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J. Biol. Chem., Vol. 279, Issue 30, 31687-31696, July 23, 2004

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Extracellular Zn²⁺ Activates Epithelial Na⁺ Channels by Eliminating Na⁺ Self-inhibition^*

Shaohu Sheng, Clint J. Perry, and Thomas R. Kleyman¶

From the Renal-Electrolyte Division, Department of Medicine, and ^¶Department of Cell Biology and Physiology, School of Medicine, University of Pittsburgh, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261

Received for publication, May 11, 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Inhibition of epithelial Na⁺ channel (ENaC) activity by high concentrations of extracellular Na⁺ is referred to as Na⁺ self-inhibition. We investigated the effects of external Zn²⁺ on whole cell Na⁺ currents and on the Na⁺ self-inhibition response in Xenopus oocytes expressing mouse ENaC. Na⁺ self-inhibition was examined by analyzing inward current decay from a peak current to a steady-state current following a fast switching of a low Na⁺ (1 mM) bath solution to a high Na⁺ (110 mM) solution. Our results indicate that external Zn²⁺ rapidly and reversibly activates ENaC in a dose-dependent manner with an estimated EC₅₀ of 2 µM. External Zn²⁺ in the high Na⁺ bath also prevents or reverses Na⁺ self-inhibition with similar affinity. Zn²⁺ activation is dependent on extracellular Na⁺ concentration and is absent in ENaCs containing H239 mutations that eliminate Na⁺ self-inhibition and in S580C following covalent modification by a sulfhydryl-reactive reagent that locks the channels in a fully open state. In contrast, external Ni²⁺ inhibition of ENaC currents appears to be additive to Na⁺ self-inhibition when Ni²⁺ is present in the high Na⁺ bath. Pretreatment of oocytes with Ni²⁺ in a low Na⁺ bath also prevents the current decay following a switch to a high Na⁺ bath but rendered the currents below the control steady-state level measured in the absence of Ni²⁺ pretreatment. Our results suggest that external Zn²⁺ activates ENaC by relieving the channel from Na⁺ self-inhibition, and that external Ni²⁺ mimics or masks Na⁺ self-inhibition.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Epithelial Na⁺ channels (ENaC)¹ mediate Na⁺ transport across apical membranes of high resistance epithelia. The regulation of Na⁺ transport via ENaC has an important role in the maintenance of extracellular fluid volume homeostasis and the control of blood pressure in humans (1). Alterations in channel activity have been associated with several disorders, including Liddle's syndrome, pseudohypoaldosteronism type 1, and cystic fibrosis (2).

ENaC activity is regulated by a variety of both intracellular and extracellular factors, including selected hormones, cations, enzymes, and other channel proteins (3, 4). Na⁺ exhibits two types of inhibitory effects on ENaC activity: self-inhibition and feedback inhibition that are due to increases in either extracellular or intracellular Na⁺ concentration, respectively. These regulatory phenomena have been proposed to provide a mechanism to prevent sudden or excessive increases in intracellular Na⁺ concentration (5, 6).

Although most studies on Na⁺ self-inhibition have utilized native Na⁺-transporting tissues, including frog skin, toad urinary bladder, and kidney collecting tubule, Na⁺ self-inhibition has also been observed in Xenopus oocytes expressing ENaCs (7–9). Extracellular cations have been reported to affect ENaC activity in native tissues that may reflect changes in Na⁺ self-inhibition (3, 5). We and others previously reported that external Ni²⁺ blocks whole cell currents of ENaCs expressed in oocytes (10, 11). In contrast, external Ni²⁺ is reported to stimulate Na⁺ currents in A6 cells by relieving ENaC from Na⁺ self-inhibition (12). Recent work suggested that external Zn²⁺ is a voltage-dependent blocker of ENaC (13). In this report, we examined the effects of external Zn²⁺ on amiloride-sensitive Na⁺ currents in oocytes expressing mouse ENaC (mENaC), and the effects of external Zn²⁺ and Ni²⁺ on Na⁺ self-inhibition of mENaC. We report that external Zn²⁺ reversibly activates ENaC by eliminating Na⁺ self-inhibition, whereas external Ni²⁺ appears to mimic Na⁺ self-inhibition.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Site-directed Mutagenesis—All ENaC clones used in this study are mouse ENaC subunits whose cDNAs were inserted into pBluescript SK-(Stratagene, La Jolla, CA) (14). Point mutations were generated previously by using a PCR-based method (11).

ENaC Expression and Two-electrode Voltage Clamp—ENaC expression in Xenopus oocytes and two-electrode voltage clamp were performed as previously reported (11). Stage V and VI oocytes free of follicle cell layers were injected with 1–4 ng of cRNA for each mENaC subunit per oocyte and incubated at 18 °C in modified Barth's saline (MBS, 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO₃, 15 mM HEPES, 0.3 mM Ca (NO₃)₂, 0.41 mM CaCl₂, 0.82 mM MgSO₄, 10 µg/ml sodium penicillin, 10 µg/ml streptomycin sulfate, 100 µg/ml gentamycin sulfate, pH 7.4). All experiments were performed at room temperature (20–24 °C). Oocytes were continuously clamped at -60 or -100 mV in most experiments. Current-voltage relationships were determined by clamping oocytes at holding potentials in the range of -140 to 60 mV in 20-mV increments.

The responses of Na⁺ self-inhibition were examined as previously reported (7, 9). A current decay from a peak current to a relatively steady-state current was considered the response for Na⁺ self-inhibition. The current decay was initiated by rapidly replacing a low Na⁺ bath solution (NaCl-1: containing 1 mM NaCl, 109 mM N-methyl-D-glucamine, 2 mM KCl, 2 mM CaCl₂, 10 mM HEPES, pH 7.4) with a high Na⁺ bath solution (NaCl-110: containing 110 mM NaCl, 2 mM KCl, 2 mM CaCl₂, 10 mM HEPES, pH 7.4). Rapid solution exchange was performed with a 6-channel Teflon valve perfusion system from Warner Instruments (Hamden, CT). At the end of an experiment, 10 µM amiloride was added to the bath to obtain the amiloride-insensitive current. Whole cell currents in the presence of 10 µM amiloride were generally less than 200 nA at -60 or -100 mV. Results from oocytes that showed unusually large amiloride-insensitive currents (>5% of total currents) were discarded to minimize current contamination from endogenous channels and membrane leak. The Na⁺ self-inhibition response was described with two parameters, a time constant () and the ratio of the steady-state current (I_ss) and the peak current (I_peak) (see Ref. 9).

The time course of Na⁺ self-inhibition and the effects of Zn²⁺ and Ni²⁺ on this process were analyzed as previously described (9). Briefly, the first 40 s of current decay (or increase) were fitted with an exponential equation by Clampfit 9.0 (Axon Instruments Inc.). The concentration at which half-maximal effects were observed (EC₅₀) were estimated by non-linear least square curve fitting of the dose response data with the Hill equation: , in which R is the relative response, C is the concentration, and n is the Hill coefficient.

Michaelis constants (K_m) for Na⁺ concentration-current relationships were obtained by a best fitting of the data according to the following equation with non-linear least square curve fitting: I = V_max · C/(C + K_m), in which I is the relative I_peak or I_ss, and C refers to the Na⁺ concentration used to initiate self-inhibition. The apparent inhibitory constant (K_i) of Na⁺ self-inhibition was estimated from a best fitting of the data with the equation: in which C and n are the concentration of Na⁺ and Hill coefficient, respectively.

Superpure NiCl₂ and ZnCl₂ (>99.999%) were purchased from Sigma-Aldrich and dissolved at 1 M in water and diluted to the desired concentrations in bath solutions. The addition of NiCl₂ or ZnCl₂ to the bath solutions at the highest concentrations (1 mM for NiCl₂ and 5 mM for ZnCl₂) used in this study did not alter the pH of the solutions or form precipitates.

Statistical Analysis—Data are presented as mean ± S.E. Significance comparisons between groups were performed using Student's t test. Curve fittings were performed with Clampfit 9.0 (Axon Instruments Inc., Union City, CA) and Origin Pro 7.0 (OriginLab Corp., Northampton, MA).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
External Zn²⁺ Activates mENaC Expressed in Xenopus Oocytes—The effect of extracellular Zn²⁺ on mENaC expressed in Xenopus oocytes was examined by comparing amiloride-sensitive Na⁺ currents prior to and following the addition of ZnCl₂ into the bath solution. Oocytes expressing wild type (WT) mENaC were clamped from a holding potential equaling the measured membrane potential to a series of voltages from -140 to 60 mV in 20-mV increments. Whole cell Na⁺ currents were recorded prior to and 60 s following the addition of varying concentrations of ZnCl₂ to a bath solution containing 110 mM Na⁺ (NaCl-110). 100 µM ZnCl₂ increased amiloride-sensitive Na⁺ currents by 1.66 ± 0.06-fold (Fig. 1, A–C). External Zn²⁺ did not alter the current-voltage relationship, indicating a lack of voltage dependence of Zn²⁺ activation (Fig. 1B). The Zn²⁺ stimulation of whole cell currents was completely reversed following washout of Zn²⁺, with currents returning to the original level prior to Zn²⁺ addition (Fig. 1, A, B, and E). The Zn²⁺ dose response was analyzed with increasing concentrations of ZnCl₂ (10^-8, 10^-7, 10^-6, 10^-5, 10^-4, 10^-3, and 5 x 10^-3 M). Stimulatory effects on the amiloride-sensitive Na⁺ currents measured at -60 mV were observed in the range of 10^-6 to 10^-3 M in a dose-dependent manner with a maximal activation at 10^-4 M. Currents in the presence of Zn²⁺ at concentrations higher than 100 µM tended to return to the basal level, and 5 mM Zn²⁺ did not affect whole cell currents (Fig. 1C). To exclude the possibility of an influence of a prior Zn²⁺ exposure on channel activity in the presence of 5 mM Zn²⁺, whole cell currents were measured prior to and following 5 mM Zn²⁺ in oocytes that had not been previously exposed to Zn²⁺. In agreement with results presented in Fig. 1C, the current ratio in the presence of 5 mM Zn²⁺, relative to current prior to Zn²⁺ addition, was 1.00 ± 0.06 (n = 7). The apparent affinity for Zn²⁺ activation of ENaC currents was estimated by analyzing dose-response data with Zn²⁺ concentrations varying from 10^-8 to 10^-4 M (Fig. 1D). The EC₅₀, Hill coefficient, and correlation coefficient from a best fitting using Hill equation were 1.99 ± 0.28 µM (n = 10), 0.87 ± 0.10 (n = 10), and 0.97 ± 0.01 (n = 10), respectively.

View larger version (34K): [in this window] [in a new window]   FIG. 1. External Zn2+ activates mENaC. A, representative recordings of whole cell currents in the absence, presence, and after washout of 100 µM ZnCl2 in NaCl-110. The oocyte expressing mENaC was clamped from -140 to 60 mV in 20-mV increments. The currents recorded in the presence of 10 µM amiloride in bath solution NaCl-110 are shown on the right. The recording is representative of 15 experiments. B, current-voltage (I-V) relationship curves were obtained by plotting the currents measured at 0.4 s following the initiation of the voltage step from recordings in (A) against clamping voltages. C, dose-response curve of the Zn2+ effect. Relative currents are the ratios of amiloride-sensitive Na+ currents measured at -60 mV in the presence of increasing concentrations of ZnCl2 in NaCl-110, relative to the amiloride-sensitive currents measured prior to addition of ZnCl2. D, re-plot of the Zn2+ dose-response data in C to determine the half-maximal effective concentration (EC50). The relative responses represent the increases in current in response to 10-8, 10-7, 10-6, 10-5, or 10-4 M Zn2+, normalized to the current increase observed with 10-4 M Zn2+ at which maximal Zn2+ activation was observed. The curve is a best fit of the data by non-linear least-square fitting with a Hill equation: Response = , where "Response" represents the relative response and "n" is the Hill coefficient. The fitting parameters are: EC50, 1.74 µM; Hill coefficient, 0.77, and correlation coefficient (R2), 0.9954. E, time course of Zn2+ activation. An mENaC-expressing oocyte was bathed in NaCl-110 solution as indicated by the black bar and continuously clamped at -60 mV. The gray bar indicates the period of time when the oocyte was perfused with NaCl-110 containing 100 µM ZnCl2. The arrow indicates the time when 10 µM amiloride was introduced into the oocyte chamber. By convention, the negative values represent inward whole cell currents. The trace is representative of six experiments.

Xenopus oocytes express several types of endogenous channels that may conduct Na⁺ (15–18). To exclude the possibility that the observed increase in whole cell currents by extracellular Zn²⁺ was due to activation of an endogenous channel, we examined the effect of 100 µM ZnCl₂ on the whole cell currents in H₂O-injected oocytes. The currents measured from six oocytes at -100 mV in NaCl-110 NaCl-110 with 100 µM ZnCl₂, and NaCl-110 with 10 µM amiloride were -66.7 ± 27.9, -50.0 ± 12.9, and -83.2 ± 16.7 nA (p > 0.05), respectively, suggesting that 100 µM external Zn²⁺ does not activate endogenous currents. Furthermore, no changes in whole cell currents were observed in oocytes expressing mENaC when 10 µM ZnCl₂ was externally applied in the presence of 10 µM amiloride. The relative currents (i.e. currents normalized to values in the absence of amiloride) in the presence of amiloride alone and amiloride with Zn²⁺ were 1.9 ± 0.6 and 1.7 ± 0.3% (n = 6), respectively (p > 0.05). The results suggest that the stimulatory effect of external Zn²⁺ on currents in oocytes expressing ENaC results from activation of ENaC. These findings differ from the reported blocking effect of external Zn²⁺ in oocytes expressing rat ENaCs and in A6 cells that express endogenous ENaC (13).

External Zn²⁺ Eliminates Na⁺ Self-inhibition—The stimulation of the amiloride-sensitive whole cell currents by extracellular Zn²⁺ may result from an increase in unitary current, open probability, or number of active channels in oocyte membranes. Because the extracellular Na⁺ concentration was constant and Zn²⁺ did not change the reversal potentials (Fig. 1B), it is unlikely that Zn²⁺ had a significant effect on the driving forces for generating the whole cell currents. The rapid time course of Zn²⁺ activation and recovery following washout of Zn²⁺ suggest a direct effect of Zn²⁺ on single channel properties rather than a change in surface channel density. Several extracellular divalent cations are known to stimulate Na⁺ transport in model epithelia such as frog skin and toad bladder, possibly through interfering with Na⁺ self-inhibition (5). To investigate the mechanism of ENaC activation by external Zn²⁺, we examined whether extracellular Zn²⁺ altered the Na⁺ self-inhibition response of mENaC expressed in Xenopus oocytes. Na⁺ self-inhibition was studied by monitoring changes in whole cell Na⁺ currents measured at either -60 or -100 mV by two-electrode voltage clamp during a rapid increase in extracellular Na⁺ concentration. We previously reported that mENaCs exhibit a Na⁺ self-inhibition response that is similar to that of rat, human, and Xenopus ENaC (7–9). The effects of external Zn²⁺ on Na⁺ self-inhibition were studied with two sets of experiments. First, we examined the effect of the presence of Zn²⁺ in the high Na⁺ bath solution (NaCl-110). A typical response for Na⁺ self-inhibition of mENaC is shown in Fig. 2A. The current reached a maximal level (termed as I_peak) and declined to a relatively steady level (termed as I_ss) following a fast solution exchange that increased the extracellular Na⁺ concentration from 1 to 110 mM. The current decay reflects the Na⁺ self-inhibition response. After successful observation of a typical response of Na⁺ self-inhibition in oocytes expressing mENaC, we examined the self-inhibition response when the low Na⁺ solution (NaCl-1) was replaced by the NaCl-110 solution containing 100 µM ZnCl₂. No current decay was observed, and the current remained at a level slightly higher than the I_peak observed in the previous test, as shown in Fig. 2A. The Zn²⁺-activated currents were completely blocked by 10 µM amiloride or by reducing bath Na⁺ to 1 mM (Fig. 2, A and B), indicating that all the currents in the presence of Zn²⁺ were conducted through ENaC. These results indicate that external Zn²⁺ at 100 µM completely prevented Na⁺ self-inhibition. This Zn²⁺ effect was also reversible, because washout of Zn²⁺ quickly restored the typical self-inhibition response (Fig. 2A). We also examined whether the addition of Zn²⁺ was able to reverse current inhibition by extracellular Na⁺ by adding 100 µM Zn²⁺ to NaCl-110 when the currents reached a steadystate level following the Na⁺ self-inhibition response. As seen in Fig. 2C, Zn²⁺ increased the current to a level near the I_peak observed in the absence of Zn²⁺, and washout of Zn²⁺ returned the current to the inhibited level. Therefore, external Zn²⁺ not only prevents Na⁺ self-inhibition, it also reverses Na⁺ self-inhibition.

View larger version (14K): [in this window] [in a new window]   FIG. 2. External Zn2+ eliminates Na+ self-inhibition of mENaC expressed in oocytes. All recording traces in this figure were obtained from oocytes that were clamped at -60 mV and are representative of at least six experiments. The current decay following a rapid switch from the NaCl-1 to the NaCl-110 solution is considered the Na+ self-inhibition response. Bath Na+ concentrations are indicated by open (1 mM)or solid bars (110 mM). The arrow indicates the time when 10 µM amiloride was added. The gray bars indicate the periods of time when 100 µM ZnCl2 was present in NaCl-110 or NaCl-1 bath solution. A, a trace showing three responses of Na+ self-inhibition that were obtained before, during, and following perfusion with a 100 µM Zn2+, NaCl-110 solution. B, a recording showing 10 µM amiloride block of the current in the presence of 100 µM Zn2+. C, a trace showing a reversible increase in the steady-state current following a self-inhibition response. D, a trace showing the effect of Zn2+ pretreatment on Na+ self-inhibition. Following a typical self-inhibition response, the l mM Na+ solution was replaced by a 100 µM ZnCl2, 1 mM Na+ solution. The bath Na+ concentration was rapidly increased to 110 mM by replacing 100 µM ZnCl2, NaCl-1 with Zn2+-free NaCl-110. After the current reached a steady state, 100 µM Zn2+ was added to the NaCl-110 solution. At the end of the recording, the oocyte was perfused with NaCl-110 containing 10 µM amiloride.

To estimate the apparent affinity for Zn²⁺ elimination of Na⁺ self-inhibition, we examined Na⁺ self-inhibition responses in the presence of increasing concentrations of ZnCl₂ in the NaCl-110 solution (Table I). A typical experiment is shown in Fig. 3A. The effect of external Zn²⁺ on preventing Na⁺ self-inhibition was dose-dependent (Fig. 3B and Table I). The estimated EC₅₀ for Zn²⁺ was 1.3 µM by a best fitting of the dose-response relationship (Fig. 3B, inset). The EC₅₀ value was almost identical to that obtained from analysis of the Zn²⁺ dose-response curve for activation of ENaC currents (Fig. 1D), suggesting a link between ENaC activation and loss of Na⁺ self-inhibition by external Zn²⁺.

View this table: [in this window] [in a new window]   TABLE I Characterizations of Na+ self-inhibition in the absence and presence of external Zn2+ or Ni2+ The time constants () for Na+ self-inhibition were obtained by exponential fitting of the current decay at the clamping voltage of -60 mV following a rapid increase of bath Na+ concentrations from 1 to 110 mM. Both Ipeak and Iss are amiloride-sensitive inward Na+ currents expressed as negative values by convention and are not compared due to different batches of oocytes used in the experiments. Fitting for 100 µM Zn2+ and 1 mM Ni2+ pretreatment failed to generate a time constant due to a absence of current decay. Zn2+ and Ni2+ were added to either the NaCl-110 solution or the NaCl-1 solution (pretreatment).

View larger version (20K): [in this window] [in a new window]   FIG. 3. Dose-dependent effect of Zn2+ on Na+ self-inhibition. A, a typical recording shows Na+ self-inhibition responses in the presence of 1, 10, or 100 µM ZnCl2 in NaCl-110. A control Na+ self-inhibition response was performed before the Zn2+ effect was examined. Na+ concentrations are indicated as open (1 mM)or black bars (110 mM). The trace is representative of experiments performed in six oocytes. B, dose-response curve of Zn2+ effect on Na+ self-inhibition. The maximal inward currents measured following quick increase of bath Na+ from 1 to 110 mM are referred to as the peak current (Ipeak), and the current measured at 40 s after the Ipeak measurement was referred to as the steady state current (Iss). The Iss/Ipeak was used as a parameter to describe the magnitude of Na+ self-inhibition. A value of 1 indicates a lack of self-inhibition. The inset shows a best fitting of the mean relative response versus Zn2+ concentrations with the Hill equation to estimate the EC50. The relative response was the difference between the mean Iss/Ipeak values from B in the presence and absence of Zn2+ that was normalized to the difference between the mean Iss/Ipeak values in the presence of 100 µM Zn2+ (which eliminated Na+ self-inhibition) and the absence of Zn2+. The fitting parameters are shown in the inset.

View this table: [in this window] [in a new window]   TABLE II Fitting parameters for the relationships between Na+ concentrations and peak currents (Ipeak), steady-state currents (Iss), and current ratios (Iss/Ipeak) for mENaCs The current decays were examined in the same oocytes as external Na+ was increased rapidly from 1 mM to 110, 60, 30, 10, or 3 mM. The Km and Ki values were obtained by fitting the Ipeak or Iss values against Na+ concentrations with equations described under `Experimental Procedures.'

View larger version (27K): [in this window] [in a new window]   FIG. 4. Dependence of the Zn2+ effect on the Na+ concentration. Zn2+ effects were examined in oocytes expressing mENaCs. A, the effects of 1 and 10 µM ZnCl2 on the Iss/Ipeak representing the amplitude of the Na+ self-inhibition responses initiated by different Na+ concentrations. Numbers of oocytes used in the experiments were 7, 8, and 7 for the 0 Zn2+, 1 µM Zn2+, and 10 µM Zn2+ groups, respectively. B, the dose response of Zn2+ on amiloride-sensitive Na+ currents measured with a 10 mM Na+ bath solution. Oocytes injected with WT mENaC cRNAs were kept in either regular MBS with 88 mM Na+ (open circle) or low Na+ MBS with 10 mM Na+ and 78 mM N-methyl-D-glucamine (open square). A clamping voltage of -100 mV was used in this experiment. Relative currents have the same meaning as in Fig. 1C. The Zn2+ effects were examined in a low Na+ bath solution (10 mM). Amiloride (100 µM) was used to define amiloride-insensitive currents. Numbers of observations were 5 for the MBS group and 14 for the 10 mM Na+ MBS group. C, a representative recording showing the effect of 10 µM Zn2+ on the steady-state current in a 10 mM Na+ bath solution. The oocyte was clamped to -100 mV. The trace is representative of five experiments. D, a recording showing the effect of 10 µM Zn2+ on Na+ self-inhibition initiated by 10 mM Na+. The oocyte was clamped to -100 mV. After a control self-inhibition response to 10 mM Na+, ZnCl2 (10 µM) was included in the 10 mM Na+ solution for the second test. During the third test, NaCl-1 was rapidly changed to NaCl-110 to verify that the channels exhibit a typical Na+ self-inhibition response. In C and D, open, dashed, and solid bars indicate 1, 10, or 110 mM Na+ concentrations in the bath, respectively. The gray bar indicates the period in the presence of 10 µM ZnCl2 in the 10 mM Na+ bath solution.

Mutations at H239 That Disrupt Na⁺ Self-inhibition Diminish Zn²⁺ Activation—We recently reported that mutations of a His residue within the extracellular loop (ECL) of mENaC (H239R, H239D, and H239C) eliminated Na⁺ self-inhibition (9). If the stimulatory effect of external Zn²⁺ depends on the presence of Na⁺ self-inhibition, Zn²⁺ should have no effect on channels with H239 mutations. No current decay was observed in oocytes expressing H239R following a fast increase in bath Na⁺ concentration to 110 mM in the absence or presence of 100 µM Zn²⁺ (Fig. 5A). In experiments using the same protocol as in Fig. 1C for WT ENaCs, whole cell amiloride-sensitive Na⁺ currents in oocytes expressing H239R also remained constant in the presence of increasing concentrations of external Zn²⁺ (Fig. 5B). These data suggest that external Zn²⁺ activation of ENaC depends on the presence of Na⁺ self-inhibition. In contrast to the effect of H239 mutations, we previously reported that H282D channels show an enhanced Na⁺ self-inhibition response (9). Addition of Zn²⁺ to the NaCl-110 solution blunted, but did not eliminate Na⁺ self-inhibition in oocytes expressing H282D mENaCs (Fig. 5C). The I_ss was much higher than the I_ss observed in the absence of Zn²⁺. Addition of 100 µM ZnCl₂ into high Na⁺ bath significantly increased the I_ss following a self-inhibition response but did not fully restore the current to the I_peak (Fig. 5D). These results suggest that external Zn²⁺ results in a "partial" activation of H282D mENaCs, in contrast to a "full" activation of WT.

View larger version (26K): [in this window] [in a new window]   FIG. 5. Zn2+ activation is absent in oocytes expressing mENaCs with H239 mutations. Open, solid, and gray bars have the same meanings as in other figures. Application of 10 µM amiloride is indicated by an arrow. All experiments in this figure have been repeated at least six times, and the results are indistinguishable. A, lack of effect of 100 µM Zn2+ on the Na+ self-inhibition response in an oocyte expressing H239R. B, dose-response curve for external Zn2+ effect on amiloride-sensitive Na+ currents measured at -60 mV and normalized to control values in oocytes expressing H239R. Experiments were performed with the same protocol as for Fig. 1C. C, a recording with a protocol similar to A in an oocyte expressing H282D mENaC. D, a representative recording shows the effect of 100 µM Zn2+ on the Na+ current following a Na+ self-inhibition response in an oocyte expressing H282D.

View larger version (15K): [in this window] [in a new window]   FIG. 6. Effect of external Zn2+ on ENaC is absent when channels are gated open. The tracing is representative of six experiments from oocytes expressing S580C. The oocyte was continuously clamped at -60 mV throughout the recording. Bath Na+ concentrations are indicated by open (1 mM) or solid (110 mM) bars. External applications of Zn2+ are indicated by gray bars, and of 1 mM MTSET is indicated by a double-arrowed line.

View larger version (23K): [in this window] [in a new window]   FIG. 7. Effects of external Ni2+ on Na+ self-inhibition of mENaC. Oocytes were injected with cRNAs for WT mENaC subunits and clamped at -60 mV. External applications of 1 mM NiCl2 are indicated by bold dashed lines. Black arrows indicate the addition of 10 µM amiloride. All recordings are representative of at least six independent observations. A, a representative recording showing the effect of Ni2+ added in NaCl-110 on Na+ self-inhibition. Following a typical Na+ self-inhibition response, NaCl-1 was quickly replaced by NaCl-110 containing 1 mM NiCl2. The dashed arrow shows the difference between the control Ipeak and the Iss in the presence of 1 mM NiCl2, which represents the total inhibition by the simultaneous application of Ni2+ and 110 mM Na+. B, a recording showing the inhibitory effect of Ni2+ on the steady-state current. NiCl2 was added after the Na+ current reached a steady state and was then washed out to show the poor reversibility of Ni2+ block. The dashed arrow indicates the difference between Ipeak in the absence of 1 mM NiCl2 and the current measured after addition of 1 mM NiCl2 into the NaCl-110 bath, which represents the inhibition due to sequential application of high Na+ and Ni2+. C, a trace showing the effect of Ni2+ pretreatment on Na+ self-inhibition. The oocyte was bathed in NaCl-1 containing 1 mM NiCl2 for 1 min and then perfused with Ni2+-free NaCl-110. Following washout of Ni2+, two consecutive responses for Na+ self-inhibition were tested to show recovery from the Ni2+ effect.

View larger version (20K): [in this window] [in a new window]   FIG. 8. Effects of external Ni2+ on the Na+ self-inhibition response of mutant ENaCs. Oocytes were injected with cRNAs for one mutant subunit with other two WT subunits and clamped at -60 mV. A, a representative recording showing effects of Ni2+ on Na+ self-inhibition in an oocyte expressing H282D. NiCl2 (1 mM) was applied in either the high or low Na+ bath solution as indicated by a dashed line. The tracing is representative of five experiments. B, a recording showing responses of Na+ self-inhibition prior to, in the presence of, and following washout of 1 mM NiCl2 in NaCl-110. The tracing is representative of six experiments. Amiloride at 10 µM was added as indicated by an arrow.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The stimulatory effect of external Zn²⁺ on ENaC currents that we observed contrasts with the voltage-dependent block of the endogenous and expressed ENaCs by external Zn²⁺ that was recently reported by Amuzescu and co-workers (13). It is not clear why opposite effects of Zn²⁺ on ENaC activity were observed. The stimulatory effect of external Zn²⁺ on ENaC is dependent on the magnitude of Na⁺ self-inhibition and on the extracellular Na⁺ concentration (Fig. 4). External Zn²⁺ only stimulates ENaC currents measured in bath Na⁺ concentration greater than the minimal concentration required for Na⁺ self-inhibition (10 mM). The blocking effect of Zn²⁺ on ENaC currents reported by Amuzescu et al. was observed in oocytes bathed in a solution containing 10 mM Na⁺ (13) at Zn²⁺ concentrations of 1–10 µM. We have not observed an inhibitory effect of extracellular Zn²⁺ in the concentration range of 10 nM to 1 mM when oocytes were bathed in a solution containing 10 mM Na⁺ (Fig. 4B). Amuzescu et al. also reported that 100 µM Zn²⁺ stimulated rENaC, in agreement with our findings (13). Although ENaCs derived from different species were used in these two studies, it is unlikely that the conflicting results are due to species differences, because rat and mouse ENaCs have >95% amino acid identity (14). Furthermore, we observed similar activation of the whole cell Na⁺ currents and elimination of Na⁺ self-inhibition by external Zn²⁺ in oocytes expressing human ENaCs.² Our results are also consistent with a previous report (22) demonstrating a lack of an effect of external Zn²⁺ at the concentrations up to 1 mM on inward Li⁺ currents in oocytes expressing rENaC and bathed in a low Li⁺ (20 mM) bath.

Our results suggest that external Zn²⁺ activates ENaC by reversing or preventing Na⁺ self-inhibition, although the mechanism by which Zn²⁺ alters the Na⁺ self-inhibition response is unclear. Na⁺ self-inhibition appears to be dependent on an external Na⁺ sensor or receptor. Zn²⁺ may interfere with Na⁺ binding to its receptor by competing with Na⁺ for the same binding site or blocking Na⁺ access to the site. If so, the conformational changes in association with Na⁺ binding that result in a decrease in channel P_o do not occur when Zn²⁺ is bound to this site. Alternatively, Zn²⁺ binding to the channel may interfere with the conformational changes that are induced by Na⁺ binding to an external site and are required for Na⁺ self-inhibition. It is also possible that Zn²⁺ increases ENaC P_o through a mechanism that is distinct from Na⁺ self-inhibition. Several observations support the possibility that Zn²⁺ and Na⁺ interact with the channel at a common external site, including (i) the dependence of Zn²⁺ activation on the external Na⁺ concentration, (ii) the rapid activation of ENaC currents ( of 5 s) by external Zn²⁺ and the rapid reversal of this effect following the removal of Zn²⁺, and (iii) a Zn²⁺-dependent rightward shift in Na⁺ concentration versus I_ss/I_peak curves.

Alkali metal ions such as Na⁺ favor oxygen atoms as a binding ligand, whereas Zn²⁺ is an intermediate divalent cation that prefers nitrogen atoms from imidazoles and sulfur atoms from Cys side chains as binding ligands. Because the coordination chemistries of Zn²⁺ and Na⁺ differ, the presence of a common (or overlapping) binding site for both Zn²⁺ and Na⁺ would impose certain constraints, such that some residues participate in the coordination of both Zn²⁺ and Na⁺, whereas other residues would participate in coordination of either Zn²⁺ or Na⁺.

We recently reported that a His residue within the extracellular loop of mENaC (H239) is required for Na⁺ self-inhibition, because substitutions of H239 with Arg, Asp, or Cys eliminated Na⁺ self-inhibition (9). Our current study suggests that H239 is also required for Zn²⁺ activation of ENaC. Based on these observations, we propose that H239 may provide a binding ligand for both Na⁺ and Zn²⁺. A direct -interaction between Na⁺ and an imidazole nitrogen atom has been observed in resolved structures (23). A Na⁺ is coordinated by three imidazole nitrogen atoms and one water oxygen atom in the structure of a mannose-binding protein (PDB ID, 1BCH [PDB] , see Fig. 9A). Given the fact that the most preferred coordination numbers of Na⁺ are 6 and 8, we speculate that Na⁺ may be coordinated by a number of different ligands, including the H239 nitrogen, as well as oxygen atoms from other residues or from the solvent.

View larger version (23K): [in this window] [in a new window]   FIG. 9. Working models for the mechanisms of Na+ self-inhibition and Zn2+ activation of ENaC. A, the Na+ coordination shell is displayed in ball-and-cylinder mode from coordinates of a structure of mannose-binding protein (PDB ID, 1BCH [PDB] ). The colors are cyan for carbon, red for oxygen, and blue for nitrogen. A Na+ is shown as a magenta sphere, and a water molecule is shown as a red sphere. The Na+ is coordinated by a water molecule, and three -nitrogen atoms from histidine residues that are located in three helices from three identical peptide chains A, B, and C. B, working model 1 illustrating overlapping putative Na+ and Zn2+ binding sites. ENaC pore is shown in blue, and a bound Na+ is displayed as a magenta sphere with putative coordinating oxygen atoms in red. The yellow and green portions represent parts of the extracellular loops of ENaC subunits with an emphasis on the hypothesized extracellular allosteric regulatory site (EARS). A putative Zn2+ binding site depicted as a green dashed circle is formed by two imidazole nitrogen atoms from H239 and an unidentified histidine, a sulfur atom from a unidentified cysteine residue, and an oxygen atom from the backbone or side chain of an unidentified residue. The Na+ binding site depicted as a magenta dashed circle is constituted by one imidazole nitrogen atom from H239 and several oxygen atoms from solvent or unidentified residues. The blue arrows indicate potential movements of the outer pore in ENaC gating as suggested by us and other investigators (19, 24, 25). The green arrows indicate possible movements that transmit the local conformational changes induced by Na+ binding to its receptor onto a putative gate. C, working model 2 showing H239 as a key residue in a putative gate that is in a location distinct from the Na+ and Zn2+ binding sites. The blue and yellow portions represent the pore and the EARS within ECL of ENaC, respectively. A putative gate within ENaC is shown as a black rod whose movement during channel closing is indicated as a black arched arrow. In the model, Zn2+ and Na+ binding sites are proposed within the EARS and may or may not overlap. The directions of conformational changes induced by Na+ and Zn2+ are shown as magenta and green arrows, respectively. Part of the EARS is proposed to interact with the putative gate during channel gating.

We and others previously reported that external Ni²⁺ inhibits whole cell amiloride-sensitive Na⁺ currents due to a decrease in ENaC open probability (10, 11). Although Ni²⁺ and Zn²⁺ share several properties such as size, charge, and binding ligands, they exhibit opposing effects on Na⁺ self-inhibition. Pretreatment of oocytes expressing mENaC with 1 mM Ni²⁺ in the low Na⁺ bath solution prevented the typical current decay following an increase in bath Na⁺ concentration, and the steady-state currents were below the I_ss levels measured prior to Ni²⁺ pretreatment and following Ni²⁺ washout (Fig. 7C). A more rapid and deeper Na⁺ self-inhibition response was observed when Ni²⁺ was present only in the NaCl-110 solution. This response likely reflects additive inhibitory effects of Na⁺ and Ni²⁺. Although Zn²⁺ activation appears to be dependent on Na⁺ self-inhibition, the effect of Ni²⁺ on ENaC activity is independent of Na⁺ self-inhibition, because Ni²⁺ inhibits ENaC activity when added to either the NaCl-1 or NaCl-110 solution. We previously reported that H282D is not blocked by Ni²⁺ (11). In agreement with our previous observations, the addition of Ni²⁺ to the NaCl-110 solution did not alter the Na⁺ self-inhibition response of H282D (9). It is possible that Ni²⁺ binds to a site that overlaps with Zn²⁺ and Na⁺ binding sites. Other investigators have suggested that Ni²⁺ competes with Na⁺ for a common binding site (12). Our results suggest that Ni²⁺-bound ENaC has a low open probability (11) regardless of the external Na⁺ concentration and that the Na⁺ self-inhibition response is "masked" by Ni²⁺. Na⁺ self-inhibition and Ni²⁺ inhibition of ENaC may share a common mechanism.

Epithelial Na⁺ channels are expressed in the distal nephron, distal colon, and within both the airway and alveolae. The concentrations of Na⁺ in the distal nephron where ENaCs are expressed are highly variable. For example, human urine Na⁺ concentrations vary in a wide range from <10 mM to >100 mM and are altered by changes in dietary Na⁺ intake as well as by changes in extracellular fluid volume (26). Luminal Na⁺ concentrations in rat late distal tubules are reported in the range of 24–48 mM (27). Colonic fluid Na⁺ concentrations are likely variable as well. Recent studies suggest that Na⁺ concentrations in distal airway surface liquid are high (>100 mM) (28). Na⁺ concentrations in the lumen of ENaC-expressing tissues are frequently above the minimal concentration (30 mM) at which Na⁺ self-inhibition is observed, suggesting that Na⁺ self-inhibition may participate in the physiological regulation of transepithelial Na⁺ transport (6). These observations, together with our data indicating that external Zn²⁺ at concentrations in the low micromolar range enhances Na⁺ influx via ENaC by relieving Na⁺ self-inhibition, suggest that extracellular Zn²⁺ may be a physiological regulator of ENaC. Urinary Zn²⁺ concentrations of 5 µM have been reported in humans (29, 30), and given the low concentration of proteins and amino acids in urine that could potentially bind Zn²⁺, free urinary Zn²⁺ concentrations likely reach levels that affect ENaC activity. Because increases in rates of urinary Zn²⁺ excretion may be associated with increases in ENaC activity in collecting ducts by relieving Na⁺ self-inhibition, we propose that increases in the urinary concentration of Zn²⁺ may increase the risk of developing salt-sensitive hypertension. Angiotensin-converting enzyme has a key role in blood pressure regulation and is a Zn²⁺-dependent protease (31). An association between increases in erythrocyte Zn²⁺ and essential hypertension has been reported (32). A relationship between urinary Zn²⁺ excretion and blood pressure has not been established, although studies with small numbers of patients have suggested an association between increases in urinary Zn²⁺ excretion and hypertension (33, 34).

In summary, our results demonstrate that extracellular Zn²⁺ activates mouse ENaCs expressed in oocytes bathed in high Na⁺ by eliminating Na⁺ self-inhibition. Our previous results and work from other investigators suggest that ENaC gating is regulated in an allosteric manner by extracellular cues (5, 9, 11). These observations raise the possibility that ENaC is a ligand-gated channel, similar to ATP-sensitive inward rectifier K⁺ channels (K_ATP) (35), cyclic nucleotide-gated channels (36), and glutamate receptor ion channels (37). Extracellular Na⁺ would serve as a ligand whose binding to ENaC reduces channel open probability, and Zn²⁺ prevents or reverses Na⁺ self-inhibition as a potential physiological regulator or ligand.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grant DK54354 and by Cystic Fibrosis Foundation Grant Kleyma03PO. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Renal-Electrolyte Division, University of Pittsburgh, 3550 Terrace St., Pittsburgh, PA 15261. Tel.: 412-648-9295; Fax: 412-383-8956; E-mail: shaohu{at}pitt.edu.

¹ The abbreviations used are: ENaC, epithelial Na⁺ channel; mENaC, mouse ENaC; P_o, open probability; WT, wild type; K_i, inhibitory constant; ECL, extracellular loop; MBS, modified Barth's saline; EARS, extracellular allosteric regulatory site; cRNA, complementary RNA; MTSET, 2-(trimethylammonium) ethyl methanethiosulfonate bromide.

² S. Sheng, C. J. Perry, and T. R. Kleyman, unpublished data.

	ACKNOWLEDGMENTS

 
We thank James B. Bruns for helpful discussions.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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