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【摘要】 Epithelial Na + channels (ENaC) are inhibited by extracellular Na +, a process referred to as Na + self-inhibition. We previously demonstrated that mutation of key residues within two furin cleavage consensus sites in, or one site in, blocked subunit proteolysis and inhibited channel activity when mutant channels were expressed in Xenopus laevis oocytes (Hughey RP, Bruns JB, Kinlough CL, Harkleroad KL, Tong Q, Carattino MD, Johnson JP, Stockand JD, and Kleyman TR. J Biol Chem 279: 18111-18114, 2004). Cleavage of subunits was also blocked by these mutations when expressed in Madin-Darby canine kidney cells, and both subunit cleavage and channel activity were blocked when wild-type subunits were expressed in furin-deficient Chinese hamster ovary cells. We now report that channels with mutant -subunits lacking either one or both furin cleavage sites exhibited a marked enhancement of the Na + self-inhibition response, while channels with a mutant -subunit showed a modestly enhanced Na + self-inhibition response. Analysis of Na + self-inhibition at varying [Na + ] indicates that channels containing mutant -subunits exhibit an increased Na + affinity. At the single-channel level, channels with a mutant -subunit had a low open probability ( P o ) in the presence of a high external [Na + ] in the patch pipette. P o dramatically increased when trypsin was also present, or when a low external [Na + ] was in the patch pipette. Our results suggest that furin cleavage of ENaC subunits activates the channels by relieving Na + self-inhibition and that activation requires that the -subunit be cleaved twice. Moreover, we demonstrate for the first time a clear relationship between ENaC P o and extracellular [Na + ], supporting the notion that Na + self-inhibition reflects a P o reduction due to high extracellular [Na + ].
【关键词】 amiloride open probability voltage clamp Xenopus laevis oocyte mutagenesis
EPITHELIAL NA + CHANNELS (ENaC) have an essential role in mediating Na + transport across high-resistance epithelia and in the regulation of extracellular fluid and blood pressure ( 25, 30 ). Typically, these highly selective Na + channels are formed by three homologous subunits,,, and, with a tetrameric or higher order stoichiometry ( 13, 24, 31 - 33 ). The channel activity is regulated by many intracellular and extracellular factors through alterations of either channel density, open probability ( P o ), or both. Changes in channel expression or activity have been observed in several human disorders, including Liddle's syndrome, pseudohypoaldosteronism type I, and cystic fibrosis ( 25 ). A potential link between ENaC and essential hypertension has also been suggested ( 22, 34 ).
Among proteins that have been implicated in the regulation of ENaC activity are several serine proteases including prostasin, furin, and elastase ( 1, 9, 19, 25, 35, 37 ). We recently reported that furin, a protease that resides primarily in the trans -Golgi network (TGN), regulates ENaC activity by cleaving specific sites within the extracellular loops (ECL) of the - and -subunits of ENaC. Inhibition of cleavage by mutation of consensus sequences for furin-dependent proteolysis (Arg-X-X-Arg, where X is any residue) within - and -subunits reduced whole cell amiloride-sensitive currents in Xenopus laevis oocytes compared with comparable expression of wild-type channels. Following trypsin treatment, levels of ENaC activity in oocytes expressing either wild-type or mutant channels were similar, consistent with comparable levels of surface expression of channels. The reduced currents observed with mutant channels likely reflected a decreased channel P o ( 19 ).
Na + self-inhibition represents the rapid decrease in Na + currents from a peak current that occurs following a sudden increase in the extracellular Na + concentration. Previous studies of native channels in model epithelia and of cloned ENaCs in heterologous expression systems suggest that ENaC P o is controlled, in part, by external Na + via Na + self-inhibition ( 8, 15, 27, 39 ). Several lines of evidence suggest that Na + self-inhibition is a response to extracellular Na + and is not related to changes in intracellular [Na + ]. Increased concentrations of intracellular Na + result in an inhibition of ENaC activity due to a reduction in channel density in the plasma membrane, a process referred to as feedback inhibition ( 8, 15 ). Experimentally, Na + self-inhibition can be easily distinguished from feedback inhibition. The former has a very rapid response with a time constant in seconds and is readily reversed upon a decrease in the extracellular Na + concentration ( 8, 27 ).
Recent studies have raised the possibility that common regulatory mechanisms may be involved in the changes in ENaC activity that occur in response to proteolysis and changes in external [Na + ]. Chraibi and Horisberger ( 8 ) demonstrated that external trypsin blunts the Na + self-inhibition response. While trypsin cleavage sites within ENaC subunits have not been identified, furin cleavage sites have been identified within the proximal portion of the ECL of the - and -subunits ( 19 ). Based on analyses of Na + self-inhibition responses of / chimeras, Babini and co-workers ( 2 ) suggested that the proximal portion of the ECL primarily determines the differences in Na + self-inhibition of and channels. We recently reported that mutations of two homologous His residues within - and -ECLs either eliminate ( H239) or enhance ( H282) the response of Na + self-inhibition, suggesting an involvement of the His residues in the mechanism of Na + self-inhibition ( 27 ). Given the proximity of the furin cleavage sites and these His residues ( Fig. 1 ), and that ENaC proteolysis and Na + self-inhibition both affect channel gating, we hypothesized that furin cleavage may regulate ENaC activity by affecting the Na + self-inhibition response. In this report, we examined the Na + self-inhibition response and single-channel properties of mouse ENaCs with mutations within the furin cleavage sites. Our results suggest that, in the absence of furin-dependent proteolysis, the reduced ENaC activity is associated with a markedly enhanced Na + self-inhibition response.
Fig. 1. Features of the Na + self-inhibition response. A : model of an ENaC subunit. The membrane-spanning domains (M1 and M2) are shown as cylinders while the cytoplasmic NH 2 terminus, COOH terminus, and extracellular loop (ECL) are displayed as thick black lines. The 2 hydrophobic segments following M1 (H1) and preceding M2 (P) are shown as gray lines. The conserved 16 Cys residues that are clustered within 2 cysteine rich domains (CRD-I and CRD-II) within the ECL are indicated in circles. HG, DEG, and PY identify an NH 2 -terminal gating domain, degeneration site, and COOH-terminal Pro-Tyr motif, respectively. The pentagon shows the location of H282 or H239, residues that have been implicated in Na + self-inhibition. The previously identified furin cleavage sites, 2 within and one within, are shown in bold letters with residue numbers in superscript. The bars under the sequences indicate a minimal consensus site for furin cleavage, RXXR (Arg-X-X-Arg). B : representative Na + self-inhibition response of wild-type mouse ENaC expressed in a Xenopus laevis ooctye. An oocyte was continuously clamped at -60 mV when bath [Na + ] was rapidly increased from 1 to 110 mM. The I peak and I ss represent the peak current and steady-state current, respectively. The time constant ( ) was obtained by fitting the current decay with an exponential equation. C : current-voltage ( I-V ) traces from the same oocyte as in B. The oocyte was clamped every second with a series of voltage steps lasting 100 ms from -140 to 60 mV in 40-mV increments to obtain I-V relationships during the change in bath [Na + ]. I-V curves obtained with 1 mM bath Na +, at 3, 6, 9, and 20 s after switching to 110 mM Na + are shown with identifying numbers 1, 2, 3, 4, and 5, respectively, within circles. These corresponding time points are also marked in B.
MATERIALS AND METHODS
Site-directed mutagenesis. The mutants R143A, R205A, R231A, and the triple mutant R205A/R208A/R231A were previously generated by site-directed mutagenesis using a PCR-based approach and characterized ( 19 ). Wild-type and mutant constructs with both NH 2 (HA)- and COOH (V5)-terminal epitope tags were previously generated and characterized ( 21 ). The use of individual subunits with an epitope tag is indicated throughout the text by an asterisk (*).
Oocyte expression. cRNAs for,, and -mENaC (wild-type and mutant) subunits were synthesized with T3 or T7 mMessage mMachine (Ambion, Austin, TX). Stage V-VI X. laevis oocytes were pretreated with 1.5 mg/ml type IV collagenase and injected with 1-2 ng of cRNA/subunit. Injected oocytes were maintained at 18°C in modified Barth's saline [88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO 3, 15 mM HEPES, 0.3 mM Ca(NO 3 ) 2, 0.41 mM CaCl 2, 0.82 mM MgSO 4, pH 7.4] supplemented with 10 µg/ml sodium penicillin, 10 µg/ml streptomycin sulfate, and 100 µg/ml gentamicin sulfate.
Procedures for observing Na + self-inhibition. To examine Na + self-inhibition, a low [Na + ] bath solution (1 mM NaCl, 109 mM NMDG, 2 mM KCl, 2 mM CaCl 2, 10 mM HEPES, pH 7.4) was rapidly replaced by a high [Na + ] bath solution (110 mM NaCl, 2 mM KCl, 2 mM CaCl 2, 10 mM HEPES, pH 7.4) while the oocytes were continuously clamped at -60 or -100 mV. Bath solution exchange was performed with a 16-channel Teflon valve perfusion system from AutoMate Scientific (San Francisco, CA). At the end of the experiment, 10 µM amiloride was added to the bath to determine the amiloride-insensitive component of the whole cell current. Currents remaining in the presence of 10 µM amiloride were generally less than 200 nA. Results from oocytes that showed unusually large amiloride-insensitive 5% of total currents) were discarded to minimize current contamination from endogenous channels and membrane leak.
The first 40 s of current decay were fit with a double-exponential equation by Clampfit 9.0 (Axon Instruments) to obtain time constants. The peak current ( I peak ) was the measured maximal inward current immediately following bath solution exchange from low [Na + ] to high [Na + ] concentration. The steady-state current ( I ss ) represented the measured current at 40 s post- I peak. The ratio I ss / I peak was used as a measure of the Na + self-inhibition response, with a smaller value indicating greater Na + self-inhibition.
To estimate the Michaelis constants ( K m ) for Na + concentration-current relationship, both I peak and I ss were measured in the same cell after the bath Na + concentration was raised from 1 to 3, 10, 30, 60, 90, or 110 mM. I peak and I ss were plotted against [Na + ]. K m and V max (maximal current) were obtained by best fit of the current-concentration data according to the following equation, with least squares nonlinear curve fitting using Origin Pro 7.0 (OriginLab, Northampton, MA): I = V max ·C/(C + K m ). In the equation, 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 also determined. I ss / I peak obtained from the Na + self-inhibition response at the different [Na + ] was plotted against the external [Na + ]. The value K i was estimated from a best fit of the data with the Hill equation ( 40 ): I ss / I peak = K i n /(C n + K i n ), where C is the [Na + ] and n is the Hill coefficient.
Single-channel recordings. Oocytes were placed in a hypertonic solution (bath solution supplemented with 200 mM sucrose) for 5-10 min and the vitelline membranes were removed manually. Oocytes were transferred to a recording chamber with bath solution containing (in mM) NaCl 110, KCl 2, CaCl 2 1.54, HEPES 10, pH 7.4, and maintained for at least 10 min at room temperature (22-25°C). Patch pipettes with a tip resistance of 5-12 M were used. Single-channel experiments were performed with two different pipette solutions, a high Na + solution (same as bath solution) or a low Na + solution containing (in mM) 1 NaCl, 109 NMDG, 2 KCl, 1.54 CaCl 2, 10 HEPES, pH 7.4. Currents were recorded in the cell-attached mode with the membrane potential clamped at 60 mV, using an Axopatch 200B Amplifier (Axon Instruments) and a DigiData 1322A interface (Axon Instruments) connected to a Pentium 4 PC (Gateway). Single-channel recordings were acquired at 4 kHz, filtered at 1,000 Hz by a 4-pole low-pass Bessel Filter built in the amplifier, and stored on the hard disk. Single-channel currents were further filtered at 100 Hz with a Gaussian filter for display and analysis. Recordings were analyzed with pClamp 6 (Axon Instruments). In some experiments, the membrane potential was clamped between -100 and +100 mV, to obtain a current-voltage relationship. Only recordings with duration of at least 3 min, and a number of channels in the patch equal or less than three, were used to estimate the P o. Voltages were not corrected by the junction potential which was predicted as -5.7 mV with the low Na + solution in the pipette.
Statistical analysis. Data are presented as means ± SE. Significance comparisons between groups were performed with unpaired Student's t -test or unpaired t -test with Welch correction. A P value of <0.05 was considered statistically different. Statistical comparisons were performed using Excel 2003 (Microsoft) or GraphPad Instant (GraphPad Software, San Diego, CA).
RESULTS
Na + self-inhibition of ENaC. ENaC subunits have a common topology, with two membrane-spanning domains separated by a large ECL. We recently identified two furin cleavage sites in the ECL of the -subunit and one site within the ECL of the -subunit ( 19 ). The sites for furin cleavage immediately follow R205, R231, and R143 and are located between the first and second conserved Cys residues present in the first cysteine-rich domain (CRD) within the ECL ( Fig. 1 A ). A third potential furin cleavage site, present within the -subunit immediately following R208, does not appear to be processed in oocytes or Madin-Darby canine kidney (MDCK) cells by furin ( 19 ).
A typical Na + self-inhibition response is shown in Fig. 1 B. Na + self-inhibition is defined as a current decay from I peak to a relatively I ss that is observed following rapid changing of the bath solution from 1 to 110 mM Na + ( 8, 27 ). To demonstrate that the current decay following a switch to a high [Na + ] bath solution is not due to a reduction in the chemical driving force as a result of an increase in the intracellular [Na + ], we monitored the current-voltage relationship curves as bath [Na + ] was increased. A change in the [Na + ] gradient was readily detected within 1 s (from point 1 to point 2 in Fig. 1 B ), as indicated by a 100-mV shift in reversal potential as shown in Fig. 1 C. Subsequent recordings revealed a progressive reduction in the slope of the current-voltage relationships (whole cell conductance), with no change in the reversal potential. The results are consistent with the notion that the current decay, or Na + self-inhibition response, is not related to a change in the chemical driving force, but rather represents a reduction in whole cell conductance that likely reflects a decrease in channel P o ( 8, 16, 36 ).
Enhanced Na + self-inhibition is observed for channels that lack furin-dependent cleavage. We previously demonstrated that mutations of key Arg residues within furin cleavage sites were associated with a large (mutant -subunit) or modest (mutant -subunit) reduction in ENaC activity. Mutation of the Arg residues immediately preceding the three putative furin cleavage sites in the -subunit ( R205A-R208A-R231A) prevented cleavage of the -subunit in both X. laevis oocytes and MDCK cells ( 19 ). We determined the Na + self-inhibition response of wild-type ENaCs, or channels with mutations of the key Arg residues immediately preceding the -subunit furin cleavage sites ( *R205A-R208A-R231A ), expressed in X. laevis oocytes (* denotes an epitope-tagged subunit). Channels with mutant -subunits had a current decay that was significantly more rapid and greater in magnitude than that observed with the wild-type channels ( *; Fig. 2 ). The decay was best fit with a double exponential equation. Both time constants ( 1 and 2 ) were significantly less than the time constants for current decay observed with wild-type ENaC ( Table 1 ). The ratio, I ss / I peak, represents the magnitude of Na + self-inhibition, with a smaller ratio corresponding to greater Na + self-inhibition. The average I ss / I peak for the *R205A-R208A-R231A mutant was 0.23 ± 0.01 ( n = 11), significantly lower than the ratio observed with corresponding * control channels (0.59 ± 0.02, n = 8; Table 1 ). The reduced I ss / I peak of the channel with a mutant -subunit reflected a lower I ss ( P < 0.05 vs. * ), as the I peak for mutant and wild-type channels was similar ( P 0.05 vs. * ). The enhanced Na + self-inhibition response of the channel with a mutant -subunit is consistent with the reduced whole cell Na + currents we previously observed with this mutant when compared with the current observed in oocytes expressing wild-type ENaC ( 19 ).
Fig. 2. Mutation of the consensus sites for furin cleavage enhance Na + self-inhibition. Oocytes expressing wild-type or mutant ENaCs were clamped at -100 mV. The Na + self-inhibition response was determined as described under MATERIALS AND METHODS. Open and filled bars indicate the period when cells were bathed in a 1 or 110 mM [Na + ] solution, respectively, as labeled in A. Each recording is representative of at least 8 independent experiments. Subunits with NH 2 (HA)- and COOH (V5)-terminal epitope tags are indicated by *.
Table 1. Parameters for Na + self-inhibition
The R143A mutation prevents furin-dependent cleavage of -ENaC in oocytes and MDCK cells ( 19 ). An enhanced Na + self-inhibition was observed for channels containing this mutant subunit ( *R143A or * *R143A), as evidenced by a more rapid current decay and a reduced I ss / I peak, when compared with wild-type channels ( * or * *) ( Fig. 2 and Table 1 ). Channels with both mutant - and -subunits ( *R205A-R208A-R231A *R143A) showed faster and stronger Na + self-inhibition compared with that of channel with only a mutant -subunit ( * *R143A) ( Fig. 2 H and Table 1 ), as expected. The I ss / I peak of * *R143A was 71% of the I ss / I peak observed with * *. The I ss was in agreement with the whole cell Na + current that we previously reported with this mutant, relative to that of the wild-type control (65 ± 6%) ( 19 ). These results suggest that the enhanced Na + self-inhibition observed with the mutant channels is responsible for the reduced levels of steady-state current expression observed.
Unexpectedly, the I ss / I peak ratios of the control channels with epitope tags on either the - and -subunits ( * *), or only the -subunit ( *), were also significantly lower than that of wild-type ENaC ( ) or channels with an epitope-tagged -subunit ( * ) ( P < 0.05). However, the Na + self-inhibition response of * was not significantly different from that of ( Table 1 ). These results suggest that the epitope-tagged -subunit contributes to the enhanced Na + self-inhibition response.
-Subunit must be cleaved twice to relieve Na + self-inhibition. Our results suggested that furin-dependent cleavage of the -subunit dramatically altered the Na + self-inhibition response. As the -subunit has two furin cleavage sites, we examined whether channels with a mutation of only one of the furin cleavage sites, R205A or R231A, still exhibited a Na + self-inhibition response that was similar to wild-type channels. As shown in Fig. 2 and Table 1, the Na + self-inhibition response observed with both *R205A and *R231A was indistinguishable from that of *R205A-R208A-R231A. These data suggest that the -subunit must be cleaved twice to relieve channels of the Na + self-inhibition response.
Na + self-inhibition of the -furin cleavage site mutants is associated with an increased apparent affinity of ENaC for extracellular Na +. Na + self-inhibition represents allosteric regulation of ENaC activity ( 15, 39 ). This regulatory phenomenon has strong temperature dependence, consistent with a conformational change initiated by the binding of extracellular Na + to the channel ( 7, 8 ). Three potential mechanisms may be responsible for the enhanced Na + self-inhibition response observed with the furin site mutants: 1 ) an enhanced Na + binding affinity to an extracellular Na + "receptor" site; 2 ) a facilitated conformational change that is initiated by Na + binding to an extracellular Na + "receptor" site and transmitted to the channel gate; or 3 ) or a "sensitized" gate that intrinsically favors an inhibited state. To address the first potential mechanism, we estimated the apparent Na + affinity for self-inhibition by analyzing Na + saturation curves for both peak and steady-state currents at varying concentrations of extracellular Na +. The Na + self-inhibition response of *R205-R208A-R231A as well as * was repeatedly examined in the same oocyte by increasing bath [Na + ] from 1 to either 110, 90, 60, 30, 10, or 3 mM ( Fig. 3, A and B ). A current decay indicating the presence of Na + self-inhibition was observed with all concentrations of bath Na + above 3 mM in the mutant channels, in contrast to * channels ( Fig. 3 ) or ( 27 ) which showed a clear current decay only when bath [Na + ] is raised to 30 mM or higher. The I peak and I ss were plotted against bath [Na + ] in Fig. 3 C. These data were fit with the Michaelis-Menten equation and the K m values resulting from best fitting are listed in Table 2. The I peak of both mutant and control channels, as well as the I ss of the control, fit well with the equation as evidenced by correlation coefficients of 0.97. In contrast, the fit of the I ss data of the mutant channels with the equation was not as robust (correlation coefficient of 0.73), due to a modest decline of I ss at [Na + ] higher than 30 mM. The poor fit indicates that the conventional Michaelis-Menten equation is not sufficient to describe the saturation behavior of the steady-state current of the mutants and the I ss was likely suppressed at high [Na + ] due to emerging Na + self-inhibition. The atypical relationship between I ss and external [Na + ] appeared to be similar to substrate inhibition of an enzyme. Thus we analyzed our data with an equation that describes substrate inhibition ( 26 ), I ss = V max ·C/( K m + C + C 2 / K i s ), where " K i s " is an inhibitory constant for Na + that reflects Na + self-inhibition. We fixed the K m at the I peak K m from the same cell in the fitting, as K m of I peak likely reflects the true saturation of channel currents or conductances and should represent I ss as well, given the same source for both I peak and I ss in the same oocyte. The resulting K i s values for both control and mutant channels were comparable to the K i values that were estimated from fitting the I ss / I peak data with the Hill equation as previously reported ( 27 ) ( Fig. 3 E and Table 2 ). Both K i and K i s of *R205A-R208A-R231A were significantly smaller than those of *, suggesting that the enhanced Na + self-inhibition observed with the furin site mutant channels was due to an increased Na + binding affinity. The estimated Hill coefficient of 0.94 ± 0.02 for * mENaCs suggests little cooperativity, if any, in Na + binding. Although the Hill coefficient of the mutants was significantly different from that of the control channels, the difference was relatively small ( Table 2 ). The K m values for both I peak and I ss of the mutant mENaCs were found significantly smaller than those of control mENaCs ( P < 0.05). Although the exact cause for the difference in K m of I peak is not clear, given the greatly accelerated current decays in oocytes expressing the mutant channels, it is possible that the I peak values at high bath [Na + ] was underestimated, as was the I peak of the mutant channel. The parameters in Table 2 obtained in oocytes expressing * are in good agreement with those previously reported for mENaC ( 27 ).
Fig. 3. Mutation of the -subunit furin cleavage consensus sites increases Na + affinity and self-inhibition. Oocytes expressing wild-type * ( A ) or mutant *R205A-R208A-R231A ( B ) channels were continuously clamped to -100 mV, and bath [Na + ] was increased from 1 mM (open bars) to various values as indicated by the numbers near the peak currents. A representative recording of whole cell current, while the bath [Na + ] is being systematically altered, is shown for ooctyes expressing * ( A ) or *R205A-R208A-R231A ( B ). Traces are representative of 8 independent experiments for each group. C : I peak and I ss vs. bath [Na + ] are plotted. The lines were derived from the best fit of the data with the Michaelis-Menten equation and represent wild-type channel I peak ( ), mutant channel I peak ( ), wild-type channel I ss ( ), and mutant channel I ss ( ). D : I ss data were fit with the substrate inhibition equation (see RESULTS ). Data from analysis of wild-type and mutant channels are shown as squares and triangles, respectively. E : plot of I ss / I peak vs. [Na + ]. The line was derived from the best fit of the data with the Hill equation. Data for wild-type channels are displayed as circles and for mutant channels as squares. C, D, and E : data are shown as means ± SE ( n = 8) for both groups.
Table 2. Fitting parameters for the dose-response of Na + self-inhibition
Analysis of Na + self-inhibition at the single-channel level. Previous studies have suggested that Na + self-inhibition reflects a reduction in channel P o ( 8, 15, 27, 38, 39 ). However, a reduction in channel P o in response to an increase in extracellular [Na + ] has not been demonstrated at a single-channel level. The enhanced Na + self-inhibition response observed with the furin site mutants provided an opportunity to observe Na + self-inhibition at a single-channel level. Intracellular [Na + ] of oocytes expressing wild-type ENaC and maintained in modified Barth's saline for 24 to 48 h increases to a concentration that is similar to external [Na + ], as whole cell current-voltage recordings have demonstrated a reversal potential of 0 mV (see Fig. 1 C ). Cell-attached patch clamp was performed with oocytes expressing *R205A-R208A-R231A, with either 1 or 110 mM Na + in the patch pipette and 110 mM Na + in the bath. The membrane potential was clamped at 60 mV and outward Na + currents were recorded. Representative recordings are shown in Fig. 4. With 110 mM Na + in the patch pipette, channels remained largely in the closed state, as shown in the all points histogram ( Fig. 4 B ). The estimated P o was 0.07 ± 0.02 ( n = 5) in 110 mM Na +, assuming that all the channels present in the patch opened simultaneously at least once during the recording ( Figs. 4 B and 5 A ). In contrast, the P o was significantly greater when 1 mM Na + was present in the patch pipette [0.38 ± 0.06 ( n = 6); P < 0.002; Figs. 4 A and 5 A; unpaired t -test with Welch correction].
Fig. 4. Single-channel recordings of *R205A-R208A-R231A. Single-channel tracings were obtained from oocytes expressing *R205A-R208A-R231A channels as described under MATERIALS AND METHODS. The closed state is indicated by C. Recordings were performed in the cell-attached mode with high-[Na + ] solution in the bath, with a clamped membrane potential of 60 mV. A and B : continuous recordings of single-channel activity recorded with low (1 mM, n = 9)- or high (110 mM, n = 8)-[Na + ] solution in the patch pipette, respectively. C : continuous recording of single-channel activity obtained with a high-[Na + ] solution (110 mM) in the patch pipette that also contained 2 µg/ml trypsin ( n = 4). Right : Normalized amplitude histograms presented for each recording.
Fig. 5. Single-channel properties of *R205A-R208A-R231A determined with low (1 mM) or high (110 mM) [Na + ] in the patch pipette. A : estimated open probability (applied pipette potential -60 mV) was 0.07 ± 0.02 ( n = 5) with a high external [Na + ]. In contrast, the open probability was significantly greater [0.38 ± 0.06 ( n = 6); P < 0.002; unpaired t -test with Welch correction] when 1 mM [Na + ] was present in the patch pipette. B : single-channel currents plotted as function of the membrane voltage for *R205A-R208A-R231A channels obtained with a low ( ) or high ( ) [Na + ] in the patch pipette, or with both a high [Na + ] and trypsin ( ). Single-channel conductances, estimated by linear regression, were 3.5 ± 0.2 pS (1 mM Na +, n = 11), 4.0 ± 0.2 pS (110 mM Na +, n = 18), and 4.2 ± 0.2 pS (110 mM Na + + trypsin, n = 8), respectively.
We previously showed that extracellular trypsin activates *R205A-R208A-R231A, presumably by cleaving the channel at sites within the ECLs ( 19 ). When 2 µg/ml trypsin were included in a patch pipette with 110 mM Na +, channel transitions were observed that were similar to those observed with a 1 mM Na + pipette solution in the absence of trypsin ( Fig. 4 C ). These data suggest that trypsin-dependent cleavage relieved *R205A-R208A-R231A of an enhanced Na + self-inhibition response.
The unitary conductance of *R205A-R208A-R231A with 1 mM Na +, 110 mM Na +, or 110 mM Na + plus trypsin in the patch pipette was estimated by linear fitting of the unitary currents at multiple holding potentials. These values were 3.5 ± 0.2 pS ( n = 11), 4.0 ± 0.2 pS ( n = 18), and 4.2 ± 0.2 pS ( n = 8), respectively ( Fig. 5 B ). These observations provide a clear demonstration of inhibition of channel P o by extracellular Na +.
DISCUSSION
Mutating a critical Arg residue within even a single furin cleavage consensus sequence Arg-X-X-Arg in the ECLs of the - or -subunit greatly enhanced ENaC Na + self-inhibition ( Fig. 2 and Table 1 ). These mutations prevent proteolytic processing of ENaC subunits ( 19 ). The increase in the Na + self-inhibition response observed with *R205A-R208A-R231A and * *R143A paralleled the reduction in whole cell Na + currents we previously reported with these mutants ( 19 ). As the whole cell currents are routinely measured in a bath solution with a high Na + concentration, these currents correspond to the steady-state current ( I ss ) observed in a Na + self-inhibition response.
The enhanced Na + self-inhibition response observed with channels that lacked one or both of the furin cleavage sites within the -subunit was similar in magnitude. These data suggest that the -subunit must be cleaved twice to exhibit "normal" gating behavior in the presence of a high external [Na + ]. The enhanced Na + self-inhibition observed with the furin site mutants reflected an increased Na + affinity compared with the wild-type channel ( Fig. 3 ). At the single-channel level, Na + self-inhibition reflected a reduction in channel P o ( Figs. 4 and 5 ).
The peak currents recorded following the switch from a low- to a high-[Na + ] solution were similar in magnitude for both wild-type ENaC and channels with furin site mutations, suggesting that both the wild-type and mutant channels were intrinsically active in the presence of a low concentration of extracellular Na + and were expressed at the membrane surface with a similar density. These results are consistent with our previous observation that trypsin treatment activated whole cell currents of wild-type ENaCs and furin cleavage site mutants to a similar level ( 19 ).
We recently reported that ENaC maturation involves both processing of Asn-linked glycans on,, and to complex type and proteolytic cleavage of the - and -subunits. Within individual channels, posttranslational processing of ENaC subunits is an all-or-none event such that two distinct pools of channels are present within cells and at the plasma membrane: 1 ) channels where all subunits have undergone posttranslational processing and 2 ) channels where none of the subunits have undergone posttranslational processing. We proposed that nonprocessed channels provided a reserve pool of inactive channels that could be activated by proteases ( 20 ). Our results suggest that a markedly enhanced Na + self-inhibition response accounts for the low P o of nonprocessed channels and that proteolytic processing of this pool of channels relieves Na + self-inhibition ( Figs. 4 and 5 ). These results are in agreement with observations published by Caldwell and co-workers ( 5, 6 ), who recently demonstrated that both trypsin and neutrophil elastase activate ENaC by selectively converting a pool of "near silent" channels to channels that exhibit a higher P o with long open and closed times.
Caldwell and co-workers ( 5 ) also reported that external proteases do not affect the gating characteristics of active channels. Furthermore, Chraibi and Horisberger ( 8 ) observed that the Na + self-inhibition response was markedly diminished by treatment with external proteases. These results and our observations suggest that Na + self-inhibition is a response primarily due to the presence of nonprocessed channels at the plasma membrane. Two mechanisms should activate this pool of noncleaved, low P o channels: 1 ) proteolytic processing of ENaC subunits by furin, prostasin, elastase, or perhaps other cellular proteases, and 2 ) reducing the luminal Na + concentration to a level that blunts the Na + self-inhibition response. In the setting of extracellular fluid volume depletion or a reduced effective arterial volume, luminal Na + concentrations may be reduced in collecting ducts to a level such that the surface pool of nonprocessed channels would become active. In states of avid renal Na + retention, lowering of the luminal Na + concentration provides a novel mechanism to recruit noncleaved channels to enhance rates of tubular Na + reabsorption.
The enhanced Na + self-inhibition of the furin site mutants results, at least in part, from an increased affinity for Na + binding to an external site within the channel ( Fig. 3 ), as we observed a decreased K i for Na + self-inhibition with the -subunit furin cleavage site mutant compared with the wild-type channel. These results suggest that a putative Na + sensor or receptor may be located in proximity to the furin cleavage sites. Consistent with this hypothesis, previous studies of / X. laevis ENaC chimeras and mouse H282 and H239 mutants suggested that sites involved in Na + self-inhibition are in a region that is near the furin cleavage sites ( 2, 27 ). It is also possible that elimination of furin cleavage sites alters conformational changes that are related to the mechanism of Na + self-inhibition and thus enhance the inhibitory response.
Recent studies of members of the ENaC/DEG family, including ENaC, acid-sensing ion channels, and peptide-gated ion channels expressed in marine snails, suggest that the initial region of the ECL following the first membrane spanning domain harbors sites that specifically bind extracellular ions or peptides that modulate channel gating, such as Na +, H +, or the peptide Phe-Met-Arg-Phe-NH 2 ( 3, 10 - 12, 18, 23, 27 - 29 ). Ligand binding is thought to transmit a signal that modifies channel gating. Mechanosensitive ion channels in C. elegans are also members of the ENaC/DEG family. These channels are thought to activate in response to an external mechanical stimulus. An extracellular regulatory domain that affects mechanosensitive gating has been identified in the ECL between two CRDs (see Fig. 1 ), which is absent in other ENaC/DEG family members ( 4, 14, 17 ). This extracellular regulatory domain within these mechanosensitive ion channels may be homologous to the external ligand binding sites within other ENaC/DEG members.
In summary, our results suggest that furin cleavage of ENaC relieves Na + self-inhibition. Cleavage of two sites within the -subunit is required to relieve Na + self-inhibition by a mechanism that increases channel P o in the presence of a high extracellular [Na + ]. As noncleaved channels exhibit a dramatic increase in P o in the presence of a low extracellular [Na + ], these channels may be activated under conditions of avid renal Na + retention as urinary [Na + ] decreases.
GRANTS
This work was supported by National Institutes of Health Grants DK-054354 and DK-065161.
ACKNOWLEDGMENTS
We thank Dr. O. B. Kashlan for helpful discussions.
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作者单位：Departments of Medicine, Cell Biology, and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania