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Acid-sensing ion channels (ASICs) are proton-gated cation-selective channels expressed in the peripheral and central nervous systems. The ion permeation pathway of ASIC1a is defined by residues 426-450 in the second transmembrane (TM2) segment. The gate, formed by the intersection of the TM2 segments, localizes near the extracellular boundary of the plasma membrane. We explored the contribution to ion permeation and selectivity of residues in the TM2 segment of ASIC1a. Studies of accessibility with positively charged methanethiosulfonate reagents suggest that the permeation pathway in the open state constricts below the gate, restricting the passage to large ions. Substitution of residues in the intracellular vestibule at positions 437, 438, 443, or 446 significantly increased the permeability to K(+) versus Na(+). ASIC1a shows a selectivity sequence for alkali metals of Na(+)>Li(+)>K(+)≫Rb(+)>Cs(+). Alanine and cysteine substitutions at position 438 increased, to different extents, the relative permeability to Li(+), K(+), Rb(+), and Cs(+). For these mutants, ion permeation was not a function of the diameter of the nonhydrated ion, suggesting that Gly-438 encompasses an ion coordination site that is essential for ion selectivity. M437C and A443C mutants showed slightly increased permeability to K(+), Rb(+), and Cs(+), suggesting that substitutions at these positions influence ion discrimination by altering molecular sieving. Our results indicate that ion selectivity is accomplished by the contribution of multiple sites in the pore of ASIC1a.

Purpose of Review: The epithelial sodium channel/degenerin family encompasses a group of cation-selective ion channels that are activated or modulated by a variety of extracellular stimuli. This review describes findings that provide new insights into the molecular mechanisms that control the function of these channels., Recent Findings: Epithelial sodium channels facilitate Na⁺ reabsorption in the distal nephron and hence have a role in fluid volume homeostasis and arterial blood pressure regulation. Acid-sensing ion channels are broadly distributed in the nervous system where they contribute to the sensory processes. The atomic structure of acid-sensing ion channel 1 illustrates the complex trimeric architecture of these proteins. Each subunit has two transmembrane spanning helices, a highly organized ectodomain and intracellular N-terminus and C-terminus. Recent findings have begun to elucidate the structural elements that allow these channels to sense and respond to extracellular factors. This review emphasizes the roles of the extracellular domain in sensing changes in the extracellular milieu and of the residues in the extracellular-transmembrane domains interface in coupling extracellular changes to the pore of the channel., Summary: Epithelial sodium channels and acid-sensing ion channels have evolved to sense extracellular cues. Future research should be directed toward elucidating how changes triggered by extracellular factors translate into pore opening and closing events.

Acid-sensing ion channels (ASICs) are trimeric cation channels that undergo activation and desensitization in response to extracellular acidification. The underlying mechanism coupling proton binding in the extracellular region to pore gating is unknown. Here we probed the reactivity toward methanethiosulfonate (MTS) reagents of channels with cysteine-substituted residues in the outer vestibule of the pore of ASIC1a. We found that positively-charged MTS reagents trigger pore opening of G428C. Scanning mutagenesis of residues in the region preceding the second transmembrane spanning domain indicated that the MTSET-modified side chain of Cys at position 428 interacts with Tyr-424. This interaction was confirmed by double-mutant cycle analysis. Strikingly, Y424C-G428C monomers were associated by intersubunit disulfide bonds and were insensitive to MTSET. Despite the spatial constraints introduced by these intersubunit disulfide bonds in the outer vestibule of the pore, Y424C-G428C transitions between the resting, open, and desensitized states in response to extracellular acidification. This finding suggests that the opening of the ion conductive pathway involves coordinated rotation of the second transmembrane-spanning domains.

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.

Acid-sensing ion channels are proton-gated Na(+) channels expressed predominantly in neurons. How channel structure translates an environmental stimulus into changes in pore permeability remains largely undefined. The pore of ASIC1 is defined by residues in the second transmembrane domain (TM2), although a segment of the outer vestibule is formed by residues of TM1. We used the voltage clamp fluorometry technique to define the role of the region preceding TM2 (pre-TM2) in activation and desensitization of mouse ASIC1a. Oocytes expressing E425C channels labeled with Alexa Fluor 488 C5-maleimide showed a change in the emission of the fluorescent probe in response to extracellular acidification. The time course of the change in fluorescence correlated with activation but not desensitization of E425C channels. The fluorescence emission did not change following extracellular acidification in oocytes carrying an inactivating mutation (W287G/E425C), although these channels were labeled and expressed at the plasma membrane. Our data indicate that pore opening occurs in conjunction with a conformational rearrangement of the pre-TM2. We observed a change in the emission of the fluorescent probe when labeled E425C channels transition from the desensitized to the resting state. The substituted-cysteine-accessibility method was used to determine whether the pre-TM2 has different conformations in the resting and desensitized states. State-dependent changes in accessibility to 2-[(trimethylammonium)ethyl]methanethiosulfonate bromide modification were observed in oocytes expressing K421C, K422C, Y424C, and E425C channels. Our results suggest that the pre-TM2 of ASIC1a undergoes dynamic conformational rearrangements during proton-dependent gating.

Purpose of Review: Epithelial sodium channels mediate Na+ transport across high resistance, Na+-transporting epithelia. This review describes recent findings that indicate that epithelial sodium channels are activated by the proteolytic release of inhibitory peptides from the alpha and gamma subunits., Recent Findings: Non-cleaved channels have a low intrinsic open probability that may reflect enhanced channel inhibition by external Na+--a process referred to as Na+ self-inhibition. Cleavage at a minimum of two sites within the alpha or gamma subunits is required to activate the channel, presumably by releasing inhibitory fragments. The extent of epithelial sodium channel proteolysis is dependent on channel residency time at the plasma membrane, as well as on the balance between levels of expression of proteases that activate epithelial sodium channels and inhibitors of these proteases. Regulated epithelial sodium channel proteolysis has been observed in rat kidney and in human airway epithelia., Summary: Proteolysis of epithelial sodium channel subunits plays a key role in modulating epithelial sodium channel activity through changes in channel open probability.

Vectorial Na(+) absorption across the aldosterone-sensitive distal nephron plays a key role in the regulation of extracellular fluid volume and blood pressure. Within this nephron segment, Na(+) diffuses from the urinary fluid into principal cells through an apical, amiloride-sensitive, epithelial Na(+) channel (ENaC), which is considered to be the rate-limiting step for Na(+) absorption. We have reported that increases in tubular flow rate in microperfused rabbit cortical collecting ducts (CCDs) lead to increases in net Na(+) absorption and that increases in laminar shear stress activate ENaC expressed in oocytes by increasing channel open probability. We therefore examined whether flow stimulates net Na(+) absorption (J(Na)) in CCDs by increasing channel open probability or by increasing the number of channels at the apical membrane. Both baseline and flow-stimulated J(Na) in CCDs were mediated by ENaC, as J(Na) was inhibited by benzamil. Flow-dependent increases in J(Na) were observed following treatment of tubules with reagents that altered membrane trafficking by disrupting microtubules (colchicine) or Golgi (brefeldin A). Furthermore, reducing luminal Ca(2+) concentration ([Ca(2+)]) or chelating intracellular [Ca(2+)] with BAPTA did not prevent the flow-dependent increase in J(Na). Extracellular trypsin has been shown to activate ENaC by increasing channel open probability, and we observed that trypsin significantly enhanced J(Na) when tubules were perfused at a slow flow rate. However, trypsin did not further enhance J(Na) in CCDs perfused at fast flow rates. Similarly, the shear-induced increase in benzamil-sensitive J(Na) in oocytes expressing protease resistance ENaC mutants was similar to that of controls. Our results suggest the rise in J(Na) accompanying increases in luminal flow rates reflects an increase in channel open probability.

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 alpha, beta, and gamma, 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 alpha subunit and at a single site within the gamma subunit. The alpha subunits must be cleaved twice, immediately following Arg-205 and Arg-231, in order for channels to be fully active. Channels lacking alpha subunit cleavage are inactive with a very low open probability. In contrast, channels lacking both alpha subunit cleavage and the tract alphaAsp-206-Arg-231 are active when expressed in oocytes, suggesting that alphaAsp-206-Arg-231 functions as an inhibitor that stabilizes the channel in the closed conformation. A synthetic 26-mer peptide (alpha-26), corresponding to alphaAsp-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 IC(50) for amiloride block of ENaC was not affected by the presence of alpha-26, indicating that alpha-26 does not bind to or interact with the amiloride binding site. Substitution of Arg residues within alpha-26 with Glu, or substitution of Pro residues with Ala, significantly reduced the efficacy of alpha-26. The peptide inhibits ENaC by reducing channel open probability. Our results suggest that proteolysis of the alpha subunit activates ENaC by disassociating an inhibitory domain (alphaAsp-206-Arg-231) from its effector site within the channel complex.

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 alpha, or one site in gamma, 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 alpha-subunits lacking either one or both furin cleavage sites exhibited a marked enhancement of the Na+ self-inhibition response, while channels with a mutant gamma-subunit showed a modestly enhanced Na+ self-inhibition response. Analysis of Na+ self-inhibition at varying [Na+] indicates that channels containing mutant alpha-subunits exhibit an increased Na+ affinity. At the single-channel level, channels with a mutant alpha-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 alpha-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+].

The epithelial Na(+) channel (ENaC) regulates epithelial salt and water reabsorption, processes that require significant expenditure of cellular energy. To test whether the ubiquitous metabolic sensor AMP-activated kinase (AMPK) regulates ENaC, we examined the effects of AMPK activation on amiloride-sensitive currents in Xenopus oocytes and polarized mouse collecting duct mpkCCD(c14) cells. Microinjection of oocytes expressing mouse ENaC (mENaC) with either active AMPK protein or an AMPK activator inhibited mENaC currents relative to controls as measured by two-electrode voltage-clamp studies. Similarly, pharmacological AMPK activation or overexpression of an activating AMPK mutant in mpkCCD(c14) cells inhibited amiloride-sensitive short circuit currents. Expression of a degenerin mutant beta-mENaC subunit (S518K) along with wild type alpha and gamma increased the channel open probability (P(o)) to approximately 1. However, AMPK activation inhibited currents similarly with expression of either degenerin mutant or wild type mENaC. Single channel recordings under these conditions demonstrated that neither P(o) nor channel conductance was affected by AMPK activation. Moreover, expression of a Liddle's syndrome-type beta-mENaC mutant (Y618A) greatly enhanced ENaC whole cell currents relative to wild type ENaC controls and prevented AMPK-dependent inhibition. These findings indicate that AMPK-dependent ENaC inhibition is mediated through a decrease in the number of active channels at the plasma membrane (N), presumably through enhanced Nedd4-2-dependent ENaC endocytosis. The AMPK-ENaC interaction appears to be indirect; AMPK did not bind ENaC in cells, as assessed by in vivo pull-down assays, nor did it phosphorylate ENaC in vitro. In summary, these results suggest a novel mechanism for coupling ENaC activity and renal Na(+) handling to cellular metabolic status through AMPK, which may help prevent cellular Na(+) loading under hypoxic or ischemic conditions.

Previous studies have shown that epithelial Na+ channels (ENaCs) are activated by laminar shear stress (LSS). ENaCs with a high intrinsic open probability because of a mutation (betaS518K) or covalent modification of an introduced Cys residue (alphaS580C) in the pre-second transmembrane domain (pre-M2) were not activated by LSS, suggesting that the pre-M2 region participates in conformational rearrangements during channel activation. We examined the role of the pore region of the alpha-subunit in channel gating by studying the kinetics of activation by LSS of wild-type ENaC and channels with Cys mutations in the tract Ser576-Ser592. Whole cell Na+ currents were monitored in oocytes expressing wild-type or mutant ENaCs prior to and following application of LSS. Following a 2.2-s delay, a monoexponential increase in Na+ currents was observed with a time constant (tau) of 8.1 s in oocytes expressing wild-type ENaC. Cys substitutions within the alpha-subunit in the tract Ser580-Ser589 resulted in: (i) a reduction (Ser580-Trp585, Gly587) or increase (Ser589) in delay times preceding channel activation by LSS, (ii) an increase (Gln581, Leu584, Trp585, Phe586, Ser588) or decrease (Ser589) in the rate of channel activation, or (iii) a decrease in the magnitude of the response (Ser583, Gly587, Leu584). Cys substitutions at a putative amiloride-binding site (alphaSer583 or betaGly525) or within the selectivity filter (alphaGly587) resulted in a reduction in the LSS response, and exhibited a multiexponential time course of activation. The corresponding gamma-subunit mutant (alphabetagammaG542C) had a minimal response to LSS and exhibited a high intrinsic open probability. These data suggest that residues in the pore region participate in the sensing and/or transduction of the mechanical stimulus that results in channel activation and are consistent with the hypothesis that the ENaC pore region has a key role in modulating channel gating.

Epithelial Na(+) channels (ENaCs) are activated by extracellular trypsin or by co-expression with channel-activating proteases, although there is no direct evidence that these proteases activate ENaC by cleaving the channel. We previously demonstrated that the alpha and gamma subunits of ENaC are cleaved during maturation near consensus sites for furin cleavage. Using site-specific mutagenesis of channel subunits, ENaC expression in furin-deficient cells, and furin-specific inhibitors, we now report that ENaC cleavage correlates with channel activity. Channel activity in furin-deficient cells was rescued by expression of furin. Our data provide the first example of a vertebrate ion channel that is a substrate for furin and whose activity is dependent on its proteolysis.

The degenerin/epithelial Na+ channel (ENaC) superfamily is a group of structurally related ion channels that are involved in diverse biological processes, including responses to mechanical stimuli. In renal cortical collecting ducts, changes in rates of perfusion affect Na+ reabsorption through an amiloride-sensitive pathway, suggesting that ENaC may be a mechanosensitive channel. In this study, we examined whether ENaC expressed in oocytes is regulated by laminar shear stress (LSS). A 1.8-mm (internal diameter) perfusion pipette was placed within 0.5-1.0 mm of the oocyte to provide laminar flow across the oocyte surface. LSS induced a dose-dependent and reversible increase in benzamil-sensitive whole cell Na+ currents in oocytes expressing alphabetagamma ENaC. The half-time for activation by LSS was approximately 5 s. Repetitive stimulation by LSS of oocytes expressing ENaC was associated with a reduction in the response to LSS. Oocytes expressing alphabetaS518Kgamma, a pore region mutant with a high open probability, were insensitive to LSS. We demonstrated previously that channels with a Cys residue introduced at position alphaSer-580 had a low open probability, but, following modification by [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET), channels exhibited a high open probability. Oocytes expressing alphaS580Cbetagamma ENaC respond to LSS similar to wild type; however, covalent modification by MTSET largely eliminated the response to LSS. Our results suggest that shear stress activates ENaC by modifying the gating properties of the channel.

The epithelial Na+ channel (ENaC) is a tetramer of two alpha-, one beta-, and one gamma-subunit, but little is known about its assembly and processing. Because co-expression of mouse ENaC subunits with three different carboxyl-terminal epitope tags produced an amiloride-sensitive sodium current in oocytes, these tagged subunits were expressed in both Chinese hamster ovary or Madin-Darby canine kidney type 1 epithelial cells for further study. When expressed alone alpha-(95 kDa), beta-(96 kDa), and gamma-subunits (93 kDa) each produced a single band on SDS gels by immunoblotting. However, co-expression of alphabetagammaENaC subunits revealed a second band for each subunit (65 kDa for alpha, 110 kDa for beta, and 75 kDa for gamma) that exhibited N-glycans that had been processed to complex type based on sensitivity to treatment with neuraminidase, resistance to cleavage by endoglycosidase H, and GalNAc-independent labeling with [3H]Gal in glycosylation-defective Chinese hamster ovary cells (ldlD). The smaller size of the processed alpha- and gamma-subunits is also consistent with proteolytic cleavage. By using alpha- and gamma-subunits with epitope tags at both the amino and carboxyl termini, proteolytic processing of the alpha- and gamma-subunits was confirmed by isolation of an additional epitope-tagged fragment from the amino terminus (30 kDa for alpha and 18 kDa for gamma) consistent with cleavage within the extracellular loop. The fragments remain stably associated with the channel as shown by immunoblotting of co-immunoprecipitates, suggesting that proteolytic cleavage represents maturation rather than degradation of the channel.

Epithelial Na+ channels (ENaCs) are regulated by the phospholipase A2 (PLA2) product arachidonic acid. Pharmacological inhibition of PLA2 with aristolochic acid induced a significant increase in amiloride-sensitive currents in Xenopus oocytes expressing ENaC. Arachidonic acid or 5,8,11,14-eicosatetraynoic acid (ETYA), a non-metabolized analog of arachidonic acid, induced a time-dependent inhibition of Na+ transport. These effects were also observed by co-expression of a calcium-independent or a calcium-dependent PLA2. Channels with a truncated alpha, beta,or gamma C terminus were not inhibited by arachidonic acid or ETYA. Furthermore, mutation of Tyr618 in the PY motif of the beta subunit abrogated the inhibitory effect of ETYA, suggesting that intact PY motifs participate in arachidonic acid-mediated ENaC inhibition. Analyses of channels expressing a series of beta subunit C-terminal truncations revealed a second region N-terminal to the PY motif (spanning residues betaVal580-betaGly599) that allowed for ETYA-mediated ENaC inhibition. Analyses of both ENaC surface expression and ENaC trafficking with mutants that either gate channels open or closed in response to [(2-(trimethylammonium) ethyl] methanethiosulfonate bromide, or with brefeldin A, suggest that ETYA reduces channel surface expression by inhibiting ENaC exocytosis and increasing ENaC endocytosis.

The epithelial sodium channel (ENaC) is a heterotrimeric protein responsible for Na(+) absorption across the apical membranes of several absorptive epithelia. The rate of Na(+) absorption is governed in part by regulated membrane trafficking mechanisms that control the apical membrane ENaC density. Previous reports have implicated a role for the t-SNARE protein, syntaxin 1A (S1A), in the regulation of ENaC current (I(Na)). In the present study, we examine the structure-function relations influencing S1A-ENaC interactions. In vitro pull-down assays demonstrated that S1A directly interacts with the C termini of the alpha-, beta-, and gamma-ENaC subunits but not with the N terminus of any ENaC subunit. The H3 domain of S1A is the critical motif mediating S1A-ENaC binding. Functional studies in ENaC expressing Xenopus oocytes revealed that deletion of the H3 domain of co-expressed S1A eliminated its inhibition of I(Na), and acute injection of a GST-H3 fusion protein into ENaC expressing oocytes inhibited I(Na) to the same extent as S1A co-expression. In cell surface ENaC labeling experiments, reductions in plasma membrane ENaC accounted for the H3 domain inhibition of I(Na). Individually substituting C terminus-truncated alpha-, beta-, or gamma-ENaC subunits for their wild-type counterparts reversed the S1A-induced inhibition of I(Na), and oocytes expressing ENaC comprised of three C terminus-truncated subunits showed no S1A inhibition of I(Na). C terminus truncation or disruption of the C terminus beta-subunit PY motif increases I(Na) by interfering with ENaC endocytosis. In contrast to subunit truncation, a beta-ENaC PY mutation did not relieve S1A inhibition of I(Na), suggesting that S1A does not perturb Nedd4 interactions that lead to ENaC endocytosis/degradation. This study provides support for the concept that S1A inhibits ENaC-mediated Na(+) transport by decreasing cell surface channel number via direct protein-protein interactions at the ENaC C termini.