Proteolytic Processing of the Epithelial Sodium Channel γ Subunit Has a Dominant Role in Channel Activation

Abstract

Maturation of the epithelial sodium channel (ENaC) involves furin-dependent cleavage at two extracellular sites within the α subunit and at a single extracellular site within the γ subunit. Channels lacking furin processing of the α subunit have very low activity. We recently identified a prostasin-dependent cleavage site (RKRK¹⁸⁶) in the γ subunit. We also demonstrated that the tract α D206-R231, between the two furin cleavage sites in the α subunit, as well as the tract γ E144-K186, between the furin and prostasin cleavage sites in the γ subunit, are inhibitory domains. ENaC cleavage by furin, and subsequently by prostasin, leads to a stepwise increase in the open probability of the channel as a result of release of the α and γ subunit inhibitory tracts, respectively. We examined whether release of either theα orγ inhibitory tract has a dominant role in activating the channel. Co-expression of prostasin and either wild type channels or mutant channels lacking furin cleavage of the α subunit (αR205A,R208A,R231Aβγ) in Xenopus laevis oocytes led to increases in whole cell currents to similar levels. In an analogous manner and independent of the proteolytic processing of theα subunit, amiloride-sensitive currents in oocytes expressing channels carrying γ subunits with both a mutation in the furin cleavage site and a deletion of the inhibitory tract (αβγR143A,ΔE144-K186 and αR205A,R208A,R231AβγR143A, ΔE144-K186) were significantly higher than those from oocytes expressing wild type ENaC. When channels lacked the α and γ subunit inhibitory tracts, α subunit cleavage was required for channels to be fully active. Channels lacking both furin cleavage and the inhibitory tract in theγ subunit (αβγR143A,ΔE144-K186) showed a significant reduction in the efficacy of block by the syntheticα-26 inhibitory peptide representing the tract αD206-R231. Our data indicate that removal of the inhibitory tract from the γ subunit, in the absence ofα subunit cleavage, results in nearly full activation of the channel.

Epithelial sodium channels (ENaCs)2 mediate Na⁺ transport across apical membranes of cells lining the distal nephron, airway, and alveoli, and distal colon. These channels are required for the maintenance of extracellular fluid volume, blood pressure, and airway surface liquid volume. ENaCs are composed of three structurally similar subunits termed α, β, and γ (1). Each subunit has two transmembrane domains connected by a large ectodomain with short intracellular N and C termini. ENaC is assembled in the endoplasmic reticulum where the three subunits undergo Asn-linked glycosylation (2–4). The recently resolved structure of chicken acid sensing ion channel 1 (cASIC1), an ENaC-related family member, revealed that members of this family of ion channels are likely homo- or heterotrimers (5).

We previously reported that α and γ ENaC subunits are processed within their extracellular domains by furin, a serine protease that resides primarily in the trans-Golgi network (6). The α subunit is cleaved twice by furin immediately following Arg-205 and Arg-231, and the γ subunit at a single site after Arg-143 (6). Two pools of ENaC subunits are expressed at the plasma membrane, an immature pool with endoglycosidase H-sensitive non-processed N-glycans that lacks proteolytic processing, and a mature pool with endoglycosidase H-resistant terminally processed N-glycans and proteolytically processed α and γ subunits (7). Channels with non-cleaved α subunits or with α subunits that are cleaved at only one furin cleavage site display low open probability when expressed in Xenopus laevis oocytes (6, 8–10). However, channels carrying mutations in the first α subunit furin cleavage site as well as a deletion of the connecting tract (αR205A,ΔD206-R231βγ) lack cleavage in the α subunit, but are active when expressed in oocytes. These observations suggest that the release or removal of an inhibitory tract (D206-R231) from the extracellular domain of the α subunit, rather than cleavage per se is required for normal channel activity (9).

ENaC activity has been shown to be modulated by several serine proteases, including a group of “channel-activating proteases” (CAPS) such as prostasin (CAP-1), TMPRSS4 (CAP-2), and matriptase (CAP-3 or MT-SP1). Other proteases that active ENaC are chymotrypsin, neutrophil elastase, pancreatic elastase, kallikrein, and trypsin (11–20). We recently identified a prostasin-dependent cleavage site in the γ subunit of ENaC (RKRK¹⁸⁶) that is distal to the furin cleavage site (RKRR¹⁴³) (14). The tract E144-K186, between the furin and prostasin cleavage sites in the γ subunit, encompasses an inhibitory domain (14). Channels proteolytically processed by both furin and prostasin or channels lacking the γ inhibitory tract exhibit a very high open probability (14).

We have proposed that sequential release of the α and γ subunit inhibitory tracts lead to a stepwise increase in the open probability of the channel (9, 14). In this report, we investigated whether loss of either the α subunit or γ subunit inhibitory tract has a dominant role in activating ENaC. In the absence of α subunit cleavage, we found that removal of the γ subunit inhibitory domain leads to near full activation of the channel, supporting the concept that proteolysis of the γ subunit with release of its inhibitory domain has a major role in the modulation of channel gating.

EXPERIMENTAL PROCEDURES

Oocyte Expression—cRNAs for α, β, and γ mouse ENaC (wild-type and mutant) subunits and mouse prostasin were synthesized with T3 or T7 mMessage mMachine™ (Ambion, Austin, TX). α and γ subunits had N-terminal HA and C-terminal V5 epitope tags (6). Mutants of mouse ENaC subunits used in this work were previously described (6, 9, 14). Stage V-VI X. laevis oocytes were injected with 1–2 ng of cRNA for each subunit and 3 ng of prostasin cRNA.

Peptides—The peptide DLRGALPHPLQRLRTPPPPNPARSAR (α-26) was synthesized and HPLC-purified by GenScript Corporation (Piscataway, NJ).

Two-electrode Voltage Clamp (TEV)—Electrophysiological measurements were performed 20–28 h after injection. The extracellular solution was (in mm): 110 NaCl, 2 KCl, 1.54 CaCl₂, 10 HEPES, pH 7.4. The amiloride-insensitive component of the whole cell current was determined by perfusion with an extracellular solution supplemented with 10 μm amiloride. The amiloride-sensitive component of the whole cell Na⁺ current, at –60 mV, was defined as the ENaC-mediated current. The α-26 mer inhibitory peptide was dissolved in the extracellular solution, where indicated.

Data and Statistical Analyses—Each set of experiments were performed with at least two batches of oocytes obtained from different frogs. Data were not normalized and were expressed as the mean ± S.E. (n), where n equals the number of independent experiments analyzed. Statistical comparisons were performed with GraphPad 3.0 (GraphPad Software, San Diego, CA). One-way ANOVA followed by Student-Newman-Keuls test were used for multiple comparisons. Repeated measures ANOVA were used to compare multiple treatments in the same cell. p < 0.05 was considered statistically significant.

RESULTS

We previously demonstrated that sequential removal of the α and γ subunit inhibitory domains leads to a stepwise increase in ENaC open probability (9, 14). Functional changes observed at the macroscopic level by removal of subunit inhibitory domains were directly associated with changes in the open probability of the channel (8, 9, 14). To gain insight into the regulation of channel gating by proteolytic processing, we examined whether sequential removal of the α and γ inhibitory domains is required for activation of the channel by prostasin. Wild-type ENaC (αβγ) or channels carrying mutations in the α subunit furin consensus cleavage sites (αR205A,R208A,R231Aβγ (αRtripleAβγ)) were expressed in X. laevis oocytes with or without prostasin. As we previously observed, oocytes expressing channels that lacked α subunit cleavage had currents that were reduced by 92.1 ± 1.0%, compared with oocytes expressing wild type channels. However, co-expression of prostasin with channels containing either wild type or mutant α subunits led to an increase in whole cell currents to a similar level (Fig. 1). We previously demonstrated that channels carrying mutations in the γ subunit within the tract RKRK¹⁸⁶ are not activated by prostasin (14). In agreement with our previous observations, mutation of both the furin and prostasin cleavage sites in the γ subunit (γR143A,RKRK¹⁸³QQQQ) prevented prostasin-dependent activation of channels that also contained either a wild type or mutant α subunit (αRtripleA) (Fig. 2). As channels with the αRtripleA mutant α subunits retain their α inhibitory tract (6), our data suggest that removal of the γ inhibitory tract is sufficient to activate the channel in the presence or absence of the α inhibitory tract.

FIGURE 1.

Channels lacking α subunit furin cleavage are activated by prostasin. A, models for wild type and furin-insensitive (αR205A,R208A, R231A (αRtripleA)) α subunits, and wild type and prostasin processed γ subunits. The sites of furin- and prostasin-dependent cleavage are denoted in gray and mutations in black. B, prostasin activates channels carrying mutations in the α subunit furin cleavage sites. TEV was performed with oocytes expressing either wild-type (αβγ) or αRtripleAβγ channels with or without prostasin. Amiloride-sensitive currents were statistically different between oocytes expressing αβγ versus αRtripleAβγ (p < 0.001). Statistically significant differences were also observed between oocytes expressing αβγ versus αβγ and prostasin (p < 0.05), and αRtripleAβγ and prostasin (p < 0.01, One-way ANOVA followed by Student-Newman-Keuls multiple comparisons test). Amiloride-sensitive currents were not statistically different between oocytes co-expressing prostasin and either αβγ or αRtripleAβγ channels. Experiments were performed with 22 oocytes for each group. The presence or absense of an inhibitory domain is indicated by the (+) or (–), respectively.

FIGURE 2.

Mutation of the furin and prostasin cleavage sites in the γ subunit impairs prostasin activation of wild type and mutant ENaCs. A, models for wild type and furin-insensitive (αR205A,R208A,R231A (αRtripleA)) α subunits, and wild type, furin-, and prostasin-insensitive (γR143A,RKRK¹⁸⁶QQQQ (γR/A,RKRK/Q4)), and prostasin-processed γ subunits. The sites of furin- and prostasin-dependent cleavage are denoted in gray and mutations in black. B, TEV was performed with oocytes expressing either wild-type (αβγ), αR205A,R208A,R231Aβγ (αRtripleAβγ), αβγR143A,RKRK¹⁸⁶QQQQ (αβγR/A,RKRK/Q4), and αR205A,R208A, R231AβγR143A,RKRK¹⁸⁶QQQQ (αRtripleAβγR/A,RKRK/Q4) channels with and without prostasin. Statistically significant differences were observed between oocytes expressing αβγ versus αRtripleAβγ, αβγR/A,RKRK/Q4 and αRtripleAβγR/A,RKRK/Q4 channels (p < 0.001). In oocytes expressing prostasin, statistically significant differences were observed between oocytes expressing αβγ versus αβγR/A,RKRK/Q4 and αRtripleAβγR/A,RKRK/Q4 channels (p < 0.001, One-way ANOVA followed by Student-Newman-Keuls multiple comparisons test). Experiments were performed with 25–26 oocytes for each group. The presence or absense of an inhibitory domain is indicated by (+) or (–), respectively.

To further validate our findings, we expressed four different channels that had α and γ subunits with inhibitory domains that were either retained or removed (see Fig. 3). Wild type channels that are processed by furin lack the α subunit inhibitory domain, but retain the γ subunit inhibitory domain as the γ subunit is only cleaved once. Channels with α subunits lacking furin cleavage sites (αRtripleAβγ) retain both the α and γ subunit inhibitory domains. The γR143A,ΔE144-K186 mutant (γR/AΔ) lacks both the γ subunit furin cleavage site and the inhibitory tract. αβγR/AΔ channels that are processed by furin will lack both the α and γ subunit inhibitory domains. αRtripleAβγR/AΔ channels will retain the α subunit inhibitory domain, but lack the γ subunit inhibitory domain. As we previously reported, oocytes expressing channels that lacked both inhibitory tracks (αβγR/AΔ) exhibited whole cell currents that were 2.55 ± 0.29 fold significantly greater than oocytes expressing wild type αβγ (Fig. 3). Whole cell currents measured in oocytes expressing channels that lacked only the γ subunit inhibitory track (αRtripleAβγR/AΔ) were similar in magnitude to that measured in oocytes lacking both inhibitory tracts (αβγR/AΔ). These results provide additional support for the concept that release of the γ subunit inhibitory domain has a dominant role in activating the channel.

FIGURE 3.

Deletion of the γ subunit inhibitory tract E144-K186 increases the activity of channels lacking α subunit furin cleavage. A, models for wild type and furin-insensitive (αR205A,R208A,R231A (αRtripleA)) α subunits, and wild type and mutant γ subunits lacking both the furin cleavage site and the inhibitory tract E144-K186 (γR143A,ΔE144-K186 (γR/AΔ)). Furin-dependent cleavage sites are indicated in gray and mutations in black. The dotted line denotes a deletion. B, TEV was performed with oocytes expressing either wild-type (αβγ), αR205A,R208A,R231Aβγ (αRtripleAβγ), αβγR143A, ΔE144-K186 (αβγR/AΔ), or αR205A,R208A,R231AβγR143A,ΔE144-K186 (αRtripleAβγR/AΔ) channels. Statistically significant differences were observed between oocytes expressing αβγ versus αRtripleAβγ (p < 0.001), αβγR/AΔ (p < 0.001), and αRtripleAβγR/AΔ channels (p < 0.001, One-way ANOVA followed by Student-Newman-Keuls multiple comparisons test). Amiloride-sensitive currents were not statistically different between oocytes expressing αβγR/AΔ and αRtripleAβγR/AΔ channels. Experiments were performed with 17–23 oocytes for each group. The presence or absense of an inhibitory domain is indicated by (+) or (–), respectively.

We previously demonstrated that α subunit proteolysis was not necessary to activate channels lacking the α subunit inhibitory tract (9). In agreement with our previous findings, we observed that oocytes expressing wild type channels exhibited currents that were similar in magnitude to channels that lacked both furin cleavage sites and the inhibitory tract in the α subunit (αR205A,ΔD206-R231βγ (αR/AΔβγ)) (Fig. 4). The difference between the wild type α subunit and αR/AΔ is that one is cleaved (wild type), while the other is not (αR/AΔ). As both constructs lack the α subunit inhibitory tract, we postulated that expression of channels with either α subunit (wild type or αR/AΔ) with a γ subunit that lacked its inhibitory domain and furin cleavage site (γR/AΔ) would result in channels with significantly enhanced activity. Surprisingly, oocytes expressing αR/AΔβγR/AΔ had whole cell currents that were significantly lower than oocytes expressing αβγR/AΔ channels (Fig. 4). Furthermore, when the α subunit lacked its inhibitory tract while retaining a furin cleavage site (αΔD206-R231 (αΔ)), deletion of the γ subunit inhibitory tract led to a large 3.14 ± 0.31 fold increase in ENaC activity when compared with αΔβγ (Fig. 5). Oocytes expressing αΔβγR/AΔ exhibited whole cell currents that were significantly greater than currents measured in oocytes expressing αR/AΔβγR/AΔ. These results suggest that when channels lack the α and γ subunit inhibitory tracts, α subunit cleavage is required for channels to be fully active.

FIGURE 4.

Channels lacking both furin cleavage and the inhibitory tracts in the α and γ subunits display reduced activity. A, models for wild type α, furin-insensitive α (αR205A,R208A,R231A (αRtripleA)) and α subunits lacking both furin cleavage sites and the inhibitory tract D206-R231 (αR205A,ΔD206-R231(αR/AΔ)), wild type γ, furin-insensitive γ (γR143A (γR/A)), and mutant γ subunits lacking both the furin cleavage site and the inhibitory tract E144-K186 (γR143A,ΔE144-K186 (γR/AΔ)). Furin-dependent cleavage sites are indicated in gray. The dotted line denotes a deletion. B, TEV was performed with oocytes expressing either wild-type (αβγ), αR205A,R208A,R231AβγR143A (αRtripleAβγR/A), αR205A,ΔD206-R231βγ (αR/AΔβγ), αβγR143A,ΔE144-K186 (αβγR/AΔ), or αR205A,ΔD206-R231βγR143A,ΔE144-K186 (αR/AΔβγR/AΔ) channels. Statistically significant differences were observed between αβγ controls versus αRtripleAβγR/A (p < 0.001), and αβγR/AΔ (p < 0.001). Amiloride-sensitive currents were statistically different between oocytes expressing αβγR/AΔ and αR/AΔβγR/AΔ channels (p < 0.001, One-way ANOVA followed by Student-Newman-Keuls multiple comparisons test). Experiments were performed with 27 oocytes for each group. The presence or absense of an inhibitory domain is indicated by (+) or (–), respectively.

FIGURE 5.

Full activation of channels lacking the γ inhibitory tract E144-K186 requires cleavage of the α subunit. A, models for α subunits lacking the inhibitory tract D206-R231 with (αR205A,ΔD206-R231 (αR/AΔ)) or without (αΔD206-R231(αΔ)) a mutation in the furin cleavage site, and wild type and mutant γ subunits lacking both the furin cleavage site and the inhibitory tract E144-K186 (γR143A,ΔE144-K186 (γR/AΔ)). Furin cleavage sites are indicated in gray. The dotted line denotes a deletion. B, TEV was performed in oocytes expressing either αΔD206-R231βγ (αΔβγ), αΔD206-R231βγR143A,ΔE144-K186 (αΔβγR/AΔ), αR205A,ΔD206-R231βγ (αR/AΔβγ), or αR205A,ΔD206-R231βγR143A,ΔE144-K186 (αR/AΔβγR/AΔ) channels. Statistical significant differences were observed between αΔβγ versus αΔβγR/AΔ (p < 0.001), and αR/AΔβγR/AΔ (p < 0.01). Amiloride-sensitive currents were also statistically different between oocytes expressing αΔβγR/AΔ and αR/AΔβγR/AΔ channels (p < 0.001, One-way ANOVA followed by Student-Newman-Keuls multiple comparisons test). Experiments were performed with 20 oocytes for each group. The presence or absense of an inhibitory domain is indicated by (+) or (–), respectively.

We previously reported whole cell amiloride-sensitive Na⁺ currents of 9.68 ± 2.06 μA in oocytes expressing channels lacking both furin cleavage and the inhibitory tract in the γ subunit (αβγR143A,ΔE144-K186) (n = 16), whereas currents in oocytes expressing wild type ENaC were 2.63 ± 0.48 μA(n = 16) (14). Within the same experimental group, oocytes expressing channels solely lacking the γ inhibitory tract (αβγΔE144-K186) exhibited currents of 9.72 ± 2.24 μA(n = 16). Our results indicate that furin cleavage of the γ subunit does not influence the activity of channels lacking the γ inhibitory tract.

Our results suggest that channels are activated by excising an inhibitory domain from the γ subunit. In this setting, the presence of an α subunit inhibitory domain did not appear to reduce channel activity (Fig. 3). The mutant α subunit in this experiment (Fig. 3) lacked both furin sites, raising the possibility that the α subunit inhibitory domain might be in an unfavorable conformation to interact with the channel. To address this possibility, we examined the activity of channels lacking the γ subunit inhibitory tract (γR/AΔ) while retaining the α subunit inhibitory tract in the setting where the α subunit was either not cleaved (αRtripleA) or was cleaved only once (αR205A). Surprisingly, oocytes expressing αR205AβγR/AΔ had whole cell currents that were significantly lower than oocytes expressing αRtripleAβγR/AΔ (Fig. 6).

FIGURE 6.

In the absence of the γ inhibitory domain, α subunit proteolysis influences the efficacy of the α inhibitory domain. A, models for wild type and furin-insensitive (αR205A and αR205A,R208A,R231A (αRtripleA)) α subunits, and wild type and mutant γ subunits lacking both the furin cleavage site and the inhibitory tract E144-K186 (γR143A,ΔE144-K186 (γR/AΔ)). Furin-dependent cleavage sites are indicated in gray and mutations in black. The dotted line denotes a deletion. B, TEV was performed with oocytes expressing either wild-type (αβγ), αR205Aβγ (αR205Aβγ), αR205A,R208A,R231Aβγ (αRtripleAβγ), αβγR143A,ΔE144-K186 (αβγR/AΔ), αR205AβγR143A,ΔE144-K186 (αR205AβγR/AΔ), or αR205A,R208A,R231AβγR143A,ΔE144-K186 (αRtripleAβγR/AΔ) channels. Statistically significant differences were observed between oocytes expressing αβγ versus αβγR/AΔ (p < 0.001), αRtripleAβγ (p < 0.05), αRtripleAβγR/AΔ (p < 0.01) and αR205Aβγ channels (p < 0.05). Amiloride-sensitive currents were also statistically different between oocytes expressing αR205AβγR/AΔ versus αRtripleAβγR/AΔ (p < 0.05) and αβγR/AΔ (p < 0.01, One-way ANOVA followed by Student-Newman-Keuls multiple comparisons test). Amiloride-sensitive currents were not statistically different between oocytes expressing αβγR/AΔ and αRtripleAβγR/AΔ channels. Experiments were performed with 20 oocytes for each group. The presence or absense of an inhibitory domain is indicated by (+) or (–), respectively.

We previously demonstrated that wild type ENaCs expressed in oocytes are inhibited by a synthetic peptide (α-26) with the sequence of the α subunit inhibitory tract (D206-R231) in the low micromolar range (9). However, the peptide was a relatively poor blocker of transepithelial Na⁺ transport in cortical collecting duct cells (mpkCCD_c14) and human airway cells (IC₅₀ ∼50–100 μm), and of whole cell Na⁺ currents in oocytes co-expressing ENaC and prostasin (IC₅₀ > 50 μm) (9). These data suggest that release of the γ subunit inhibitory tract may affect the interaction of the α inhibitory peptide with the channel. We therefore examined whether the α-26 peptide was an effective inhibitor of channels lacking the γ subunit inhibitory tract. While whole cell currents in oocytes expressing wild type αβγ were inhibited by 1 and 10 μm α-26 in a dose-dependent manner, only a small inhibition of current was observed in oocytes expressing αβγR/AΔ, which lacked the furin cleavage site and the inhibitory tract in the γ subunit (Fig. 7).

FIGURE 7.

Deletion of the γ inhibitory tract E144-K186 reduces the efficacy of the α-26 mer peptide to block ENaC. Amiloride-sensitive currents were recorded in oocytes expressing wild type (αβγ) or αβγR143A,ΔE144-K186 (αβγR/AΔ) channels before and following 3 min of perfusion with the α-26 mer peptide. A reduction in amiloride-sensitive currents was observed in oocytes expressing αβγ channels after treatment with the α-26 inhibitory peptide at a concentration of 1 and 10 μm (p < 0.001). Amiloride-sensitive currents were significantly reduced in oocytes expressing αβγR/AΔ channels after treatment with the α-26 inhibitory peptide at a concentration of 10 μm (p < 0.01, Repeated measures ANOVA followed by Student-Newman-Keuls multiple comparisons test). Experiments were performed with 10–11 oocytes for each group.

DISCUSSION

We previously reported that both the α and γ subunits of ENaC contain inhibitory tracts that can be liberated by proteolysis. The release of the α subunit inhibitory tract led to partial channel activation, whereas the release of both inhibitory tracts resulted in channels with a very high open probability (9, 14). We now report that channels lacking furin cleavage of the α subunit are still near fully activated by co-expression with prostasin. Furthermore, we observed a significantly increased activity of channels with a retained α subunit inhibitory tract and deleted γ subunit inhibitory tract E144-K186. These observations suggest that the release or removal of the inhibitory tract in the γ subunit, even in presence of the α inhibitory tract, results in nearly full activation of the channel.

Our results suggest that pools of channels that have not been proteolytically processed, or channels that have been cleaved by furin and have lost their α subunit inhibitory tract will be fully activated by release of the γ subunit inhibitory tract. For channels with γ subunits that have been processed by furin, cleavage of the channel within the vicinity of the prostasin cleavage site should be sufficient to fully activate the channel. Several proteases have been reported to cleave the γ subunit in the region near the prostasin cleavage site. ENaC activation by the proteases neutrophil elastase, prostasin and kallikrein is associated with proteolytic processing of the γ subunit at sites located distal to the previously identified furin cleavage site at RKRR¹⁴³ (11–14). These proteases likely share a common mechanism of activation of ENaC by releasing the γ subunit inhibitory tract.

The atomic structure of cASIC1, an ENaC related family member, was recently resolved at 1.9 Å (5). The extracellular domain of the channel is organized in several subdomains termed the thumb, the finger, the β-ball, the knuckle, and the palm. The α and γ subunit inhibitory domains align within the finger domain of cASIC1, a region that is poorly conserved among members of the ENaC/degenerin family. The finger domain is exposed to the extracellular milieu, which is consistent with cleavage of the α and γ subunits by multiple proteases within this region. It is likely that conformational changes are translated from the α or γ “finger domains” to the pore of the channel. Despite the fact that proteolytic removal of inhibitory domains from the α and γ subunits constitute a key mechanism to modulate ENaC gating, the molecular rearrangements associated with this process remain poorly understood.

Our finding that currents from oocytes expressing α and γ subunits lacking their respective inhibitory tracts were significantly larger when the α subunits were cleaved was unexpected. Perhaps the absence of the tract D206-R231 in α subunits lacking furin cleavage imposes a constraint to the channel that prevents full activation, despite removal of the γ subunit inhibitory tract. Given that ENaC is likely a heterotrimer, interactions between α and γ subunits are expected to occur. It is possible that a constraint introduced by mutating the furin sites and deleting the intervening region in the α subunit affects these intersubunit interactions.

Furthermore, the activity of channels lacking the γ subunit inhibitory tract while retaining the α subunit inhibitory tract is dependent on whether the α subunit is cleaved (Fig. 6). These data suggest that, in the setting of an active channel where the γ subunit inhibitory domain has been removed, the efficacy of the α subunit inhibitory domain is enhanced when this domain is provided an increased degree of flexibility. As conformational changes are likely transmitted from the finger region downstream to the pore of the channel, it is not surprising that structural changes introduced in the finger domain of the α subunit affect channel gating behavior initiated in the finger region of the γ subunit.

We previously reported that a synthetic peptide derived from the α subunit (tract D206-R231) reversibly inhibited wild type channels expressed in oocytes with an IC₅₀ of 2.8 μm (9). However, peptide efficacy was significantly reduced in cell lines expressing endogenous ENaCs, and in oocytes co-expressing ENaC and prostasin (9). The activity of channels lacking the γ subunit inhibitory tract is, at best, moderately influenced by proteolytic processing of the α subunit. This suggests that the α inhibitory tract (D206-R231), when present within the α subunit, is unable to efficiently block channels that lack the γ subunit inhibitory tract. In agreement with this finding, we also observed a reduced efficacy of the α-26 mer peptide to inhibit channels that lacked the γ subunit inhibitory tract (Fig. 7).

In conclusion, our work indicates that the contributions of inhibitory domains within the α and γ subunits in the modulation of channel gating are not equal. Removal of the γ subunit inhibitory tract results in nearly full activation of the channel in the absence of α subunit furin processing.