RESUMO
The epithelial Na+ channel (ENaC) emerged early in vertebrates and has played a role in Na+ and fluid homeostasis throughout vertebrate evolution. We previously showed that proteolytic activation of the channel evolved at the water-to-land transition of vertebrates. Sensitivity to extracellular Na+, known as Na+ self-inhibition, reduces ENaC function when Na+ concentrations are high and is a distinctive feature of the channel. A fourth ENaC subunit, δ, emerged in jawed fishes from an α subunit gene duplication. Here, we analyzed 849 α and δ subunit sequences and found that a key Asp in a postulated Na+ binding site was nearly always present in the α subunit, but frequently lost in the δ subunit (e.g. human). Analysis of site evolution and codon substitution rates provide evidence that the ancestral α subunit had the site and that purifying selection for the site relaxed in the δ subunit after its divergence from the α subunit, coinciding with a loss of δ subunit expression in renal tissues. We also show that the proposed Na+ binding site in the α subunit is a bona fide site by conferring novel function to channels comprising human δ subunits. Together, our findings provide evidence that ENaC Na+ self-inhibition improves fitness through its role in Na+ homeostasis in vertebrates.
Assuntos
Canais Epiteliais de Sódio , Evolução Molecular , Homeostase , Seleção Genética , Sódio , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/metabolismo , Animais , Sódio/metabolismo , Humanos , Sítios de Ligação , Vertebrados/genética , Subunidades Proteicas/metabolismo , Subunidades Proteicas/genética , FilogeniaRESUMO
A key regulator of blood pressure homeostasis is the steroid hormone aldosterone, which is released as the final signaling hormone of the renin-angiotensin-aldosterone-signaling (RAAS) system. Aldosterone increases sodium (Na+) reabsorption in the kidney distal nephron to regulate blood volume. Unregulated RAAS signaling can lead to hypertension and cardiovascular disease. The serum and glucocorticoid kinase (SGK1) coordinates much of the Na+ reabsorption in the cortical collecting duct (CCD) tubular epithelial cells. We previously demonstrated that aldosterone alters the expression of microRNAs (miRs) in CCD principal cells. The aldosterone-regulated miRs can modulate Na+ transport and the cellular response to aldosterone signaling. However, the sex-specific regulation of miRs by aldosterone in the kidney distal nephron has not been explored. In this study, we report that miR-19, part of the miR-17-92 cluster, is upregulated in female mouse CCD cells in response to aldosterone activation. Mir-19 binding to the 3'-untranslated region of SGK1 was confirmed using a dual-luciferase reporter assay. Increasing miR-19 expression in CCD cells decreased SGK1 message and protein expression. Removal of this cluster using a nephron-specific, inducible knockout mouse model increased SGK1 expression in female mouse CCD cells. The miR-19-induced decrease in SGK1 protein expression reduced the response to aldosterone stimulation and may account for sex-specific differences in aldosterone signaling. By examining evolution of the miR-17-92 cluster, phylogenetic sequence analysis indicated that this cluster arose at the same time that other Na+-sparing and salt regulatory proteins, specifically SGK1, first emerged, indicating a conserved role for these miRs in kidney function of salt and water homeostasis.NEW & NOTEWORTHY Expression of the microRNA-17-92 cluster is upregulated by aldosterone in mouse cortical collecting duct principal cells, exclusively in female mice. MiR-19 in this cluster targets the serum and glucocorticoid kinase (SGK1) to downregulate both mRNA and protein expression, resulting in a decrease in sodium transport across epithelial cells of the collecting duct. The miR-17-92 cluster is evolutionarily conserved and may act as a novel feedback regulator for aldosterone signaling in females.
Assuntos
MicroRNAs , Feminino , Camundongos , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Aldosterona/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Glucocorticoides , Filogenia , Rim/metabolismo , Sódio/metabolismo , Canais Epiteliais de Sódio/metabolismoRESUMO
Epithelial Na+ channels (ENaCs) are activated by proteolysis of the α and γ subunits at specific sites flanking embedded inhibitory tracts. To examine the role of α subunit proteolysis in channel activation in vivo, we generated mice lacking the distal furin cleavage site in the α subunit (αF2M mice). On a normal Na+ control diet, no differences in ENaC protein abundance in kidney or distal colon were noted between wild-type (WT) and αF2M mice. Patch-clamp analyses revealed similar levels of ENaC activity in kidney tubules, while no physiologically relevant differences in blood chemistry or aldosterone levels were detected. Male αF2M mice did exhibit diminished ENaC activity in the distal colon, as measured by amiloride-sensitive short-circuit current (ISC). Following dietary Na+ restriction, WT and αF2M mice had similar natriuretic and colonic ISC responses to amiloride. However, single-channel activity was significantly lower in kidney tubules from Na+-restricted αF2M mice compared with WT littermates. ENaC α and γ subunit expression in kidney and distal colon were also enhanced in Na+-restricted αF2M vs. WT mice, in association with higher aldosterone levels. These data provide evidence that disrupting α subunit proteolysis impairs ENaC activity in vivo, requiring compensation in response to Na+ restriction. KEY POINTS: The epithelial Na+ channel (ENaC) is activated by proteolytic cleavage in vitro, but key questions regarding the role of ENaC proteolysis in terms of whole-animal physiology remain to be addressed. We studied the in vivo importance of this mechanism by generating a mouse model with a genetic disruption to a key cleavage site in the ENaC's α subunit (αF2M mice). We found that αF2M mice did not exhibit a physiologically relevant phenotype under normal dietary conditions, but have impaired ENaC activation (channel open probability) in the kidney during salt restriction. ENaC function at the organ level was preserved in salt-restricted αF2M mice, but this was associated with higher aldosterone levels and increased expression of ENaC subunits, suggesting compensation was required to maintain homeostasis. These results provide the first evidence that ENaC α subunit proteolysis is a key regulator of channel activity in vivo.
Assuntos
Canais Epiteliais de Sódio , Furina , Animais , Canais Epiteliais de Sódio/metabolismo , Canais Epiteliais de Sódio/genética , Camundongos , Masculino , Furina/metabolismo , Furina/genética , Sódio/metabolismo , Colo/metabolismo , Camundongos Endogâmicos C57BL , Aldosterona/metabolismo , Dieta HipossódicaRESUMO
Beyond glycemic control, SGLT2 inhibitors (SGLT2is) have protective effects on cardiorenal function. Renoprotection has been suggested to involve inhibition of NHE3 leading to reduced ATP-dependent tubular workload and mitochondrial oxygen consumption. NHE3 activity is also important for regulation of endosomal pH, but the effects of SGLT2i on endocytosis are unknown. We used a highly differentiated cell culture model of proximal tubule (PT) cells to determine the direct effects of SGLT2i on Na+-dependent fluid transport and endocytic uptake in this nephron segment. Strikingly, canagliflozin but not empagliflozin reduced fluid transport across cell monolayers and dramatically inhibited endocytic uptake of albumin. These effects were independent of glucose and occurred at clinically relevant concentrations of drug. Canagliflozin acutely inhibited surface NHE3 activity, consistent with a direct effect, but did not affect endosomal pH or NHE3 phosphorylation. In addition, canagliflozin rapidly and selectively inhibited mitochondrial complex I activity. Inhibition of mitochondrial complex I by metformin recapitulated the effects of canagliflozin on endocytosis and fluid transport, whereas modulation of downstream effectors AMPK and mTOR did not. Mice given a single dose of canagliflozin excreted twice as much urine over 24 h compared with empagliflozin-treated mice despite similar water intake. We conclude that canagliflozin selectively suppresses Na+-dependent fluid transport and albumin uptake in PT cells via direct inhibition of NHE3 and of mitochondrial function upstream of the AMPK/mTOR axis. These additional targets of canagliflozin contribute significantly to reduced PT Na+-dependent fluid transport in vivo.NEW & NOTEWORTHY Reduced NHE3-mediated Na+ transport has been suggested to underlie the cardiorenal protection provided by SGLT2 inhibitors. We found that canagliflozin, but not empagliflozin, reduced NHE3-dependent fluid transport and endocytic uptake in cultured proximal tubule cells. These effects were independent of SGLT2 activity and resulted from inhibition of mitochondrial complex I and NHE3. Studies in mice are consistent with greater effects of canagliflozin versus empagliflozin on fluid transport. Our data suggest that these selective effects of canagliflozin contribute to reduced Na+-dependent transport in proximal tubule cells.
Assuntos
Canagliflozina , Túbulos Renais Proximais , Inibidores do Transportador 2 de Sódio-Glicose , Trocador 3 de Sódio-Hidrogênio , Animais , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/enzimologia , Trocador 3 de Sódio-Hidrogênio/metabolismo , Canagliflozina/farmacologia , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Camundongos , Masculino , Transportador 2 de Glucose-Sódio/metabolismo , Endocitose/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Albuminas/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Compostos Benzidrílicos , GlucosídeosRESUMO
Transmembrane proteins have unique requirements to fold and integrate into the endoplasmic reticulum (ER) membrane. Most notably, transmembrane proteins must fold in three separate environments: extracellular domains fold in the oxidizing environment of the ER lumen, transmembrane domains (TMDs) fold within the lipid bilayer, and cytosolic domains fold in the reducing environment of the cytosol. Moreover, each region is acted upon by a unique set of chaperones and monitored by components of the ER associated quality control machinery that identify misfolded domains in each compartment. One factor is the ER lumenal Hsp70-like chaperone, Lhs1. Our previous work established that Lhs1 is required for the degradation of the unassembled α-subunit of the epithelial sodium channel (αENaC), but not the homologous ß- and γENaC subunits. However, assembly of the ENaC heterotrimer blocked the Lhs1-dependent ER associated degradation (ERAD) of the α-subunit, yet the characteristics that dictate the specificity of Lhs1-dependent ERAD substrates remained unclear. We now report that Lhs1-dependent substrates share a unique set of features. First, all Lhs1 substrates appear to be unglycosylated, and second they contain two TMDs. Each substrate also contains orphaned or unassembled TMDs. Additionally, interfering with inter-subunit assembly of the ENaC trimer results in Lhs1-dependent degradation of the entire complex. Finally, our work suggests that Lhs1 is required for a subset of ERAD substrates that also require the Hrd1 ubiquitin ligase. Together, these data provide hints as to the identities of as-yet unconfirmed substrates of Lhs1 and potentially of the Lhs1 homolog in mammals, GRP170.
Assuntos
Degradação Associada com o Retículo Endoplasmático , Retículo Endoplasmático , Animais , Citosol , Bicamadas Lipídicas , Proteínas de Membrana/genética , MamíferosRESUMO
SIGNIFICANCE STATEMENT: Loss of function of the 2Cl - /H + antiporter ClC-5 in Dent disease causes an unknown impairment in endocytic traffic, leading to tubular proteinuria. The authors integrated data from biochemical and quantitative imaging studies in proximal tubule cells into a mathematical model to determine that loss of ClC-5 impairs endosome acidification and delays early endosome maturation in proximal tubule cells, resulting in reduced megalin recycling, surface expression, and half-life. Studies in a Dent mouse model also revealed subsegment-specific differences in the effects of ClC-5 knockout on proximal tubule subsegments. The approach provides a template to dissect the effects of mutations or perturbations that alter tubular recovery of filtered proteins from the level of individual cells to the entire proximal tubule axis. BACKGROUND: Loss of function of the 2Cl - /H + antiporter ClC-5 in Dent disease impairs the uptake of filtered proteins by the kidney proximal tubule, resulting in tubular proteinuria. Reduced posttranslational stability of megalin and cubilin, the receptors that bind to and recover filtered proteins, is believed to underlie the tubular defect. How loss of ClC-5 leads to reduced receptor expression remains unknown. METHODS: We used biochemical and quantitative imaging data to adapt a mathematical model of megalin traffic in ClC-5 knockout and control cells. Studies in ClC-5 knockout mice were performed to describe the effect of ClC-5 knockout on megalin traffic in the S1 segment and along the proximal tubule axis. RESULTS: The model predicts that ClC-5 knockout cells have reduced rates of exit from early endosomes, resulting in decreased megalin recycling, surface expression, and half-life. Early endosomes had lower [Cl - ] and higher pH. We observed more profound effects in ClC-5 knockout cells expressing the pathogenic ClC-5 E211G mutant. Alterations in the cellular distribution of megalin in ClC-5 knockout mice were consistent with delayed endosome maturation and reduced recycling. Greater reductions in megalin expression were observed in the proximal tubule S2 cells compared with S1, with consequences to the profile of protein retrieval along the proximal tubule axis. CONCLUSIONS: Delayed early endosome maturation due to impaired acidification and reduced [Cl - ] accumulation is the primary mediator of reduced proximal tubule receptor expression and tubular proteinuria in Dent disease. Rapid endosome maturation in proximal tubule cells is critical for the efficient recovery of filtered proteins.
Assuntos
Doença de Dent , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Animais , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Doença de Dent/genética , Doença de Dent/metabolismo , Endocitose , Proteinúria/patologia , Endossomos/metabolismo , Túbulos Renais Proximais/metabolismo , Modelos Animais de Doenças , Camundongos Knockout , Técnicas de Cultura de Células , AntiportersRESUMO
Proximal tubule (PT) cells retrieve albumin and a broad array of other ligands from the glomerular ultrafiltrate. Efficient uptake of albumin requires PT expression of both megalin and cubilin receptors. Although most proteins engage cubilin selectively, megalin is required to maintain robust flux through the apical endocytic pathway. Receptor-associated protein (RAP) is a chaperone that directs megalin to the cell surface, and recombinant RAP dramatically inhibits the uptake of numerous megalin and cubilin ligands. The mechanism by which this occurs has been suggested to involve competitive inhibition of ligand binding and/or conformational changes in megalin that prevent interaction with ligands and/or with cubilin. To discriminate between these possibilities, we determined the effect of RAP on endocytosis of albumin, which binds to cubilin and megalin receptors with high and low affinity, respectively. Uptake was quantified in opossum kidney (OK) cells and in megalin or cubilin (Cubn) knockout (KO) clones. Surprisingly, RAP inhibited fluid-phase uptake in addition to receptor-mediated uptake in OK cells and Cubn KO cells but had no effect on endocytosis when megalin was absent. The apparent Ki for RAP inhibition of albumin uptake was 10-fold higher in Cubn KO cells compared with parental OK cells. We conclude that in addition to its predicted high-affinity competition for ligand binding to megalin, the primary effect of RAP on PT cell endocytosis is to globally dampen megalin-dependent endocytic flux. Our data explain the complex effects of RAP on binding and uptake of filtered proteins and reveal a novel role in modulating endocytosis in PT cells.NEW & NOTEWORTHY Receptor-associated protein inhibits binding and uptake of all known endogenous ligands by megalin and cubilin receptors via unknown mechanism(s). Here, we took advantage of recently generated knockout cell lines to dissect the effect of this protein on megalin- and cubilin-mediated endocytosis. Our study reveals a novel role for receptor-associated protein in blocking megalin-stimulated endocytic uptake of fluid-phase markers and receptor-bound ligands in proximal tubule cells in addition to its direct effect on ligand binding to megalin receptors.
Assuntos
Albuminas , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Ligantes , Albuminas/metabolismo , Membrana Celular/metabolismo , Endocitose/fisiologia , Túbulos Renais Proximais/metabolismoRESUMO
Bile acids, originally known to emulsify dietary lipids, are now established signalling molecules that regulate physiological processes. Signalling targets several proteins that include the ion channels involved in regulating intestinal motility and bile viscosity. Studies show that bile acids regulate the epithelial sodium channel (ENaC) in cultured cell models and heterologous expression systems. ENaC plays both local and systemic roles in regulating extracellular fluids. Here we investigated whether bile acids regulate ENaC expressed in native tissues. We found that taurocholic acid and taurohyodeoxycholic acid regulated ENaC in both the distal nephron and distal colon. We also tested the hypothesis that regulation occurs through direct binding. Using photoaffinity labelling, we found evidence for specific binding to both the ß and γ subunits of the channel. In functional experiments, we found that the α subunit was sufficient for regulation. We also found that regulation by at least one bile acid was voltage-sensitive, suggesting that one binding site may be closely associated with the pore-forming helices of the channel. Our data provide evidence that bile acids regulate ENaC by binding to multiple sites to influence the open probability of the channel. KEY POINTS: Recent studies have shown that bile acids regulate the epithelial sodium channel (ENaC) in vitro. Here we investigated whether bile acids regulate ENaC in native tissues and whether bile acids directly bind the channel. We found that bile acids regulate ENaC expressed in the mouse cortical collecting duct and mouse colon by modulating open probability. Photoaffinity labelling experiments showed specific binding to the ß and γ subunits of the channel, while channels comprising only α subunits were sensitive to taurocholic acid in functional experiments using Xenopus oocytes. Taurocholic acid regulation of ENaC was voltage-dependent, providing evidence for binding to pore-forming helices. Our data indicate that bile acids are ENaC regulatory effectors that may have a role in the physiology and pathophysiology of several systems.
Assuntos
Ácidos e Sais Biliares , Canais Epiteliais de Sódio , Animais , Camundongos , Amilorida , Ácidos e Sais Biliares/farmacologia , Ácidos e Sais Biliares/metabolismo , Canais Epiteliais de Sódio/metabolismo , Íons/metabolismo , Oócitos/fisiologia , Sódio/metabolismo , Ácido Taurocólico/metabolismo , Xenopus laevis/metabolismo , Canais de Sódio/metabolismoRESUMO
Epithelial Na+ channels (ENaCs) are members of the ENaC/degenerin family of ion channels that evolved to respond to extracellular factors. In addition to being expressed in the distal aspects of the nephron, where ENaCs couple the absorption of filtered Na+ to K+ secretion, these channels are found in other epithelia as well as nonepithelial tissues. This review addresses mechanisms by which ENaC activity is regulated by extracellular factors, including proteases, Na+, and shear stress. It also addresses other factors, including acidic phospholipids and modification of ENaC cytoplasmic cysteine residues by palmitoylation, which enhance channel activity by altering interactions of the channel with the plasma membrane.
Assuntos
Membrana Celular/metabolismo , Canais Epiteliais de Sódio/metabolismo , Estresse Fisiológico/fisiologia , Animais , Transporte Biológico , Humanos , LipoilaçãoRESUMO
The epithelial sodium channel (ENaC) mediates Na+ transport in several epithelia, including the aldosterone-sensitive distal nephron, distal colon, and biliary epithelium. Numerous factors regulate ENaC activity, including extracellular ligands, post-translational modifications, and membrane-resident lipids. However, ENaC regulation by bile acids and conjugated bilirubin, metabolites that are abundant in the biliary tree and intestinal tract and are sometimes elevated in the urine of individuals with advanced liver disease, remains poorly understood. Here, using a Xenopus oocyte-based system to express and functionally study ENaC, we found that, depending on the bile acid used, bile acids both activate and inhibit mouse ENaC. Whether bile acids were activating or inhibiting was contingent on the position and orientation of specific bile acid moieties. For example, a hydroxyl group at the 12-position and facing the hydrophilic side (12α-OH) was activating. Taurine-conjugated bile acids, which have reduced membrane permeability, affected ENaC activity more strongly than did their more membrane-permeant unconjugated counterparts, suggesting that bile acids regulate ENaC extracellularly. Bile acid-dependent activation was enhanced by amino acid substitutions in ENaC that depress open probability and was precluded by proteolytic cleavage that increases open probability, consistent with an effect of bile acids on ENaC open probability. Bile acids also regulated ENaC in a cortical collecting duct cell line, mirroring the results in Xenopus oocytes. We also show that bilirubin conjugates activate ENaC. These results indicate that ENaC responds to compounds abundant in bile and that their ability to regulate this channel depends on the presence of specific functional groups.
Assuntos
Ácidos e Sais Biliares/farmacologia , Bilirrubina/farmacologia , Ácido Desoxicólico/farmacologia , Canais Epiteliais de Sódio/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Sódio/metabolismo , Animais , Antioxidantes/farmacologia , Colagogos e Coleréticos/farmacologia , Canais Epiteliais de Sódio/genética , Fármacos Gastrointestinais/farmacologia , Humanos , Transporte de Íons , Lipoilação , Camundongos , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Proteólise , Xenopus laevisRESUMO
Proximal tubule (PT) cells express a single saturable albumin-binding site whose affinity matches the estimated tubular concentration of albumin; however, albumin uptake capacity is greatly increased under nephrotic conditions. Deciphering the individual contributions of megalin and cubilin to the uptake of normal and nephrotic levels of albumin is impossible in vivo, as knockout of megalin in mice globally disrupts PT endocytic uptake. We quantified concentration-dependent albumin uptake in an optimized opossum kidney cell culture model and fit the kinetic profiles to identify albumin-binding affinities and uptake capacities. Mathematical deconvolution fit best to a three-component model that included saturable high- and low-affinity uptake sites for albumin and underlying nonsaturable uptake consistent with passive uptake of albumin in the fluid phase. Knockdown of cubilin or its chaperone amnionless selectively reduced the binding capacity of the high-affinity site, whereas knockdown of megalin impacted the low-affinity site. Knockdown of disabled-2 decreased the capacities of both binding sites. Additionally, knockdown of megalin or disabled-2 profoundly inhibited the uptake of a fluid phase marker, with cubilin knockdown having a more modest effect. We propose a novel model for albumin retrieval along the PT in which cubilin and megalin receptors have different functions in recovering filtered albumin in proximal tubule cells. Cubilin binding to albumin is tuned to capture normally filtered levels of the protein. In contrast, megalin binding to albumin is of lower affinity, and its expression is also essential for enabling the recovery of high concentrations of albumin in the fluid phase.
Assuntos
Albuminúria/metabolismo , Túbulos Renais Proximais/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Nefrose/metabolismo , Receptores de Superfície Celular/metabolismo , Albumina Sérica/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Albuminúria/genética , Albuminúria/fisiopatologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Endocitose , Feminino , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Túbulos Renais Proximais/fisiopatologia , Cinética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/deficiência , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Knockout , Modelos Biológicos , Nefrose/genética , Nefrose/fisiopatologia , Gambás , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genéticaRESUMO
The epithelial Na+ channel (ENaC) is a member of the ENaC/degenerin family of ion channels. In the structure of a related family member, the "thumb" domain's base interacts with the pore, and its tip interacts with the divergent "finger" domain. Between the base and tip, the thumb domain is characterized by a conserved five-rung disulfide ladder holding together two anti-parallel α helices. The ENaC α and γ subunits' finger domains harbor autoinhibitory tracts that can be proteolytically liberated to activate the channel and also host an ENaC-specific pair of cysteines. Using a crosslinking approach, we show that one of the finger domain cysteines in the α subunit (αCys-263) and both of the finger domain cysteines in the γ subunit (γCys-213 and γCys-220) lie near the dynamic finger-thumb domain interface. Our data suggest that the αCys-256/αCys-263 pair is not disulfide-bonded. In contrast, we found that the γCys-213/γCys-220 pair is disulfide-bonded. Our data also suggest that the γ subunit lacks the terminal rung in the thumb domain disulfide ladder, suggesting asymmetry between the subunits. We also observed functional asymmetry between the α and γ subunit finger-thumb domain interfaces; crosslinks bridging the α subunit finger-thumb interface only inhibited ENaC currents, whereas crosslinks bridging the γ subunit finger-thumb interface activated or inhibited currents dependent on the length of the crosslinker. Our data suggest that reactive cysteines lie at the dynamic finger-thumb interfaces of the α and γ subunits and may play a yet undefined role in channel regulation.
Assuntos
Canais Epiteliais de Sódio/química , Animais , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/metabolismo , Humanos , Domínios Proteicos , Xenopus laevisRESUMO
Epithelial Na+ channel (ENaC) maturation and activation require proteolysis of both the α and γ subunits. Cleavage at multiple sites in the finger domain of each subunit liberates their autoinhibitory tracts. Synthetic peptides derived from the proteolytically released fragments inhibit the channel, likely by reconstituting key interactions removed by the proteolysis. We previously showed that a peptide derived from the α subunit's autoinhibitory sequence (α-8) binds at the α subunit's finger-thumb domain interface. Despite low sequence similarity between the α and γ subunit finger domains, we hypothesized that a peptide derived from the γ subunit's autoinhibitory sequence (γ-11) inhibits the channel through an analogous mechanism. Using Xenopus oocytes, we found here that channels lacking a γ subunit thumb domain were no longer sensitive to γ-11, but remained sensitive to α-8. We identified finger domain sites in the γ subunit that dramatically reduced γ-11 inhibition. Using cysteines and sulfhydryl reactive cross-linkers introduced into both the peptide and the subunit, we also could cross-link γ-11 to both the finger domain and the thumb domain of the γ subunit. Our results suggest that α-8 and γ-11 occupy similar binding pockets within their respective subunits, and that proteolysis of the α and γ subunits activate the channel through analogous mechanisms.
Assuntos
Regulação Alostérica , Canais Epiteliais de Sódio/fisiologia , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/metabolismo , Animais , Sítios de Ligação , Canais Epiteliais de Sódio/metabolismo , Humanos , Oócitos , Proteólise , Xenopus laevisRESUMO
The Caenorhabditis elegans MEC-4/MEC-10 channel mediates the worm's response to gentle body touch and is activated by laminar shear stress (LSS) when expressed in Xenopus oocytes. Substitutions at multiple sites within the second transmembrane domain (TM2) of MEC-4 or MEC-10 abolish the gentle touch response in worms, but the roles of these residues in mechanosensing are unclear. The present study therefore examined the role of specific MEC-4 and MEC-10 TM2 residues in the channel's response to LSS. We found that introducing mutations within the TM2 of MEC-4 or MEC-10 not only altered channel activity, but also affected the channel's response to LSS. This response was enhanced by Cys substitutions at selected MEC-4 sites (Phe715, Gly716, Gln718, and Leu719) between the degenerin and the putative amiloride-binding sites in this subunit. In contrast, the LSS response was largely blunted in MEC-10 variants bearing single Cys substitutions in the regions preceding and following the amiloride-binding site (Gly677-Leu681), as well as with four MEC-10 touch-deficient mutations that introduced charged residues into the TM2 domain. An enhanced response to LSS was observed with a MEC-10 mutation in the putative selectivity filter. Overall, MEC-4 or MEC-10 mutants that altered the channel's LSS response are primarily clustered between the degenerin site and the selectivity filter, a region that probably forms the narrowest portion of the channel pore. Our results suggest that pore-lining residues of MEC-4 and MEC-10 have important yet different roles in tuning the channel's response to mechanical forces.
Assuntos
Aminoácidos/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Membrana/metabolismo , Estresse Mecânico , Xenopus laevis/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Células Cultivadas , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Mutação , Oócitos/citologia , Oócitos/metabolismo , Conformação Proteica , Homologia de Sequência , Xenopus laevis/crescimento & desenvolvimentoRESUMO
Epithelial Na+ channel (ENaC) subunits undergo N-linked glycosylation in the endoplasmic reticulum where they assemble into an αßγ complex. Six, 13, and 5 consensus sites (Asn-X-Ser/Thr) for N-glycosylation reside in the extracellular domains of the mouse α-, ß-, and γ-subunits, respectively. Because the importance of ENaC N-linked glycans has not been fully addressed, we examined the effect of preventing N-glycosylation of specific subunits on channel function, expression, maturation, and folding. Heterologous expression in Xenopus oocytes or Fischer rat thyroid cells with αßγ-ENaC lacking N-linked glycans on a single subunit reduced ENaC activity as well as the inhibitory response to extracellular Na+. The lack of N-linked glycans on the ß-subunit also precluded channel activation by trypsin. However, channel activation by shear stress was N-linked glycan independent, regardless of which subunit was modified. We also discovered that the lack of N-linked glycans on any one subunit reduced the total and surface levels of cognate subunits. The lack of N-linked glycans on the ß-subunit had the largest effect on total levels, with the lack of N-linked glycans on the γ- and α-subunits having intermediate and modest effects, respectively. Finally, channels with wild-type ß-subunits were more sensitive to limited trypsin proteolysis than channels lacking N-linked glycans on the ß-subunit. Our results indicate that N-linked glycans on each subunit are required for proper folding, maturation, surface expression, and function of the channel.
Assuntos
Canais Epiteliais de Sódio/metabolismo , Processamento de Proteína Pós-Traducional , Sódio/metabolismo , Animais , Canais Epiteliais de Sódio/química , Canais Epiteliais de Sódio/genética , Glicosilação , Mecanotransdução Celular , Potenciais da Membrana , Mutação , Conformação Proteica , Dobramento de Proteína , Transporte Proteico , Ratos Endogâmicos F344 , Relação Estrutura-Atividade , Tripsina/metabolismo , Xenopus laevisRESUMO
The epithelial Na(+) channel (ENaC) has a key role in the regulation of extracellular fluid volume and blood pressure. ENaC belongs to a family of ion channels that sense the external environment. These channels have large extracellular regions that are thought to interact with environmental cues, such as Na(+), Cl(-), protons, proteases, and shear stress, which modulate gating behavior. We sought to determine the molecular mechanism by which ENaC senses high external Na(+) concentrations, resulting in an inhibition of channel activity. Both our structural model of an ENaC α subunit and the resolved structure of an acid-sensing ion channel (ASIC1) have conserved acidic pockets in the periphery of the extracellular region of the channel. We hypothesized that these acidic pockets host inhibitory allosteric Na(+) binding sites. Through site-directed mutagenesis targeting the acidic pocket, we modified the inhibitory response to external Na(+). Mutations at selected sites altered the cation inhibitory preference to favor Li(+) or K(+) rather than Na(+). Channel activity was reduced in response to restraining movement within this region by cross-linking structures across the acidic pocket. Our results suggest that residues within the acidic pocket form an allosteric effector binding site for Na(+). Our study supports the hypothesis that an acidic cleft is a key ligand binding locus for ENaC and perhaps other members of the ENaC/degenerin family.
Assuntos
Canais Iônicos Sensíveis a Ácido/química , Canais Epiteliais de Sódio/química , Subunidades Proteicas/química , Sódio/química , Canais Iônicos Sensíveis a Ácido/genética , Potenciais de Ação , Regulação Alostérica , Amilorida/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bloqueadores do Canal de Sódio Epitelial/química , Canais Epiteliais de Sódio/genética , Expressão Gênica , Transporte de Íons , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Oócitos , Técnicas de Patch-Clamp , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Alinhamento de Sequência , Xenopus laevisRESUMO
The epithelial sodium channel (ENaC) is composed of three homologous subunits (α, ß, and γ) with cytoplasmic N and C termini. Our previous work revealed that two cytoplasmic Cys residues in the ß subunit, ßCys-43 and ßCys-557, are Cys-palmitoylated. ENaCs with mutant ßC43A/C557A exhibit normal surface expression but enhanced Na(+) self-inhibition and reduced channel open probability. Although the α subunit is not palmitoylated, we now show that the two cytoplasmic Cys residues in the γ subunit are palmitoylated. ENaCs with mutant γC33A, γC41A, or γC33A/C41A exhibit reduced activity compared with wild type channels but normal surface expression and normal levels of α and γ subunit-activating cleavage. These mutant channels have significantly enhanced Na(+) self-inhibition and reduced open probability compared with wild type ENaCs. Channel activity was enhanced by co-expression with the palmitoyltransferase DHHC2 that also co-immunoprecipitates with ENaCs. Secondary structure prediction of the N terminus of the γ subunit places γCys-33 within an α-helix and γCys-44 on a coil before the first transmembrane domain within a short tract that includes a well conserved His-Gly motif, where mutations have been associated with altered channel gating. Our current and previous results suggest that palmitoylation of the ß and γ subunits of ENaCs enhances interactions of their respective cytoplasmic domains with the plasma membrane and stabilizes the open state of the channel. Comparison of activities of channels lacking palmitoylation sites in individual or multiple subunits revealed that γ subunit palmitoylation has a dominant role over ß subunit palmitoylation in modulating ENaC gating.
Assuntos
Cisteína/metabolismo , Canais Epiteliais de Sódio/química , Canais Epiteliais de Sódio/metabolismo , Lipoilação , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Animais , Sítios de Ligação , Citoplasma/metabolismo , Cães , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Células Madin Darby de Rim Canino , Camundongos , Subunidades Proteicas/antagonistas & inibidores , Sódio/farmacologiaRESUMO
The epithelial sodium channel, ENaC, plays a critical role in maintaining salt and water homeostasis, and not surprisingly defects in ENaC function are associated with disease. Like many other membrane-spanning proteins, this trimeric protein complex folds and assembles inefficiently in the endoplasmic reticulum (ER), which results in a substantial percentage of the channel being targeted for ER-associated degradation (ERAD). Because the spectrum of factors that facilitates the degradation of ENaC is incomplete, we developed yeast expression systems for each ENaC subunit. We discovered that a conserved Hsp70-like chaperone, Lhs1, is required for maximal turnover of the ENaC α subunit. By expressing Lhs1 ATP binding mutants, we also found that the nucleotide exchange properties of this chaperone are dispensable for ENaC degradation. Consistent with the precipitation of an Lhs1-αENaC complex, Lhs1 holdase activity was instead most likely required to support the ERAD of αENaC. Moreover, a complex containing the mammalian Lhs1 homolog GRP170 and αENaC co-precipitated, and GRP170 also facilitated ENaC degradation in human, HEK293 cells, and in a Xenopus oocyte expression system. In both yeast and higher cell types, the effect of Lhs1 on the ERAD of αENaC was selective for the unglycosylated form of the protein. These data establish the first evidence that Lhs1/Grp170 chaperones can act as mediators of ERAD substrate selection.
Assuntos
Degradação Associada com o Retículo Endoplasmático , Canais Epiteliais de Sódio/metabolismo , Glicoproteínas/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Amilorida/farmacologia , Animais , Retículo Endoplasmático/metabolismo , Canais Epiteliais de Sódio/genética , Feminino , Glicoproteínas/genética , Células HEK293 , Proteínas de Choque Térmico HSP70/genética , Humanos , Immunoblotting , Transporte de Íons/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Mutação , Oócitos/metabolismo , Oócitos/fisiologia , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Sódio/metabolismo , XenopusRESUMO
The epithelial Na + channel (ENaC) resides on the apical surfaces of specific epithelia in vertebrates and plays a critical role in extracellular fluid homeostasis. Evidence that ENaC senses the external environment emerged well before the molecular identity of the channel was reported three decades ago. This article discusses progress toward elucidating the mechanisms through which specific external factors regulate ENaC function, highlighting insights gained from structural studies of ENaC and related family members. It also reviews our understanding of the role of ENaC regulation by the extracellular environment in physiology and disease. After familiarizing the reader with the channel's physiological roles and structure, we describe the central role protein allostery plays in ENaC's sensitivity to the external environment. We then discuss each of the extracellular factors that directly regulate the channel: proteases, cations and anions, shear stress, and other regulators specific to particular extracellular compartments. For each regulator, we discuss the initial observations that led to discovery, studies investigating molecular mechanism, and the physiological and pathophysiological implications of regulation. © 2024 American Physiological Society. Compr Physiol 14:5407-5447, 2024.