1Laboratory of Physiology, Department of Molecular Cell Biology, K. U. Leuven, Campus Gasthuisberg O & N, Leuven, Belgium; 2Division of Animal Physiology, Department of Biology/Chemistry, University of Osnabrück, Osnabrück, Germany
Submitted 25 August 2004 ; accepted in final form 30 May 2005
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ABSTRACT |
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epithelia; divalent cations; amiloride; Na+; voltage clamp
The use of Ni2+ as an inorganic Na+ channel probe is well suited to the detection of strategic cysteine and histidine residues in the Na+ channel structure. Moreover, the study of toxic effects of this heavy metal, well known to be a very harmful pathogen in technology, will allow for the assessment of risk factors arising from interactions with accessible apical membrane transporters such as the ENaC. In a previous study (8), we used A6 epithelia, an immortalized cell line derived from the distal nephron of the clawed toad Xenopus laevis, to explore the effects of external Ni2+ on Na+ transport. Ni2+ stimulates Na+ transport in A6 epithelia. Amiloride-induced current fluctuation analysis demonstrated competition between Na+ and amiloride on the one hand and between Ni2+ and Na+ as well as amiloride on the other hand. In the present study, to better understand the stimulatory mechanism of Ni2+ on ENaCs and the competition with amiloride and Na+, we investigated the effects of Ni2+ on the channel cloned from Xenopus ENaC (xENaC) A6 cells expressed in Xenopus oocytes.
Moreover, we have investigated whether the effect of Ni2+ is specific for the xENaC or whether stimulation is a general characteristic of ENaCs cloned from other organs and/or species. Therefore, we compared the effect of Ni2+ on rENaCs cloned from rat colon and expressed in Xenopus oocytes. We found that Ni2+ stimulates Na+ current and conductance in xENaCs and has an inhibitory effect in rENaCs (27). The results obtained in rENaCs are also in agreement with previous observations made regarding mENaCs (28). Most interestingly, a recent report by Sheng et al. (29) may have shown that Zn2+, in contrast to Ni2+, was able to stimulate the current through mENaCs and that a cysteine was pinned down as a possible reaction partner. In their former study of Ni2+, these authors identified key extracellular histidine residues (-His282 and
-His239) within conserved regions of the mENaC
- and
-subunits that were required for channel block. This segment (WYRFHY) represents a putative binding site for amiloride. To extend our previous experience with rENaCs and xENaCs, we compared Na+ channel characteristics and amiloride binding for the wild-type (WT) channels of both species after expression in Xenopus oocytes. Moreover, in an effort to localize the site (presumably histidine) at which Ni2+ binds in the xENaC to exert its stimulatory effect, we performed mutagenesis studies of the extracellular segment of the
-subunit of xENaC. We investigated xENaCs (WT and mutated) and WT rENaCs by means of two electrophysiological methods. One method was the classic two-microelectrode voltage-clamp (TEVC) technique. The other approach used was the transoocyte voltage-clamp (TOVC) method recently developed in our laboratory (7). The TOVC method was used for the investigation of transoocyte current (ITO), transoocyte conductance (GTO), and amiloride-induced fluctuation in current. The TOVC method is an excellent technique with which to record current fluctuation spectra, to make accurate estimates of the amiloride association rate constant (kon), and to compare this parameter obtained from the xENaC with that from the rENaC in the WT. Furthermore, we assessed the channel characteristics [i.e., current-voltage (I-V) relationships] as well as the amiloride kinetics before and after Ni2+ treatment and/or histidine mutation.
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MATERIALS AND METHODS |
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Preparation of ENaC mRNA and ENaC mutants.
Xenopus and rat ENaC cDNA encoding the -,
-, and
-subunits (gift of B. Rossier and J. D. Horisberger, University of Lausanne, Lausanne, Switzerland) were cloned into the pSDEasy and pSport 1 vectors, respectively. Point mutations were generated in
-xENaC cDNA with the sequential polymerase chain reaction (PCR) method using Pfu DNA polymerase (QuickChange site-directed mutagenesis kit; Stratagene, La Jolla, CA).
Expression of ENaCs in Xenopus oocytes.
Xenopus females were purchased from the African Xenopus Facility (Knysna, South Africa). They were anesthetized by inducing hypothermia, and the ovarian lobes containing oocytes were removed. Oocytes were defolliculated by incubation in collagenase (1 mg/ml; Serva, Mannheim, Germany) for 2 h and subsequently washed with Ca2+-free Ringer for 10 min (see below for composition). cRNA of each of the -,
-, and
-subunits were synthesized, and equal amounts of subunit cRNA (5 ng of total cRNA) were injected into oocytes.
Solutions and chemicals. Native noninjected oocytes were incubated in Ringer solution containing (in mM) 90 NaCl, 2 CaCl2, 3 KCl, and 5 HEPES, pH 7.6. For the storage of ENaC-injected oocytes, we used a low-Na+ Ringer solution that contained (in mM) 5 NaCl, 85 N-methyl-D-glucammonium Cl (NMDG-Cl), 2 CaCl2, 3 KCl, and 5 HEPES, pH 7.6. The experiments were performed in solutions with the following composition (in mM): 102 NaCl, 2.5 KHCO3, and 1 CaCl2, pH 8. NiCl2 (2 mM) was added to the solutions without osmolality adjustment.
Transoocyte voltage clamp. Transoocyte current and conductance from ENaC-expressing oocytes were measured using the TOVC technique as described previously (7). Briefly, the oocyte is mounted in a container designed to fit in an Ussing-type chamber. Figure 1A shows that one side of the oocyte was exposed to 102 mM NaCl-Ringer solution; this is referred to as the high-Na+ (HN) side, whereas the other side was exposed to Na+-free solution and is referred to as the zero-Na+ (ZN) side (NMDG-Cl-Ringer, no Na+). In the TOVC arrangement, the positive current indicates cation movement from the HN side to the ZN side.
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This technique allows measurements of transoocyte current and conductance as well as current fluctuation analysis (7). For current fluctuation analysis, the HN side of the oocyte was exposed to different amiloride concentrations for short periods. Current noise was amplified, digitized, and Fourier-transformed to yield power density spectra. We recorded noise spectra as the mean of 50 sweeps of 2-s duration, resulting in a fundamental frequency of 0.5 Hz. The interaction of amiloride with the Na+ channel induced a Lorentzian component in the power density spectra. The Lorentzian parameters, the low-frequency plateau (So), and the corner frequency (fc) were determined using nonlinear curve fitting of the spectra (see Fig. 2 and Ref. 32). The on (kon) and off (koff) rate constants of the blocking reaction were calculated from the fit of the following relationship:
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Two-microelectrode voltage-clamp technique. The measurements were performed using the standard TEVC technique (30). The oocytes were placed in a small Plexiglas chamber and continuously superfused with solutions. Microelectrodes were pulled from borosilicate glass capillaries with a thin filament (Clark Electromedical Instruments, Reading, UK) equipped with a Ag-AgCl wire. Both electrodes were filled with 3 M KCl. Using micromanipulators, the oocyte was impaled with the microelectrodes under a low-magnification stereomicroscope. We used a voltage-clamp amplifier manufactured by Warner Instruments (Hamden, CT). All transmembrane ion currents were measured as a deflection from the baseline current. The ground electrodes in the bath were also made of Ag-AgCl wires. The flow of positive charge (i.e., Na+) from the bathing solution to the oocyte cytosol is termed inward current and is conventionally expressed with a negative sign. I-V relationships were studied with voltage pulses ranging from 140 to +40 mV in increments of 20 mV. Between voltage pulses, the oocytes potential was held at 0 mV. The duration of a pulse was 400 ms, and the interval between pulses was 1,500 ms. The ENaC conductance was calculated by performing linear regression analysis of the data points between 140 and 80 mV.
Analysis of voltage-activated currents.
When a voltage pulse was applied (see above), the difference between the baseline current (zero voltage) and the current magnitude reached after 5 ms was termed instantaneous current (Iinst), representing a prompt reaction to the pulse (see Fig. 1B). Starting from this point, the subsequent slow voltage activation in the xENaC current was fitted with an exponential function whose amplitude was termed the voltage-activated current (IV).
Statistics. Results are expressed as means ± SE. N is the number of animal donors, and n represents the number of experiments (oocytes).
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RESULTS |
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The amiloride-induced fluctuation in ITO was analyzed in oocytes expressing xENaCs and rENaCs under conditions in which transoocyte currents were partly inhibited with the diuretic on the HN side. Power density spectra could be recorded with amiloride concentrations ranging from 1 to 20 µM. In this concentration range, Lorentzian noise was markedly greater than instrumentation noise. Figure 2C shows power density spectra recorded from xENaCs for two amiloride concentrations. The averaged values of fc were 13.0 ± 1.5 Hz at 3 µM and 30.7 ± 0.7 Hz at 8 µM amiloride concentration (N = 3, n = 10). The association (kon) and dissociation (koff) rate constants were determined by performing linear regression analysis of the 2fc amiloride concentration data (Fig. 2D and Eq. 1). The strong reduction of kon at higher [Na+] confirms competition between Na+ and amiloride as suggested in Fig. 2B and as shown in Fig. 2D.
Obviously, Na+ entry at the HN side occurs through the ENaC and is amiloride sensitive. On the other hand, Na+ exit from the cell side to the ZN side could occur not only through the ENaC but also via the endogenous Na+-K+-ATPase (20). The effect of amiloride on outward Na+ currents has been studied in frog skin (31), and the amiloride inhibition constant was calculated from noise analysis data as 0.31 µM for outward Na+ currents compared with 0.19 µM for inward Na+ currents. Outward Na+ currents were also measured using the TEVC method in human ENaC (hENaC)-expressing oocytes (6). These currents could be inhibited by large (50 µM) doses of amiloride, but Ki for amiloride was not determined. We tried using amiloride from the ZN side, but we could measure amiloride-sensitive currents at zero potential in only 4 of 20 experiments. Figure 3A shows an experiment in xENaCs, in which, upon the addition of 100 µM amiloride to the ZN side, ITO was blocked by 50%. The inability of amiloride to block Na+ exit observed in 16 of 20 experiments may have been caused by different factors. A first possibility is that the exit of Na+ occurs only via the Na+-K+-ATPases on the ZN side. To verify this hypothesis, we added 100 µM ouabain to the ZN side. To avoid Na+ loading, the oocytes were incubated in Na+-free solutions on both sides and were exposed for only brief periods to high [Na+]HN (Fig. 3B). The ITO remained unaffected despite treatment with 100 µM ouabain for
30 min. This result suggests that the exit of Na+ to the ZN side was not via Na+/K+ pumps. Another explanation could be that the exit of Na+ on the ZN membrane is mediated through other native Na+ transporters in the oocyte membrane. A voltage-dependent and amiloride-insensitive Na+ channel activated by long depolarization was found in the Xenopus oocyte membrane (4). Because we expected cell depolarization during exposure to high [Na+] on the HN side, it is likely that in our experimental arrangement, this Na+ channel was opened and mediated the exit of Na+. Of course, other transporters, if Na+ coupled, could mediate the efflux of Na+ at the ZN side as well.
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Figure 4 shows the effects of a saturating dose of Ni2+ (2 mM) on current and conductance measured using the TOVC method for xENaCs and for a submaximal concentration of 2 mM [Ni2+] in the case of rENaCs. Here, too, the HN side contained 102 mM Na+, whereas on the ZN side, the oocyte was bathed in 102 mM NMDG-Cl solution. Figure 4, A and B, shows ITO and GTO in response to a voltage step from 0 to +40 mV (referenced to the ZN side) and the effect of amiloride. In xENaC-expressing oocytes, addition of 2 mM [Ni2+] to the HN side solutions augmented the amiloride-sensitive conductance by 50 ± 1% (n = 6, N = 4). The striking similarity of the degree of stimulation in xENaC-expressing oocytes and A6 cells (8) suggests that Ni2+ acts on the channel itself and not via cellular signaling mechanisms that most likely differ in these cellular systems. Therefore, the interaction of Ni2+ with other native proteins in A6 does not seem to cause the activation of the current.
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The amiloride-induced Lorentzian components in the ITO noise spectra were recorded in the presence and in the absence of Ni2+. The addition of Ni2+ to the amiloride-containing solutions in 102 mM [Na+]HN on the HN side caused a marked diminution of kon in both xENaCs and rENaCs. Figure 5A demonstrates the influence of 2 mM [Ni2+] on the amiloride kon for xENaCs and rENaCs. For xENaCs, the control kon was 17.8 ± 0.4 µM1·s1 and is comparable to the value previously recorded in A6 cells as 20.2 µM1·s1 (8). In rENaC-expressing oocytes, kon was 21.6 ± 1.2 µM1·s1 and thus compares well with both the kon value obtained from A6 cells and that obtained from xENaC-expressing oocytes. Although Ni2+ exerts an opposite effect on the conductance of rENaCs vs. xENaCs, we thus found a similar, competition-like effect on the binding of amiloride. A comparable reduction of kon was obtained for both species, amounting to 59.7 ± 10.2% (n = 5, N = 2) and 59.2 ± 19.5% (n = 6, N = 3) for rENaCs and xENaCs, respectively. The reduction of kon in xENaCs as well as in rENaCs suggests direct competition between Ni2+ and amiloride for a common binding site. Alternatively, it is conceivable that a steric modification of the channel by Ni2+ hinders amiloride to reach its site for specific blocking farther away. Because both external Ni2+ (Fig. 5A) and high [Na+] (Fig. 2D) diminished amiloride kon, we hypothesized that the effect of both cations on amiloride binding occurs through an interaction at an analogous, if not identical, site in both channel species, thus suggesting competition between amiloride, Ni2+, and Na+.
This prompted us to examine the effect of 2 mM [Ni2+] at different [Na+]HN on the rate of amiloride binding. Current noise induced by 8 µM amiloride was recorded from xENaC- and rENaC-expressing oocytes in the [Na+] range between 30 and 102 mM. Figure 5B shows the effect of Ni2+ on the [Na+]HN dependence of the amiloride chemical binding rate. In control conditions, with xENaCs as well as with rENaCs, the amiloride reaction rate recorded with 8 µM diuretic decreased when [Na+] was elevated. Conspicuously, in the presence of 2 mM [Ni2+], the corner frequency became independent of the [Na+], an observation in support of the presumed competition between Ni2+ and Na+. Previously, for A6 cells, we suggested a competition of Ni2+ with a Na+ receptor outside the channel mouth, located in the extracellular segment and probably identical to the site of self-inhibition (8). This assumption was based on experiments similar to those shown in Fig. 5B. The resemblance to the results obtained in oocytes expressing xENaCs is clear, whereas the relationship to the inhibitory effect of Ni2+ observed in rENaC-expressing oocytes contrasts with the stimulatory effect in xENaCs, despite the obvious Na+-Ni2+-amiloride competition. In this case, a blockade of the channel pore by Ni2+ in rENaCs is conceivable if it impedes amiloride to bind to its high-affinity site located in the channel pore and thus blocks the entry of Na+. To further explore the process of the interaction of Ni2+ with both xENaCs and rENaCs, we recorded I-V curves using the TEVC method.
Oocytes were clamped at 0 mV, and step changes in the membrane voltage were applied as described in MATERIALS AND METHODS. Generally, the oocytes were incubated in low-[Na+] solution (5 mM) to avoid cell loading with Na+, and they were exposed to high (102 mM) [Na+]o solutions only for brief periods. The time dependence of the current response to the voltage pulses, as well as I-V relationships could be studied. The superimposed traces of the currents recorded during the voltage pulse protocol are shown in Fig. 6. From these records (Fig. 6A), it is clear that the membrane current in the case of xENaCs consists of two components: 1) an instantaneous current jump that reflects the conductance of the membrane at 0 mV and 2) a voltage-activated current that becomes particularly apparent at high, hyperpolarizing voltages. The instantaneous and the activated currents are completely abolished by amiloride (data not shown), demonstrating that both components represent an increase in the current flowing through ENaCs. To quantify the activation process, the amiloride-sensitive currents were fitted with a single exponent (see Fig. 1B). From this analysis, we obtained the instantaneous current (Iinst), which represents the current jump at the beginning of the voltage pulse and the voltage-activated current (IV), determined as the amplitude of the exponential function. These activated Na+ currents may be attributed to a slow (in the range of tenths of a second) process that is caused by the effect of voltage on channel gating. A similar activation of the ENaC by hyperpolarizing voltages was previously reported for both ENaCs from A6 cells (24) and of human origin (3).
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Next, we analyzed in more detail the effect of Ni2+ on Iinst and IV for xENaCs by constructing the I-V relationships in the presence and absence of the divalent cation. Figure 7, A and B, shows that Ni2+ stimulated practically only Iinst in xENaCs. Furthermore, Ni2+ stimulated Iinst independent of voltage, because the ratio of the current in the presence of Ni2+ over control (IinstNi2+/Iinst) is
2 at any voltage between 140 and 40 mV, which is shown in Fig. 7A. Together, these data are consistent with the idea of Na+ transport stimulation at a site located outside the voltage-sensing channel pore. Membrane Na+ conductance (GNa) was calculated from the slope of the linear regression analysis of Iinst between 140 and 80 mV (see Fig. 7A). After Ni2+ addition, GNa increased from 40.4 ± 2.3 to 83.8 ± 7 µS. These data confirm the results obtained from measurements in A6 cells (8). The current stimulation by Ni2+ is fast (Fig. 4A), which indicates that this voltage-insensitive site for the stimulatory action of Ni2+ must be easily accessible. Moreover, the process of stimulation of Iinst is not related to the secondary voltage activation of the channel (Fig. 7B).
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Histidine mutations in the extracellular loop of the -xENaC in the domains WYRFHis215Y and HKSWGHis416C: stimulation by Ni2+ is unaffected, although amiloride and Na+ interact differently with xENaCs.
All of the above results demonstrate that Ni2+ exerts opposite effects on Na+ current in xENaC- and rENaC-expressing oocytes, although the channels have strikingly similar biophysical properties. Moreover, we have demonstrated that in xENaCs (but also in rENaCs), amiloride binding is impaired by Ni2+ and interferes with Na+. This result is most likely related to the fact that the binding of the diuretic and the divalent cations take place in areas that are in close proximity to each other, if not in identical locations. Therefore, our attempts to localize the binding site of Ni2+ were based on data available for the binding site of amiloride. A mutagenesis screen of amino acids preceding the second transmembrane segment of
-,
-, and
-ENaC identified the residues
-Ser583,
-Gly525, and
-Gly537 that, when mutated, reduced 1,000-fold the channel affinity to amiloride (18). It has been shown that all ENaCs cloned to date share this site for amiloride inhibition. Before as well as after the identification of the critical role of the homologous
-Ser583,
-Gly525, and
-Gly537 in amiloride binding, other mutations were made in search of other residues that contribute to amiloride block. Thus the short amino acid segment WYRFHY in the mouse and rat
-ENaC domain was also proposed to participate in amiloride binding (14, 19). Deletion of this region resulted in a loss of amiloride binding to the channel. Clearly, the interaction between amiloride and the ENaC is complex and may involve other, as yet unidentified residues in the extracellular loop of the ENaC family. Within the WYRFHY segment, from
- and
-subunits of mENaCs, the mutation
-His282 to aspartate or double mutations to arginine
-His282Arg/
-His239Arg eliminated the Ni2+ block in oocytes expressing mENaCs (28). Finally, extracellular loop folding might bring the WYRFHY segment close to the
-Ser583-
-Gly525-
-Gly537 amiloride-binding motif at the pore entrance and thus form a complex drug-binding pocket.
To examine whether Ni2+ would bind to the homologous segment in xENaCs and thus contribute to Ni2+ stimulation, we substituted the corresponding histidine residue -His215 within the WYRFHY segment of the Xenopus
-subunit with aspartate and coexpressed an
-His215Asp ENaC with WT
- and
-ENaCs. We did not observe any significant changes regarding Ni2+ action on amiloride-binding rates in xENaCs in two experiments (data not shown). Not unexpectedly, 2 mM Ni2+ stimulated the amiloride-sensitive current and conductance by
40%. Assuming that histidine is important for Ni2+ binding, this suggests that the site for Ni2+ binding and stimulation of xENaCs does not involve the histidine residue from WYRFHY, which, in contrast, participates in the inhibitory Ni2+ binding in mENaCs. Even more so, deleting the entire WYRFHY stretch led to the same result (data not shown), indicating that neither His215 nor its immediate surroundings are related to the xENaC stimulation by Ni2+.
In our previous report on A6 cells (8), we attempted to chemically characterize the residues involved in xENaC stimulation by Ni2+. We found that p-chloromercuribenzoate (PCMB), a reagent that binds to cysteine but not the histidine-reactive diethyl pyrocarbonate (DEPC) mimicked the stimulatory effect of Ni2+. However, from the chemical point of view, histidine rather than cysteine may be the most preferred partner with which to form complexes with Ni2+, e.g., in enzymes such as urease (34), and it has also become an important Ni2+-complexing structure (the so-called His tag) in preparative biochemistry. In the extracellular loop of each xENaC subunit, there are several histidine residues: nine in the -subunit, eight in the
-subunit, and six in the
-subunit. However, they are not grouped into histidine-rich motifs as described for enzymes that bind Ni2+, although protein folding could bring two or more histidine residues (possibly including also cysteines), which appear distant in the sequence map, into close proximity to form the final tertiary structure.
We demonstrated with experiments in xENaCs and rENaCs that Ni2+ impaired the binding of amiloride. Therefore, a residue that is located closer to the high-affinity site for the blocker at the channel mouth is also likely to coordinate Ni2+ and might, even if linearly (on the amino acid counting scale) somewhat distant but perhaps able to fold into the amiloride site neighborhood, become a docking site for Ni2+-Na+-amiloride simultaneously. This is the reason why we chose to analyze the function of the xENaC -His416 residue that is closest to the amiloride-binding site at the beginning of the M2 domain (16). The position of the amino acid segment containing His416 is shown in Fig. 8. The sequence map shows not only another histidine (His411) in the vicinity of His416 but also an intimate neighbor, a cysteine residue (Cys417). These three residues may interact with Ni2+ to construct a high-affinity site for Ni2+ binding as shown for the permease of Escherichia coli, for example (5). As mentioned above, we previously suggested a cysteine as one possible Ni2+-binding site in the A6 ENaC (8). Figure 8 also shows that the corresponding segment in the rENaC contains no histidines, but we note that it still shares the cysteine position with Xenopus.
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We also analyzed the effect of -His416 substitution on the process of voltage-activated currents. As depicted in Figs. 6B and 7B for control (His416), Ni2+ had no consistent effect on
IV, and this was also the case for all mutations (data not shown). Therefore we shall not deal with this issue further.
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DISCUSSION |
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In the present study, we have provided the first direct comparison of the functional properties and the effect of Ni2+ in heterologously expressed xENaCs and rENaCs. We report significant differences between these channels with respect to Ni2+ sensitivity and the process of voltage activation. Ni2+ ions may become important tools for molecular identification of regulatory sites in ENaCs from various cell types and tissues.
Ni2+ exerts opposite effects on Na+ current in WT xENaCs and rENaCs.
We found in this study that Ni2+ stimulated xENaCs. The stimulatory effect of Ni2+ is in agreement with the findings of previous studies that showed that Ni2+ as well as other polyvalent cations, such as La3+, Zn2+ (36), and Cu2+ (9), stimulated Na+ uptake in frog skin. To the contrary, in toad urinary bladder, divalent cations such as Ca2+, Mg2+, or Ba2+ blocked the ENaC from the external side in a voltage-dependent manner with an estimated Ki of 200 mM (23). Because all of the above results were obtained from measurements of Na+ transport in amphibian tissues, it is clear that the reported effects might be species dependent. In view of the fact that Ni2+ is only one of many cationic agents that stimulate ENaCs, its action may occur at a site that is not very selective. However, studies conducted at our laboratory, have shown that divalent and polyvalent cations exert different influences on Na+ transport in the very same epithelial tissue. For instance, in A6 epithelia, Zn2+ inhibits Na+ transport with a Ki of 45 µM (1), whereas Ni2+ stimulated INa with a Km of 0.5 mM (8) and Mg2+ has no effect when added on the apical side (15). In rENaCs (and also mENaCs), Ni2+ is a blocker whereas Zn2+ turned out to be a stimulator in mENaCs, obviously counteracting self-inhibition (29). Clearly, the action of heavy metal ions such as Ni2+ must occur at selective binding sites. On the other hand, the action of divalent cations may be influenced by other regulatory proteins specific to each of these tissues. To circumvent this problem, we analyzed the influence of Ni2+ on oocytes expressing ENaCs. The Xenopus oocyte expression system enabled us to study the influence of Ni2+ on ENaCs cloned from A6 cells and from rat colon. In this way, we were able to compare the results obtained from xENaC- and rENaC-expressing oocytes with those from epithelial cells.
Comparison of amiloride noise (TOVC) and whole cell Na+ current (TEVC).
Previous studies of xENaCs and mENaCs have produced conflicting results regarding the relationship between extracellular divalent cations and amiloride. In the case of A6 cells, xENaC direct activation by Ni2+ and competition with amiloride was reported (8). Because the effect of Ni2+ on A6 cells was investigated using the noise analysis technique, we aimed to implement the same method to study amiloride-induced current fluctuation in intact oocytes. In early studies, noise analysis was widely used for the estimation of the amiloride rate constants in Na+-transporting epithelia. More recently, studies of oocytes expressing ENaCs revealed limits for the accurate calculation of the amiloride inhibition constant and the analysis of INa block. The reason why kon and koff rate constants of amiloride are not reported for patch-clamp experiments in ENaC-expressing oocytes may be due to the slow gating mode of ENaCs that complicates the kinetic analysis (17). The blocking events by amiloride are indistinguishable from the channel-closed state because they have about the same duration. Only one group of researchers (17) has reported amiloride rate constants calculated from blockade of rENaCs expressed in oocytes. To determine the association rate of the blocker, these authors used ENaCs formed by - and
-subunits only, because this channel is almost constantly open. Therefore, blocking events could be readily distinguished from the rare spontaneous channel closing. However, a precise estimate of the amiloride rate constants in
-,
-, and
-ENaC-expressing oocytes measured using the patch-clamp technique has not been reported to date.
We implemented the noise analysis method adapted for the TEVC method as reported by Segal et al. (26). The disadvantage of this technique is that the amplifier noise level is elevated and produces a limited bandwidth. With the development of the TOVC technique (7), we are now able to compare the amiloride rate constants obtained from A6 cells with those obtained from oocytes expressing xENaCs. We calculated kon as 17.9 ± 0. 4 µM1·s1 and koff as 9.1 ± 3.3 s1, which is in excellent agreement with the values obtained from A6 cells. Our results obtained with oocytes indicated that Ni2+ diminishes the amiloride Ki by decreasing the on rate to 7.2 ± 0.3 µM1·s1 and increasing the off rate constant to 11.6 ± 2.7 s1. It is clear that the determination of the off rate constant is relatively inaccurate (see MATERIALS AND METHODS), so more weight should be given to kon, and the conclusions derived from this analysis are fully consistent with those from the study of macroscopic Ki behavior; that is, the amiloride on rate is the parameter that is influenced by Ni2+ and/or Na+. One simple way to interpret the data shown in Fig. 5 (amiloride-Na+-Ni2+ interaction) would be that Ni2+ competes with amiloride and Na+ at the very same site on the channel protein and that this is true for xENaCs and rENaCs. However, we would then expect Ni2+ not to stimulate but rather to block xENaCs as observed with rENaCs. Alternatively, at a site outside the channel pore, Ni2+ might exert an allosteric effect on Na+-amiloride binding. This mechanism could then stimulate Na+ entry by affecting the channel in a way that hinders amiloride from reaching its site, i.e., by establishing an "apparent competition" due to mutual exclusion effects on different binding sites for Na+-amiloride and Ni2+, respectively.
Another important finding of an earlier report (27) and of our present study is that Ni2+ inhibits the amiloride-sensitive current in oocytes expressing rENaC. The blocking effect of Ni2+ is in good agreement with the observations of Sheng et al. (28), who reported that Ni2+ inhibited mENaC current, but with a Ki of 0.5 mM, or
1 order of magnitude smaller than that in rENaCs, which might suggest molecularly different blocking sites and/or Ni2+ accessibility in the two species. This is supported by the findings of Sheng et al. (28), who identified histidine in the WYRFHY section as a ligand for the inhibitory Ni2+ in mENaCs. Our data regarding xENaC-WYRFHY could exclude His215 as ligand of the stimulatory Ni2+. Contrary to the Sheng et al. report on mENaCs, our findings regarding both xENaCs and rENaCs indicate that Ni2+ diminishes amiloride kon by 59.7 ± 10.2%, again suggesting possibly allosteric competition between the blocker and the divalent cation. To gain insight into why the effect of Ni2+ on INa is different in xENaCs and rENaCs, we studied the influence of Ni2+ on INa in the absence of amiloride at rapid step voltages between 140 and +40 mV.
An interesting finding from our TEVC study is that in the xENaC, which is sensitive to voltage, only Iinst is stimulated by Ni2+ rather than the voltage-activated part, IV. The rENaC, however, behaves in a fully ohmic manner and is inhibited by Ni2+ not only in the instantaneous current jump phase but in a secondary, slow Ni2+-dependent inhibition of the current that becomes apparent and is even more effective at more negative voltages (Fig. 6). A voltage-dependent blockade by Ni2+ of rENaC can be the result of two different mechanisms. 1) Ni2+ blocks the rENaC pore by occupying a site located within the permeation pathway, where membrane voltage drops. 2) Ni2+, rather than obstructing the permeation pathway, stabilizes the closed state of the channel through a mechanism that would be independent of the blocking itself but nevertheless would be dependent on voltage. For instance, in studies of high-voltage-activated Ca2+ channels, investigators have proposed the existence of two binding sites for Ni2+: one accounting for the direct blocking and an additional one that stabilizes the Ca2+ channel-closed state (21).
The species difference in the Ni2+ effect, stimulating the xENaC but inhibiting the rENaC, could also be only an apparent difference; for instance, it might be the result of a different time lag of the processes involved. In xENaC, voltage activation is slow and thus visible in the recordings, whereas in rENaC the process could be much faster and might be terminated already during the settling time of the voltage clamp, within the first few milliseconds after the voltage step is initiated. Because of the lack of evidence, such an idea seems far fetched. We therefore propose a working model in which Ni2+ binds in the extracellular, voltage-insensitive domain of the xENaC, thereby changing the channel allosterically in such a way that amiloride and its competitor Na+ are impaired in binding to their common site. Such a site should also be available in the rENaC to allow the observed interaction of Na+, Ni2+, and amiloride (Fig. 5). At present, we cannot tell whether the sites in question in xENaCs and rENaCs share analogous chemical features. Previous studies showed that Ni2+ affects a variety of ion channels, such as voltage-gated Ca2+ channels (11), voltage-gated Na+ channels (33), P2X receptors (33), and glutamate receptors (33). The mechanism of Ni2+ effects on these membrane proteins is not always clear in detail, but both direct blocking and allosteric processes have been suggested (12, 21).
As demonstrated herein for Ni2+-induced xENaC stimulation but inhibition of rENaC and mENaC stimulation (28), other transporters are affected by Ni2+ as well, e.g., the human aquaporin 3 (37). High doses of Ni2+ predispose to asthma, lung fibrosis, lung cancer, and kidney cancer. Taken together, these observations suggest that Ni2+-related diseases may originate from epithelial effects in the first place.
Lessons from mutation experiments.
More recently, Sheng et al. (28) suggested that a histidine residue from the extracellular WYRFHY domain of the -subunit and one from the extracellular domain of the
-subunit form the inhibitory Ni2+-binding site in mENaCs. The residue from the
-subunit is highly conserved among the species mouse, rat, and Xenopus, and substitution of the homologous histidine residue from mENaCs with aspartic acid abolished the Ni2+ effect on amiloride-sensitive Na+ currents. We identified this histidine residue in the
-subunit of the xENaC as His215, and we generated the point mutation His215 to aspartate. As in the WT, in these channels, Ni2+ caused stimulation of ITO and diminution of the amiloride kon by
60% (experiments not shown). This result indicates that the mutation
-His215Asp is not involved as Ni2+-binding site in xENaCs. Moreover, complete removal of the
-WYRFHY sequence likewise did not impede stimulation by Ni2+, suggesting a different binding site for this divalent cation. Finally, we again want to shed light on a recent finding regarding A6 cells (8), in which the thiol reagent PCMB, but not the histidine tracer DEPC, stimulated xENaC-like Ni2+ ions. It therefore is not unlikely that Cys417, the neighbor of the mutated His416, may be involved in Ni2+ binding. On the other hand, there are plenty of cysteines (almost diagnostic for ENaCs) that might be possible Ni2+ targets in the extracellular loop. The world of Na+ channels and heavy metals seems much more complicated than expected.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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