na+ without (Ctrl, n = 11) or with 100 µM Cu2+ (n = 12) is plotted vs. pipette holding potential.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Properties and summary of effects of heavy metals on properties of ENaC. Because the coordination chemistry of the heavy metals is complex, it is not surprising that the metal ions used in this study produce substantially different effects on ENaC. The three effects, change of N, change of Po, and change of voltage dependence of the channel, are summarized in Table 2, along with the some of the physical chemical properties of the divalent cations. It is also important to recognize that, because of the application and measurement methods, the effects we describe are acute effects that alter only the external surface of ENaC.


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  		Table 2. Properties of heavy metals and a summary of their effects on ENaC

 
There are multiple heavy metal interaction sites in ENaC ECLs. Using single-channel recording, we have reported, for the first time, the effects of heavy metals on ENaC activity. After separating the single-channel NPo into N and Po, we found that the stimulatory effects of Zn2+, Ni2+, and Cu2+ on ENaC activity were different, which suggests that these metals coordinate with different binding sites in ENaC ECLs. The effect of Ni2+ on ENaC seems species dependent, since Ni2+ increases instantaneous current of Xenopus ENaC expressed in Xenopus oocytes at a holding potential of 0 mV (5), which is consistent with our observation that Ni2+ increases channel NPo at depolarizing membrane potentials. Mouse and rat ENaC, however, were inhibited by external Ni2+ when the respective cRNAs were expressed in Xenopus oocytes (24, 29). Sheng et al. (29) reported that, in the ECLs of mouse ENaC, two conserved His residues play crucial roles in the inhibitory effect of Ni2+. The opposing effects of Ni2+ on different ENaC homologs suggest that Ni2+ could be simultaneously coordinating with ligands other than these two conserved His residues. Since Sheng et al. recorded whole cell current, it was difficult for them to determine whether Ni2+ specifically affected N or Po. Our results, however, demonstrate that Ni2+ increased channel activity by increasing Po and that Cu2+ exerted the same effect on ENaC Po, even when extracellular His residues were covalently modified by DEPC (Fig. 9). These findings suggest that although Cu2+ and (Ni2+) may bind extracellular His residues, they stimulate Po by binding other residues that are not accessible to DEPC in the ECLs of Xenopus ENaC.

In contrast to the effect of Ni2+, the stimulatory effect of Zn2+ on ENaC does not appear to be species specific. Zn2+ increased ENaC activity in Xenopus A6 cells, as well as rat and mouse ENaC expressed in Xenopus oocytes (30). However, Amuzescu et al. (1) observed an inhibitory effect of Zn2+ on ENaC activity in A6 cells. The discrepancy between their observation and our data may be due to differences in the experimental approach. Amuzescu et al. depolarized A6 cell membrane potential by incubating cells in a high-K+ bath solution before recording. Our data, however, show that transmembrane potential affects Na+ channel activity, with the most profound effects of Zn2+ at hyperpolarized potentials. Our results also demonstrate that the stimulatory effect of Zn2+ can be mimicked by the Cys-modifying reagent MTSET. Zn2+ and MTSET increased channel number in a patch to a similar extent but had no effect on Po. Among different species, the conservation of Cys residues in ECLs of ENaC is >90%; this might be the reason that Zn2+ has similar effects on ENaC from different species.

Cys residues are also a favored ligand for Hg2+ and Cd2+; the interactions between Cys residues and these two metals are almost as strong as covalent interactions (8). However, the inhibitory effects of these two ions seem inconsistent with the stimulatory effect of Zn2+. The physical properties of Cd2+ and Hg2+ are similar to but different from those of Zn2+, Ni2+, and Cu2+. The radius of Cd2+ and Hg2+ is ~40% larger than the radius of Zn2+, Ni2+, and Cu2+, which means that their hydrated radius is smaller (Table 2). In addition, the electronic configuration of Cd2+ and Hg2+ implies that these ions are very polarizable, which, along with the reduced hydrated radius, will allow easier access of these ions to more Cys residues in ENaC ECLs and within the channel pore.

A three-residue tract conserved in all three subunits of the ENaC/degenerin family has been identified as a key structure forming the channel selectivity filter. In the {gamma}-subunit, there is a highly conserved Cys residue within this tract (14, 15, 27). Cd2+ was reported to have no effect on mouse ENaC activity when mouse ENaC genes are expressed in Xenopus oocytes, suggesting that this Cys residue is facing away from the Na+ conductivity pathway (28). However, Hg2+, an ion similar in size to Cd2+ but much more polarizable, inhibited mouse ENaC activity in the same oocyte expression system. Human ENaC expressed in Xenopus oocytes is inhibited by a small thiol-modifying reagent, methylthiosulfonate ethylammonium, but not by the large MTSET reagent. Furthermore, this inhibition is due to methylthiosulfonate ethylammonium binding to the Cys residue within the tract (33). Hence, the strong inhibitory effect of Hg2+ and Cd2+ on A6 ENaC is quite possibly due to binding of these two ions to the same Cys residue in the selectivity filter of the {gamma}-subunit, with Hg2+ having easier access and stronger binding. These metal cations, therefore, may interact with the same sites as Zn2+, but in addition, they also interact with sites in the channel pore inaccessible to the larger Zn2+. When Hg2+ and Cd2+ interact with the sites in the pore, they destabilize the open state and, thereby, reduce Po. In a previous study, 100 µM Cd2+ was reported to reduce frog skin transepithelial resistance but increase SCC (10); in this study, the effect of Cd2+ on the frog skin was imitated by a Ca2+ chelator, EDTA. In another report, Cd2+ disrupted cadherin structure by replacing its binding Ca2+ (22). Hence, the stimulatory effect of Cd2+ on the whole frog skin may be due to disruption of the tight epithelial connection or, perhaps, might even affect a channel type other than ENaC, since there are different types of channels in frog skin.

Pb2+ has no statistically significant effect on ENaC activity. However, the power of the statistical tests was such that we are reluctant to conclude that there is no effect, since the intrinsic variability of the patch data might hide a small increase in activity (Fig. 3D). There may be other reasons for our failure to observe an effect of Pb2+. Pb2+ inhibits the neuronal nicotinic acetylcholine receptor ion channel only at submicromolar concentration. This inhibitory effect is reduced when Pb2+ concentrations are increased to 10–100 µM (20). It is not clear whether the effect of Pb2+ on ENaC is also dose dependent. If it is, then the concentration of Pb2+ used in our study may be not optimum. On the other hand, the outer shell structure of Pb2+ is different from that of all the other heavy metal ions examined and, therefore, may not coordinate with Cys residues as well as it may coordinate with Hg2+ and Cd2+.

ENaC voltage sensor is in the ECLs. In the presence of steroid hormones, ENaC is constitutively active and Po is mildly voltage dependent. The voltage dependence of ENaC is much weaker than that of a typical voltage-gated channel. The potential sensor of a voltage-gated channel consists of a cluster of positively charged amino acids spaced every other three or four amino acids in the transmembrane domain; however, there is no such cluster within ENaC transmembrane domains of all three subunits. ENaC voltage sensitivity is abolished when the cell's apical membrane is exposed to DEPC. Because DEPC is slightly membrane permeable, His residues in ENaC transmembrane domains, as well as in ECLs, may act as the voltage "sensor." A comparison of all the amino acids in the transmembrane domains of Xenopus, rat, and mouse ENaC revealed the existence of a His residue conserved in the second transmembrane domain of the {alpha}-subunit of Xenopus and mouse ENaC. However, Xenopus, but not mouse, ENaC is voltage dependent (5). Thus the conserved His in the transmembrane region is not likely to be the source of the ENaC voltage sensor. Additionally, since Ni2+ and Cu2+ are membrane impermeable, yet they could still affect voltage sensitivity (Fig. 3B), it is unlikely that the voltage sensor is in the transmembrane domains. On the basis of our studies, the voltage sensor is most likely in the ECLs. This implies that the external vestibule of the channel formed by the ECLs must be sufficiently resistive to allow the potential field of the membrane to extend beyond the lipid head group surface of the apical membrane.

His is negatively charged at neutral pH. The values for channel voltage dependence, {alpha}, imply that the voltage-sensing charged residue senses very little of the overall membrane field (Fig. 5). If there is only one charge, it would need to sense only ~30% of the membrane field, and two or more charges would sense proportionately less, so it is reasonable to suggest that some portion of the field could extend into the external vestibule (considering the highly resistive, low unit conductance nature of the channel).

Heavy metal stimulation of ENaC activity is due to reduction of Na+ self-inhibition. Na+ self-inhibition refers to ENaC activity that is inhibited by high extracellular Na+ concentration. All cloned ENaCs from species that have been tested display Na+ self-inhibition. Heavy metal-stimulated ENaC activity was examined after Na+ self-inhibition was reduced. Zn2+ can only stimulate mouse ENaC activity (in an oocyte expression system) with high external Na+ concentrations (30). As indicated by transepithelial SCC, Ni2+ increases native ENaC in A6 cells by releasing Na+ self-inhibition (6). By recording single-channel activity in A6 cells using similar procedures, we found that Zn2+ increases N, Ni2+ increases Po, and Cu2+ stimulates N and Po. These results imply that there are at least two Na+ self-inhibition sites in the ECLs: one is involved in determining Po, and the other is involved in switching channels on or off. This suggestion is further supported by another set of experiments (Fig. 8B) in which, at low extracellular Na+ concentration, Cu2+ no longer was capable of altering N or Po.

Na+ self-inhibition has been proven to be an intrinsic characteristic of the channel. Two His residues in ECLs of mouse ENaC {alpha}- and {gamma}-subunits are considered to play opposite roles in the Na+ self-inhibition process: mutation of the {alpha}-subunit His282 enhances, whereas mutation of the {gamma}-subunit His239 eliminates, Na+ self-inhibition (26). Recent studies demonstrate that Na+ self-inhibition is related in some way to proteolytic maturation of ENaC. According to a biochemical analysis of ENaC {alpha}-, beta-, and {gamma}-subunits, two pools of ENaC coexist in the plasma membrane: one is completely postranslationally processed, with high Po, and the other is incompletely processed, with low or very low Po (12). Proteolytic cleavage of ENaC {alpha}- and {gamma}-subunit ECLs is a critical process in conversion of channels with low activity to channels with high activity (13). Chraibi and Horisberger (4) observed that Na+ self-inhibition was markedly diminished by extracellular application of proteases. Furthermore, Sheng et al. (25) reported that expression of mouse ENaC in Xenopus oocytes with furin cleavage sites on the {alpha}- or {gamma}-subunit eliminated by mutation greatly enhanced Na+ self-inhibition. Na+ self-inhibition appears to be a primary response of noncleaved channels in the plasma membrane. The noncleaved channel has higher affinity to Na+ than cleaved channels. At low extracellular Na+ concentrations, both channel types have similar activity, but at high external Na+ concentration the noncleaved channels may bind Na+ and allosterically change the ECLs of ENaC and, thus, change the channel gating (25). When a furin cleavage site on the {gamma}-subunit was eliminated, Po of mouse ENaC was significantly reduced at high external Na+ concentration, which is consistent with our observation that Cu2+ stimulates Po only at high external Na+ concentration. It is quite possible that binding of Cu2+ (and, probably, Ni2+) to ECLs prevents Na+ from binding to the self-inhibition site and, thus, reduces Na+ self-inhibition. If this is true, then the furin cleavage site, the Na+ self-inhibition site, and the Cu2+-binding site are likely physically close to one another.

Interpreting how heavy metals affect human health. In summary, all the metals we examined, except Pb2+, exerted significant effects on ENaC in A6 cells. Hg2+ tends to inhibit the channel activity at concentrations as low as 2 µM, whereas Cu2+ increases ENaC activity by severalfold. The effect of these metals on ENaC activity is mainly through Cys and His residues of ECLs of ENaC; the conservation of Cys residues between Xenopus and human ENaC is 100% in {alpha}- and {gamma}-subunits, whereas in the beta-subunit it is ~90%. The heavy metal concentrations we used in our experiments (except Ni2+) are approximately within the physiological range of those found in human kidney cortex tissue in cases of heavy metal poisonings (16). Therefore, the effects of these metals on ENaC in A6 cells are likely to be similar to their effects on ENaC in human kidneys. Since the stimulatory functions of heavy metals on ENaC require high external Na+ concentrations, in those at risk of heavy metal toxicity, a low-Na+ diet may help prevent heavy metal-induced hypertension.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. Yu, Emory Univ. School of Medicine, Dept. of Physiology, Whitehead Biomedical Research Bldg., 615 Michael St., Atlanta, GA 30322 (e-mail: lyu{at}physio.emory.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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