Interacting domains in the epithelial sodium channel that mediate proteolytic activation.
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ABSTRACT:
SUBMITTER: Berman JM
PROVIDER: S-EPMC4826093 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The epithelial sodium channel (ENaC) is probably a heterotrimer with three well characterized subunits (alphabetagamma). In humans an additional delta-subunit (delta-hENaC) exists but little is known about its function. Using the Xenopus laevis oocyte expression system, we compared the functional properties of alphabetagamma- and deltabetagamma-hENaC and investigated whether deltabetagamma-hENaC can be proteolytically activated. The amiloride-sensitive ENaC whole-cell current (DeltaI(ami)) was about 11-fold larger in oocytes expressing deltabetagamma-hENaC than in oocytes expressing alphabetagamma-hENaC. The 2-fold larger single-channel Na(+) conductance of deltabetagamma-hENaC cannot explain this difference. Using a chemiluminescence assay, we demonstrated that an increased channel surface expression is also not the cause. Thus, overall channel activity of deltabetagamma-hENaC must be higher than that of alphabetagamma-hENaC. Experiments exploiting the properties of the known betaS520C mutant ENaC confirmed this conclusion. Moreover, chymotrypsin had a reduced stimulatory effect on deltabetagamma-hENaC whole-cell currents compared with its effect on alphabetagamma-hENaC whole-cell currents (2-fold versus 5-fold). This suggests that the cell surface pool of so-called near-silent channels that can be proteolytically activated is smaller for deltabetagamma-hENaC than for alphabetagamma-hENaC. Proteolytic activation of deltabetagamma-hENaC was associated with the appearance of a delta-hENaC cleavage product at the cell surface. Finally, we demonstrated that a short inhibitory 13-mer peptide corresponding to a region of the extracellular loop of human alpha-ENaC inhibited DeltaI(ami) in oocytes expressing alphabetagamma-hENaC but not in those expressing deltabetagamma-hENaC. We conclude that the delta-subunit of ENaC alters proteolytic channel activation and enhances base-line channel activity.
| S-EPMC2781449 | biostudies-literature
Project description:The epithelial Na(+) channel (ENaC) functions as a pathway for Na(+) absorption in the kidney and lung, where it is crucial for Na(+) homeostasis and blood pressure regulation. However, the basic mechanisms that control ENaC gating are poorly understood. Here we define a role in gating for residues forming interfaces between the extracellular domains of the three ENaC subunits. Using cysteine substitution combined with chemical cross-linking, we determined that residues located at equivalent positions in the three subunits (αK477, βE446, and γE455) form interfaces with residues in adjacent subunits (βV85, γV87, and αL120, respectively). Cross-linking of these residues altered ENaC activity in a length-dependent manner; long cross-linkers increased ENaC current by increasing its open probability, whereas short cross-linkers reduced ENaC open probability. Cross-linking also disrupted ENaC gating responses to extracellular pH and Na(+), signals which modulate ENaC activity during shifts in volume status. Introduction of charged side chains at the interfacing residues altered ENaC activity in a charge-dependent manner. Current increased when like charges were present at both interfacing residues, whereas opposing charges reduced current. Together, these data indicate that conformational changes at intersubunit interfaces participate in ENaC transitions between the open and closed states; movements that increase intersubunit distance favor the open state, whereas the closed state is favored when the distance is reduced. This provides a mechanism to modulate ENaC gating in response to changing extracellular conditions that threaten Na(+) homeostasis.
| S-EPMC4178938 | biostudies-literature
Project description:Proteolytic activation is a unique feature of the epithelial sodium channel (ENaC). However, the underlying molecular mechanisms and the physiologically relevant proteases remain to be identified. The serine protease trypsin I can activate ENaC in vitro but is unlikely to be the physiologically relevant activating protease in ENaC-expressing tissues in vivo. Herein, we investigated whether human trypsin IV, a form of trypsin that is co-expressed in several extrapancreatic epithelial cells with ENaC, can activate human ENaC. In Xenopus laevis oocytes, we monitored proteolytic activation of ENaC currents and the appearance of γENaC cleavage products at the cell surface. We demonstrated that trypsin IV and trypsin I can stimulate ENaC heterologously expressed in oocytes. ENaC cleavage and activation by trypsin IV but not by trypsin I required a critical cleavage site (Lys-189) in the extracellular domain of the γ-subunit. In contrast, channel activation by trypsin I was prevented by mutating three putative cleavage sites (Lys-168, Lys-170, and Arg-172) in addition to mutating previously described prostasin (RKRK(178)), plasmin (Lys-189), and neutrophil elastase (Val-182 and Val-193) sites. Moreover, we found that trypsin IV is expressed in human renal epithelial cells and can increase ENaC-mediated sodium transport in cultured human airway epithelial cells. Thus, trypsin IV may regulate ENaC function in epithelial tissues. Our results show, for the first time, that trypsin IV can stimulate ENaC and that trypsin IV and trypsin I activate ENaC by cleavage at distinct sites. The presence of distinct cleavage sites may be important for ENaC regulation by tissue-specific proteases.
| S-EPMC4081944 | biostudies-literature
Project description:Activity of the epithelial Na(+) channel (ENaC) is modulated by Na(+) self-inhibition, an allosteric down-regulation of channel open probability by extracellular Na(+). We searched for determinants of Na(+) self-inhibition by analyzing changes in this inhibitory response resulting from specific mutations within the extracellular domains of mouse ENaC subunits. Mutations at gammaMet(438) altered the Na(+) self-inhibition response in a substitution-specific manner. Fourteen substitutions (Ala, Arg, Asp, Cys, Gln, Glu, His, Ile, Phe, Pro, Ser, Thr, Tyr, and Val) significantly suppressed Na(+) self-inhibition, whereas three mutations (Asn, Gly, and Leu) moderately enhanced the inhibition. Met to Lys mutation did not alter Na(+) self-inhibition. Mutations at the homologous site in the alpha subunit (G481A, G481C, and G481M) dramatically increased the magnitude and speed of Na(+) self-inhibition. Mutations at the homologous betaAla(422) resulted in minimal or no change in Na(+) self-inhibition. Low, high, and intermediate open probabilities were observed in oocytes expressing alphaG481Mbetagamma, alphabetagammaM438V, and alphaG481M/betagammaM438V, respectively. This pair of residues map to thealpha5 helix in the extracellular thumb domain in the chicken acid sensing ion channel 1 structure. Both residues likely reside near the channel surface because both alphaG481Cbetagamma and alphabetagammaM438C channels were inhibited by an externally applied and membrane-impermeant sulfhydryl reagent. Our results demonstrate that alphaGly(481) and gammaMet(438) are functional determinants of Na(+) self-inhibition and of ENaC gating and suggest that the thumb domain contributes to the channel gating machinery.
| S-EPMC2658069 | biostudies-literature
Project description:The epithelial sodium channel (ENaC) mediates sodium absorption in lung, kidney, and colon epithelia. Channels in the ENaC/degenerin family possess an extracellular region that senses physicochemical changes in the extracellular milieu and allosterically regulates the channel opening. Proteolytic cleavage activates the ENaC opening, by the removal of specific segments in the finger domains of the ?- and ? ENaC-subunits. Cleavage causes perturbations in the extracellular region that propagate to the channel gate. However, it is not known how the channel structure mediates the propagation of activation signals through the extracellular sensing domains. Here, to identify the structure-function determinants that mediate allosteric ENaC activation, we performed MD simulations, thiol modification of residues substituted by cysteine, and voltage-clamp electrophysiology recordings. Our simulations of an ENaC heterotetramer, ?1??2?, in the proteolytically cleaved and uncleaved states revealed structural pathways in the ?-subunit that are responsible for ENaC proteolytic activation. To validate these findings, we performed site-directed mutagenesis to introduce cysteine substitutions in the extracellular domains of the ?-, ?-, and ? ENaC-subunits. Insertion of a cysteine at the ?-subunit Glu557 site, predicted to stabilize a closed state of ENaC, inhibited ENaC basal activity and retarded the kinetics of proteolytic activation by 2-fold. Our results suggest that the lower palm domain of ?ENaC is essential for ENaC activation. In conclusion, our integrated computational and experimental approach suggests key structure-function determinants for ENaC proteolytic activation and points toward a mechanistic model for the allosteric communication in the extracellular domains of the ENaC/degenerin family channels.
| S-EPMC5846138 | biostudies-literature
Project description:The molecular bases of heteromeric assembly and link between Na+ self-inhibition and protease-sensitivity in epithelial sodium channels (ENaCs) are not fully understood. Previously, we demonstrated that ENaC subunits - ?, ?, and ? - assemble in a counterclockwise configuration when viewed from outside the cell with the protease-sensitive GRIP domains in the periphery (Noreng et al., 2018). Here we describe the structure of ENaC resolved by cryo-electron microscopy at 3 Å. We find that a combination of precise domain arrangement and complementary hydrogen bonding network defines the subunit arrangement. Furthermore, we determined that the ? subunit has a primary functional module consisting of the finger and GRIP domains. The module is bifurcated by the ?2 helix dividing two distinct regulatory sites: Na+ and the inhibitory peptide. Removal of the inhibitory peptide perturbs the Na+ site via the ?2 helix highlighting the critical role of the ?2 helix in regulating ENaC function.
| S-EPMC7413742 | biostudies-literature
Project description:The voltage-gated sodium channel (VGSC) interacting peptide is of special interest for both basic research and pharmaceutical purposes. In this study, we established a yeast-two-hybrid based strategy to detect the interaction(s) between neurotoxic peptide and the extracellular region of VGSC. Using a previously reported neurotoxin JZTX-III as a model molecule, we demonstrated that the interactions between JZTX-III and the extracellular regions of its target hNav1.5 are detectable and the detected interactions are directly related to its activity. We further applied this strategy to the screening of VGSC interacting peptides. Using the extracellular region of hNav1.5 as the bait, we identified a novel sodium channel inhibitor SSCM-1 from a random peptide library. This peptide selectively inhibits hNav1.5 currents in the whole-cell patch clamp assays. This strategy might be used for the large scale screening for target-specific interacting peptides of VGSCs or other ion channels.
| S-EPMC3972499 | biostudies-literature
Project description:RATIONALE:Alveolar liquid clearance is regulated by Na(+) uptake through the apically expressed epithelial sodium channel (ENaC) and basolaterally localized Na(+)-K(+)-ATPase in type II alveolar epithelial cells. Dysfunction of these Na(+) transporters during pulmonary inflammation can contribute to pulmonary edema. OBJECTIVES:In this study, we sought to determine the precise mechanism by which the TIP peptide, mimicking the lectin-like domain of tumor necrosis factor (TNF), stimulates Na(+) uptake in a homologous cell system in the presence or absence of the bacterial toxin pneumolysin (PLY). METHODS:We used a combined biochemical, electrophysiological, and molecular biological in vitro approach and assessed the physiological relevance of the lectin-like domain of TNF in alveolar liquid clearance in vivo by generating triple-mutant TNF knock-in mice that express a mutant TNF with deficient Na(+) uptake stimulatory activity. MEASUREMENTS AND MAIN RESULTS:TIP peptide directly activates ENaC, but not the Na(+)-K(+)-ATPase, upon binding to the carboxy-terminal domain of the ? subunit of the channel. In the presence of PLY, a mediator of pneumococcal-induced pulmonary edema, this binding stabilizes the ENaC-PIP2-MARCKS complex, which is necessary for the open probability conformation of the channel and preserves ENaC-? protein expression, by means of blunting the protein kinase C-? pathway. Triple-mutant TNF knock-in mice are more prone than wild-type mice to develop edema with low-dose intratracheal PLY, correlating with reduced pulmonary ENaC-? subunit expression. CONCLUSIONS:These results demonstrate a novel TNF-mediated mechanism of direct ENaC activation and indicate a physiological role for the lectin-like domain of TNF in the resolution of alveolar edema during inflammation.
| S-EPMC4214088 | biostudies-literature
Project description:Limited proteolysis, accomplished by endopeptidases, is a ubiquitous phenomenon underlying the regulation and activation of many enzymes, receptors, and other proteins synthesized as inactive precursors. Serine proteases make up one of the largest and most conserved families of endopeptidases involved in diverse cellular activities, including wound healing, blood coagulation, and immune responses. Heteromeric α,β,γ-epithelial sodium channels (ENaC) associated with diseases like cystic fibrosis and Liddle's syndrome are irreversibly stimulated by membrane-anchored proteases (MAPs) and furin-like convertases. Matriptase/channel activating protease-3 (CAP3) is one of the several MAPs that potently activate ENaC. Despite identification of protease cleavage sites, the basis for the enhanced susceptibility of α- and γ-ENaC to proteases remains elusive. Here, we elucidate the energetic and structural bases for activation of ENaC by CAP3. We find a region near the γ-ENaC furin site that has previously not been identified as a critical cleavage site for CAP3-mediated stimulation. We also report that CAP3 mediates cleavage of ENaC at basic residues downstream of the furin site. Our results indicate that surface proteases alone are sufficient to fully activate uncleaved ENaC and explain how ENaC in epithelia expressing surface-active proteases can appear refractory to soluble proteases. Our results support a model in which proteases prime ENaC for activation by cleaving at the furin site, and cleavage at downstream sites is accomplished by membrane surface proteases or extracellular soluble proteases. On the basis of our results, we propose a dynamics-driven "anglerfish" mechanism that explains less stringent sequence requirements for substrate recognition and cleavage by matriptase than by furin.
| S-EPMC3404201 | biostudies-literature
Project description:The epithelial sodium channel (ENaC) is activated upon endoproteolytic cleavage of specific segments in the extracellular domains of the ?- and ?-subunits. Cleavage is accomplished by intracellular proteases prior to membrane insertion and by surface-expressed or extracellular soluble proteases once ENaC resides at the cell surface. These cleavage events are partially regulated by intracellular signaling through an unknown allosteric mechanism. Here, using a combination of computational and experimental techniques, we show that the intracellular N terminus of ?-ENaC undergoes secondary structural transitions upon interaction with phosphoinositides. From ab initio folding simulations of the N termini in the presence and absence of phosphatidylinositol 4,5-bisphosphate (PIP2), we found that PIP2 increases ?-helical propensity in the N terminus of ?-ENaC. Electrophysiology and mutation experiments revealed that a highly conserved cluster of lysines in the ?-ENaC N terminus regulates accessibility of extracellular cleavage sites in ?-ENaC. We also show that conditions that decrease PIP2 or enhance ubiquitination sharply limit access of the ?-ENaC extracellular domain to proteases. Further, the efficiency of allosteric control of ENaC proteolysis is dependent on Tyr(370) in ?-ENaC. Our findings provide an allosteric mechanism for ENaC activation regulated by the N termini and sheds light on a potential general mechanism of channel and receptor activation.
| S-EPMC4132802 | biostudies-literature