Shaohu Sheng

Epithelial Na+ channels are fully activated by furin- and prostasin-dependent release of an inhibitory peptide from the gamma-subunit.

Epithelial sodium channels (ENaC) are expressed in the apical membrane of high resistance Na+ transporting epithelia and have a key role in regulating extracellular fluid volume and the volume of airway surface liquids. Maturation and activation of ENaC subunits involves furin-dependent cleavage of the ectodomain at two sites in the α subunit and at a single site within the γ subunit. We now report that the serine protease prostasin further activates ENaC by inducing cleavage of the γ subunit at a site distal to the furin cleavage site. Dual cleavage of the γ subunit is predicted to release a 43-amino acid peptide. Channels with a γ subunit lacking this 43-residue tract have increased activity due to a high open probability. A synthetic peptide corresponding to the fragment cleaved from the γ subunit is a reversible inhibitor of endogenous ENaCs in mouse cortical-collecting duct cells and in primary cultures of human airway epithelial cells. Our results suggest that multiple proteases cleave ENaC γ subunits to fully activate the channel.

The epithelial Na+ channel is inhibited by a peptide derived from proteolytic processing of its alpha subunit.

Epithelial sodium channels (ENaCs) mediate Na+ entry across the apical membrane of high resistance epithelia that line the distal nephron, airway and alveoli, and distal colon. These channels are composed of three homologous subunits, termed α, β, and γ, which have intracellular amino and carboxyl termini and two membrane-spanning domains connected by large extracellular loops. Maturation of ENaC subunits involves furin-dependent cleavage of the extracellular loops at two sites within the α subunit and at a single site within the γ subunit. The α subunits must be cleaved twice, immediately following Arg-205 and Arg-231, in order for channels to be fully active. Channels lacking α subunit cleavage are inactive with a very low open probability. In contrast, channels lacking both α subunit cleavage and the tract αAsp-206-Arg-231 are active when expressed in oocytes, suggesting that αAsp-206-Arg-231 functions as an inhibitor that stabilizes the channel in the closed conformation. A synthetic 26-mer peptide (α-26), corresponding to αAsp-206-Arg-231, reversibly inhibits wild-type mouse ENaCs expressed in Xenopus oocytes, as well as endogenous Na+ channels expressed in either a mouse collecting duct cell line or primary cultures of human airway epithelial cells. The IC50 for amiloride block of ENaC was not affected by the presence of α-26, indicating that α-26 does not bind to or interact with the amiloride binding site. Substitution of Arg residues within α-26 with Glu, or substitution of Pro residues with Ala, significantly reduced the efficacy of α-26. The peptide inhibits ENaC by reducing channel open probability. Our results suggest that proteolysis of the α subunit activates ENaC by disassociating an inhibitory domain (αAsp-206-Arg-231) from its effector site within the channel complex.

External Nickel Inhibits Epithelial Sodium Channel by Binding to Histidine Residues within the Extracellular Domains of α and γ Subunits and Reducing Channel Open Probability

Epithelial sodium channels (ENaC) are regulated by various intracellular and extracellular factors including divalent cations. We studied the inhibitory effect and mechanism of external Ni2+ on cloned mouse α-β-γ ENaC expressed inXenopus oocytes. Ni2+ reduced amiloride-sensitive Na+ currents of the wild type mouse ENaC in a dose-dependent manner. The Ni2+ block was fast and partially reversible at low concentrations and irreversible at high concentrations. ENaC inhibition by Ni2+ was accompanied by moderate inward rectification at concentrations higher than 0.1 mm. ENaC currents were also blocked by the histidine-reactive reagent diethyl pyrocarbonate. Pretreatment of the oocytes with the reagent reduced Ni2+inhibition of the remaining current. Mutations at αHis282and γHis239 located within the extracellular loops significantly decreased Ni2+ inhibition of ENaC currents. The mutation αH282D or double mutations αH282R/γH239R eliminated Ni2+ block. All mutations at γHis239eliminated Ni2+-induced inward current rectification. Ni2+ block was significantly enhanced by introduction of a histidine at αArg280. Lowering extracellular pH to 5.5 and 4.4 decreased or eliminated Ni2+ block. Although αH282C-β-γ channels were partially inhibited by the sulfhydryl-reactive reagent [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), α-β-γ H239C channels were insensitive to MTSET. From patch clamp studies, Ni2+ did not affect unitary current but decreased open probability when perfused into the recording pipette. Our results suggest that external Ni2+ reduces ENaC open probability by binding to a site consisting of αHis282 and γHis239 and that these histidine residues may participate in ENaC gating.

Functional Role of Extracellular Loop Cysteine Residues of the Epithelial Na+ Channel in Na+ Self-inhibition

The epithelial Na+ channel (ENaC) is typically formed by three homologous subunits (α, β, and γ) that possess a characteristic large extracellular loop (ECL) containing 16 conserved cysteine (Cys) residues. We investigated the functional role of these Cys residues in Na+ self-inhibition, an allosteric inhibition of ENaC activity by extracellular Na+. All 16 Cys residues within α and γ ECLs and selected β ECL Cys residues were individually mutated to alanine or serine residues. The Na+ self-inhibition response of wild type and mutant channels expressed in Xenopus oocytes was determined by whole cell voltage clamp. Individual mutation of eight α (Cys-1, -4, -5, -6, -7, -10, -13, or -16), one β (Cys-7), and nine γ (Cys-3, -4, -6, -7, -10, -11, -12, -13, or -16) residues significantly reduced the magnitude of Na+ self-inhibition. Na+ self-inhibition was eliminated by simultaneous mutations of either the last three α ECL Cys residues (Cys-14, -15, and -16) or Cys-7 within both α and γ ECLs. By analyzing the Na+ self-inhibition responses and the effects of a methanethiosulfonate reagent on channel currents in single and double Cys mutants, we identified five Cys pairs within the αECL (αCys-1/αCys-6, αCys-4/αCys-5, αCys-7/αCys-16, αCys-10/αCys-13, and αCys-11/αCys-12) and one pair within the γECL (γCys-7/γCys-16) that likely form intrasubunit disulfide bonds. We conclude that approximately half of the ECL Cys residues in the α and γ ENaC subunits are required to establish the tertiary structure that ensures a proper Na+ self-inhibition response, likely by formation of multiple intrasubunit disulfide bonds.

Novel determinants of epithelial sodium channel gating within extracellular thumb domains

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 γMet438 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 α subunit (G481A, G481C, and G481M) dramatically increased the magnitude and speed of Na+ self-inhibition. Mutations at the homologous βAla422 resulted in minimal or no change in Na+ self-inhibition. Low, high, and intermediate open probabilities were observed in oocytes expressing αG481Mβγ, αβγM438V, and αG481M/βγM438V, respectively. This pair of residues map to theα5 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 αG481Cβγ and αβγM438C channels were inhibited by an externally applied and membrane-impermeant sulfhydryl reagent. Our results demonstrate that αGly481 and γMet438 are functional determinants of Na+ self-inhibition and of ENaC gating and suggest that the thumb domain contributes to the channel gating machinery.

Base of the thumb domain modulates epithelial sodium channel gating

The activity of the epithelial sodium channel (ENaC) is modulated by multiple external factors, including proteases, cations, anions and shear stress. The resolved crystal structure of acid-sensing ion channel 1 (ASIC1), a structurally related ion channel, and mutagenesis studies suggest that the large extracellular region is involved in recognizing external signals that regulate channel gating. The thumb domain in the extracellular region of ASIC1 has a cylinder-like structure with a loop at its base that is in proximity to the tract connecting the extracellular region to the transmembrane domains. This loop has been proposed to have a role in transmitting proton-induced conformational changes within the extracellular region to the gate. We examined whether loops at the base of the thumb domains within ENaC subunits have a similar role in transmitting conformational changes induced by external Na+ and shear stress. Mutations at selected sites within this loop in each of the subunits altered channel responses to both external Na+ and shear stress. The most robust changes were observed at the site adjacent to a conserved Tyr residue. In the context of channels that have a low open probability due to retention of an inhibitory tract, mutations in the loop activated channels in a subunit-specific manner. Our data suggest that this loop has a role in modulating channel gating in response to external stimuli, and are consistent with the hypothesis that external signals trigger movements within the extracellular regions of ENaC subunits that are transmitted to the channel gate.

Extracellular Allosteric Regulatory Subdomain within the γ Subunit of the Epithelial Na+ Channel

The activity of the epithelial Na+ channel (ENaC) is modulated by Na+ self-inhibition, a down-regulation of the open probability of ENaC by extracellular Na+. A His residue within the extracellular domain of γENaC (γHis239) was found to have a critical role in Na+ self-inhibition. We investigated the functional roles of residues in the vicinity of this His by mutagenesis and analyses of Na+ self-inhibition responses in Xenopus oocytes. Significant changes in the speed and magnitude of Na+ self-inhibition were observed in 16 of the 47 mutants analyzed. These 16 mutants were distributed within a 22-residue tract. We further characterized this scanned region by examining the accessibility of introduced Cys residues to the sulfhydryl reagent MTSET. External MTSET irreversibly increased or decreased currents in 13 of 47 mutants. The distribution patterns of the residues where substitutions significantly altered Na+ self-inhibition or/and conferred sensitivity to MTSET were consistent with the existence of two helices within this region. In addition, single channel recordings of the γH239F mutant showed that, in the absence of Na+ self-inhibition and with an increased open probability, ENaCs still undergo transitions between open and closed states. We conclude that γHis239 functions within an extracellular allosteric regulatory subdomain of the γ subunit that has an important role in conferring the response of the channel to external Na+.

External Cu2+ Inhibits Human Epithelial Na+ Channels by Binding at a Subunit Interface of Extracellular Domains

Epithelial Na+ channels (ENaCs) play an essential role in the regulation of body fluid homeostasis. Certain transition metals activate or inhibit the activity of ENaCs. In this study, we examined the effect of extracellular Cu2+ on human ENaC expressed in Xenopus oocytes and investigated the structural basis for its effects. External Cu2+ inhibited human αβγ ENaC with an estimated IC50 of 0.3 μm. The slow time course and a lack of change in the current-voltage relationship were consistent with an allosteric (non pore-plugging) inhibition of human ENaC by Cu2+. Experiments with mixed human and mouse ENaC subunits suggested that both the α and β subunits were primarily responsible for the inhibitory effect of Cu2+ on human ENaC. Lowering bath solution pH diminished the inhibition by Cu2+. Mutations of two α, two β, and two γ His residues within extracellular domains significantly reduced the inhibition of human ENaC by Cu2+. We identified a pair of residues as potential Cu2+-binding sites at the subunit interface between thumb subdomain of αhENaC and palm subdomain of βhENaC, suggesting a counterclockwise arrangement of α, β, and γ ENaC subunits in a trimeric channel complex when viewed from above. We conclude that extracellular Cu2+ is a potent inhibitor of human ENaC and binds to multiple sites within the extracellular domains including a subunit interface.

Gain-of-function variant of the human epithelial sodium channel

Epithelial Na(+) channel (ENaC) mutations are associated with several human disorders, underscoring the importance of these channels in human health. Recent human genome sequencing projects have revealed a large number of ENaC gene variations, several of which have been found in individuals with salt-sensitive hypertension, cystic fibrosis, and other disorders. However, the functional consequences of most variants are unknown. In this study, we used the Xenopus oocyte expression system to examine the functional properties of a human ENaC variant. Oocytes expressing αβγL511Q human ENaCs showed 4.6-fold greater amiloride-sensitive currents than cells expressing wild-type channels. The γL511Q variant did not significantly alter channel surface expression. Single channel recordings revealed that the variant had fourfold higher open probability than wild type. In addition, γL511Q largely eliminated the Na(+) self-inhibition response, which reflects a downregulation of ENaC open probability by extracellular Na(+). Moreover, γL511Q diminished chymotrypsin-induced activation of the mutant channel. We conclude that γL511Q is a gain-of-function human ENaC variant. Our results suggest that γL511Q enhances ENaC activity by increasing channel open probability and dampens channel regulation by extracellular Na(+) and proteases.

Probing the Structural Basis of Zn2+ Regulation of the Epithelial Na+ Channel

Background: Extracellular Zn2+ regulates epithelial Na+ channel (ENaC) activity. Results: Specific mutations selectively weaken either the stimulatory or inhibitory effect of Zn2+. Conclusion: External Zn2+ regulates ENaC by interacting with multiple extracellular sites within the γ-subunit. Significance: This report provides novel insights into the structural basis of Zn2+ regulation of ENaC. Extracellular Zn2+ activates the epithelial Na+ channel (ENaC) by relieving Na+ self-inhibition. However, a biphasic Zn2+ dose response was observed, suggesting that Zn2+ has dual effects on the channel (i.e. activating and inhibitory). To investigate the structural basis for this biphasic effect of Zn2+, we examined the effects of mutating the 10 extracellular His residues of mouse γENaC. Four mutations within the finger subdomain (γH193A, γH200A, γH202A, and γH239A) significantly reduced the maximal Zn2+ activation of the channel. Whereas γH193A, γH200A, and γH202A reduced the apparent affinity of the Zn2+ activating site, γH239A diminished Na+ self-inhibition and thus concealed the activating effects of Zn2+. Mutation of a His residue within the palm subdomain (γH88A) abolished the low-affinity Zn2+ inhibitory effect. Based on structural homology with acid-sensing ion channel 1, γAsp516 was predicted to be in close proximity to γHis88. Ala substitution of the residue (γD516A) blunted the inhibitory effect of Zn2+. Our results suggest that external Zn2+ regulates ENaC activity by binding to multiple extracellular sites within the γ-subunit, including (i) a high-affinity stimulatory site within the finger subdomain involving His193, His200, and His202 and (ii) a low-affinity Zn2+ inhibitory site within the palm subdomain that includes His88 and Asp516.