RESUMO
We examined the interaction of a membrane-associated protein, MARCKS-like Protein-1 (MLP-1), and an ion channel, Epithelial Sodium Channel (ENaC), with the anionic lipid, phosphatidylinositol 4, 5-bisphosphate (PIP2). We found that PIP2 strongly activates ENaC in excised, inside-out patches with a half-activating concentration of 21 ± 1.17 µM. We have identified 2 PIP2 binding sites in the N-terminus of ENaC ß and γ with a high concentration of basic residues. Normal channel activity requires MLP-1's strongly positively charged effector domain to electrostatically sequester most of the membrane PIP2 and increase the local concentration of PIP2. Our previous data showed that ENaC covalently binds MLP-1 so PIP2 bound to MLP-1 would be near PIP2 binding sites on the cytosolic N terminal regions of ENaC. We have modified the charge structure of the PIP2 -binding domains of MLP-1 and ENaC and showed that the changes affect membrane localization and ENaC activity in a way consistent with electrostatic theory.
RESUMO
Hypertension is characterized by increased sodium (Na+) reabsorption along the aldosterone-sensitive distal nephron (ASDN) as well as chronic systemic inflammation. Interleukin-6 (IL-6) is thought to be a mediator of this inflammatory process. Interestingly, increased Na+ reabsorption within the ASDN does not always correlate with increases in aldosterone (Aldo), the primary hormone that modulates Na+ reabsorption via the mineralocorticoid receptor (MR). Thus, understanding how increased ASDN Na+ reabsorption may occur independent of Aldo stimulation is critical. Here, we show that IL-6 can activate the MR by activating Rac1 and stimulating the generation of reactive oxygen species (ROS) with a consequent increase in thiazide-sensitive Na+ uptake. Using an in vitro model of the distal convoluted tubule (DCT2), mDCT15 cells, we observed nuclear translocation of eGFP-tagged MR after IL-6 treatment. To confirm the activation of downstream transcription factors, mDCT15 cells were transfected with mineralocorticoid response element (MRE)-luciferase reporter constructs; then treated with vehicle, Aldo, or IL-6. Aldosterone or IL-6 treatment increased luciferase activity that was reversed with MR antagonist cotreatment, but IL-6 treatment was reversed by Rac1 inhibition or ROS reduction. In both mDCT15 and mpkCCD cells, IL-6 increased amiloride-sensitive transepithelial Na+ current. ROS and IL-6 increased 22Na+ uptake via the thiazide-sensitive sodium chloride cotransporter (NCC). These results are the first to demonstrate that IL-6 can activate the MR resulting in MRE activation and that IL-6 increases NCC-mediated Na+ reabsorption, providing evidence for an alternative mechanism for stimulating ASDN Na+ uptake during conditions where Aldo-mediated MR stimulation may not occur.
Assuntos
Aldosterona , Receptores de Mineralocorticoides , Aldosterona/farmacologia , Interleucina-6 , Espécies Reativas de Oxigênio , Túbulos Renais Distais , Néfrons , Sódio , TiazidasRESUMO
The epithelial sodium channel (ENaC) regulates blood pressure by fine-tuning distal nephron sodium reabsorption. Our previous work has shown that ENaC gating is regulated by anionic phospholipid phosphates, including phosphatidylinositol 4,5-bisphosphate (PIP2). The PIP2-dependent regulation of ENaC is mediated by the myristoylated alanine-rich protein kinase C substrate-like protein-1 (MLP-1). MLP-1 binds to and is a reversible source of PIP2 at the plasma membrane. We examined MLP-1 regulation of ENaC in distal convoluted tubule clonal cell line DCT-15 cells. Wild-type MLP-1 runs at an apparent molecular mass of 52 kDa despite having a predicted molecular mass of 21 kDa. Native MLP-1 consists of several distinct structural elements: an effector domain that is highly positively charged, sequesters PIP2, contains serines that are the target of PKC, and controls MLP-1 association with the membrane; a myristoylation domain that promotes association with the membrane; and a multiple homology 2 domain of previously unknown function. To further examine MLP-1 in DCT-15 cells, we constructed several MLP-1 mutants: WT, a full-length wild-type protein; S3A, three substitutions in the effector domain to prevent phosphorylation; S3D mimicked constitutive phosphorylation by replacing three serines with aspartates; and GA replaced the myristoylation site glycine with alanine, so GA could not be myristoylated. Each mutant was tagged with either NH2-terminal 3XFLAG or COOH-terminal mCherry or V5. Transfection with MLP mutants modified ENaC activity in DCT-15 cells: activity was highest in S3A and lowest in S3D, and the activity after transfection with either construct was significantly different from WT. In Western blots, when transfected with 3XFLAG-tagged MLP-1 mutants, the expression of the full length of MLP-1 at 52 kDa increased in mutant S3A-MLP-1-transfected DCT-15 cells and decreased in S3D-MLP-1-transfected DCT-15 cells. Several lower molecular mass bands were also detected that correspond to potential presumptive calpain cleavage products. Confocal imaging shows that the different mutants localize in different subcellular compartments consistent with their preferred location in the membrane or in the cytosol. Activation of protein kinase C increases phosphorylation of endogenous MLP-1 and reduces ENaC activity. Our results suggest a complicated role for proteolytic processing in MLP-1 regulation of ENaC.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Canais Epiteliais de Sódio/metabolismo , Proteínas dos Microfilamentos/metabolismo , Néfrons/metabolismo , Animais , Proteínas de Ligação a Calmodulina/genética , Linhagem Celular , Membrana Celular/metabolismo , Camundongos , Proteínas dos Microfilamentos/genética , Fosfatidilinositóis/metabolismo , Fosforilação , Proteína Quinase C/metabolismoRESUMO
It has been suggested that voltage-dependent anion channels (VDACs) control the release of superoxide from mitochondria. We have previously shown that reactive oxygen species (ROS) such as superoxide (O2ÌÌ) and hydrogen peroxide (H2O2) stimulate epithelial sodium channels (ENaCs) in sodium-transporting epithelial tissue, including cortical collecting duct (CCD) principal cells. Therefore, we hypothesized that VDACs could regulate ENaC by modulating cytosolic ROS levels. Herein, we find that VDAC3-knockout(KO) mice can maintain normal salt and water balance on low-salt and high-salt diets. However, on a high-salt diet for 2 weeks, VDAC3-KO mice had significantly higher systolic blood pressure than wildtype mice. Consistent with this observation, after a high-salt diet for 2 weeks, ENaC activity in VDAC3-KO mice was significantly higher than wildtype mice. EM analysis disclosed a significant morphological change of mitochondria in the CCD cells of VDAC3-KO mice compared with wildtype mice, which may have been caused by mitochondrial superoxide overload. Of note, compared with wildtype animals, ROS levels in VDAC3-KO animals fed a normal or high-salt diet were consistently and significantly increased in renal tubules. Both the ROS scavenger 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine (TEMPOL) and the mitochondrial ROS scavenger (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (mito-TEMPO) could reverse the effect of high-salt on ENaC activity and systolic blood pressure in the VDAC3-KO mice. Mito-TEMPO partially correct the morphological changes in VDAC3-KO mice. Our results suggest that knocking out mitochondrial VDAC3 increases ROS, alters renal sodium transport, and leads to hypertension.
Assuntos
Canais Epiteliais de Sódio/metabolismo , Peróxido de Hidrogênio/metabolismo , Rim/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/deficiência , Sódio/metabolismo , Superóxidos/metabolismo , Canais de Ânion Dependentes de Voltagem/deficiência , Animais , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/genética , Óxidos N-Cíclicos/farmacologia , Canais Epiteliais de Sódio/genética , Hipertensão/genética , Hipertensão/metabolismo , Hipertensão/patologia , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/genética , Rim/patologia , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Compostos Organofosforados/farmacologia , Piperidinas/farmacologia , Marcadores de Spin , Canais de Ânion Dependentes de Voltagem/metabolismoRESUMO
Distal sodium transport is a final step in the regulation of blood pressure. As such, understanding how the two main sodium transport proteins, the thiazide-sensitive sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC), are regulated is paramount. Both are expressed in the late distal nephron; however, no evidence has suggested that these two sodium transport proteins interact. Recently, we established that these two sodium transport proteins functionally interact in the second part of the distal nephron (DCT2). Given their co-localization within the DCT2, we hypothesized that NCC and ENaC interactions might be modulated by aldosterone (Aldo). Aldo treatment increased NCC and αENaC colocalization (electron microscopy) and interaction (coimmunoprecipitation). Finally, with co-expression of the Aldo-induced protein serum- and glucocorticoid-inducible kinase 1 (SGK1), NCC and αENaC interactions were increased. These data demonstrate that Aldo promotes increased interaction of NCC and ENaC, within the DCT2 revealing a novel method of regulation for distal sodium reabsorption.
Assuntos
Aldosterona/farmacologia , Canais Epiteliais de Sódio/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Animais , Linhagem Celular , Canais Epiteliais de Sódio/ultraestrutura , Córtex Renal/metabolismo , Córtex Renal/ultraestrutura , Camundongos , Subunidades Proteicas/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/ultraestruturaRESUMO
We previously demonstrated a role for the myristoylated alanine-rich C kinase substrate (MARCKS) to serve as an adaptor protein in the anionic phospholipid phosphate-dependent regulation of the epithelial sodium channel (ENaC). Both MARCKS and ENaC are regulated by proteolysis. Calpains are a family of ubiquitously expressed intracellular Ca2+-dependent cysteine proteases involved in signal transduction. Here we examine the role of calpain-2 in regulating MARCKS and ENaC in cultured renal epithelial cells and in the mouse kidney. Using recombinant fusion proteins, we show that MARCKS, but not the ENaC subunits, are a substrate of calpain-2 in the presence of Ca2+ Pharmacological inhibition of calpain-2 alters MARCKS protein expression in light-density sucrose gradient fractions from cell lysates of mouse cortical collecting duct cells. Calpain-dependent cleaved products of MARCKS are detectable in cultured renal cells. Ca2+ mobilization and calpain-2 inhibition decrease the association between ENaC and MARCKS. The inhibition of calpain-2 reduces ENaC activity as demonstrated by single-channel patch-clamp recordings and transepithelial current measurements. These results suggest that calpain-2 proteolysis of MARCKS promotes its interaction with lipids and ENaC at the plasma membrane to allow for the phosphatidylinositol 4,5-bisphosphate (PIP2)-dependent regulation of ENaC activity in the kidney.
Assuntos
Calpaína/genética , Canais Epiteliais de Sódio/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Potenciais de Ação/efeitos dos fármacos , Amilorida/farmacologia , Animais , Cálcio/metabolismo , Calpaína/metabolismo , Fracionamento Celular , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Citocalasina D/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Canais Epiteliais de Sódio/metabolismo , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/efeitos dos fármacos , Túbulos Renais Coletores/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Substrato Quinase C Rico em Alanina Miristoilada , Técnicas de Patch-Clamp , Proteólise/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Xenopus laevisRESUMO
A thin fluid layer in alveoli is normal and results from a balance of fluid entry and fluid uptake by transepithelial salt and water reabsorption. Conventional wisdom suggests the reabsorption is via epithelial Na+ channels (ENaC), but if all Na+ reabsorption were via ENaC, then amiloride, an ENaC inhibitor, should block alveolar fluid clearance (AFC). However, amiloride blocks only half of AFC. The reason for failure to block is clear from single-channel measurements from alveolar epithelial cells: ENaC channels are observed, but another channel is present at the same frequency that is nonselective for Na+ over K+, has a larger conductance, and has shorter open and closed times. These two channel types are known as highly selective channels (HSC) and nonselective cation channels (NSC). HSC channels are made up of three ENaC subunits since knocking down any of the subunits reduces HSC number. NSC channels contain α-ENaC since knocking down α-ENaC reduces the number of NSC (knocking down ß- or γ-ENaC has no effect on NSC, but the molecular composition of NSC channels remains unclear). We show that NSC channels consist of at least one α-ENaC and one or more acid-sensing ion channel 1a (ASIC1a) proteins. Knocking down either α-ENaC or ASIC1a reduces both NSC and HSC number, and no NSC channels are observable in single-channel patches on lung slices from ASIC1a knockout mice. AFC is reduced in knockout mice, and wet wt-to-dry wt ratio is increased, but the percentage increase in wet wt-to-dry wt ratio is larger than expected based on the reduction in AFC.
Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Canais Epiteliais de Sódio/metabolismo , Alvéolos Pulmonares/metabolismo , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Animais , Líquido da Lavagem Broncoalveolar , Células Cultivadas , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Oxigênio/farmacologia , Ligação Proteica/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Alvéolos Pulmonares/efeitos dos fármacos , Venenos de Serpentes/toxicidade , Água/metabolismoRESUMO
Regulation of the epithelial sodium channel (ENaC), which regulates fluid homeostasis and blood pressure, is complex and remains incompletely understood. The TIP peptide, a mimic of the lectin-like domain of TNF, activates ENaC by binding to glycosylated residues in the extracellular loop of ENaC-α, as well as to a hitherto uncharacterized internal site. Molecular docking studies suggested three residues, Val567, Glu568, and Glu571, located at the interface between the second transmembrane and C-terminal domains of ENaC-α, as a critical site for binding of the TIP peptide. We generated Ala replacement mutants in this region of ENaC-α and examined its interaction with TIP peptide (3M, V567A/E568A/E571A; 2M, V567A/E568A; and 1M, E571A). 3M and 2M ENaC-α, but not 1M ENaC-α, displayed significantly reduced binding capacity to TIP peptide and to TNF. When overexpressed in H441 cells, 3M mutant ENaC-α formed functional channels with similar gating and density characteristics as the WT subunit and efficiently associated with the ß and γ subunits in the plasma membrane. We subsequently assayed for increased open probability time and membrane expression, both of which define ENaC activity, following addition of TIP peptide. TIP peptide increased open probability time in H441 cells overexpressing wild type and 1M ENaC-α channels, but not 3M or 2M ENaC-α channels. On the other hand, TIP peptide-mediated reduction in ENaC ubiquitination was similar in cells overexpressing either WT or 3M ENaC-α subunits. In summary, this study has identified a novel site in ENaC-α that is crucial for activation of the open probability of the channel, but not membrane expression, by the lectin-like domain of TNF.
Assuntos
Agonistas do Canal de Sódio Epitelial/farmacologia , Canais Epiteliais de Sódio/metabolismo , Peptídeos Cíclicos/farmacologia , Linhagem Celular Tumoral , Canais Epiteliais de Sódio/química , Canais Epiteliais de Sódio/genética , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Mutação Puntual , Domínios Proteicos/efeitos dos fármacos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
The thiazide-sensitive sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC) are two of the most important determinants of salt balance and thus systemic blood pressure. Abnormalities in either result in profound changes in blood pressure. There is one segment of the nephron where these two sodium transporters are coexpressed, the second part of the distal convoluted tubule. This is a key part of the aldosterone-sensitive distal nephron, the final regulator of salt handling in the kidney. Aldosterone is the key hormonal regulator for both of these proteins. Despite these shared regulators and coexpression in a key nephron segment, associations between these proteins have not been investigated. After confirming apical localization of these proteins, we demonstrated the presence of functional transport proteins and native association by blue native PAGE. Extensive coimmunoprecipitation experiments demonstrated a consistent interaction of NCC with α- and γ-ENaC. Mammalian two-hybrid studies demonstrated direct binding of NCC to ENaC subunits. Fluorescence resonance energy transfer and immunogold EM studies confirmed that these transport proteins are within appropriate proximity for direct binding. Additionally, we demonstrate that there are functional consequences of this interaction, with inhibition of NCC affecting the function of ENaC. This novel finding of an association between ENaC and NCC could alter our understanding of salt transport in the distal tubule.
Assuntos
Canais Epiteliais de Sódio/metabolismo , Simportadores de Cloreto de Sódio/metabolismo , Animais , Linhagem Celular , Transferência Ressonante de Energia de Fluorescência , Córtex Renal/metabolismo , Camundongos , Microscopia Confocal , Ligação Proteica , Técnicas do Sistema de Duplo-HíbridoRESUMO
The renal epithelial sodium channel (ENaC) provides regulated sodium transport in the distal nephron. The effects of intracellular calcium ([Ca(2+)]i) on this channel are only beginning to be elucidated. It appears from previous studies that the [Ca(2+)]i increases downstream of ATP administration may have a polarized effect on ENaC, where apical application of ATP and the subsequent [Ca(2+)]i increase have an inhibitory effect on the channel, whereas basolateral ATP and [Ca(2+)]i have a stimulatory effect. We asked whether this polarized effect of ATP is, in fact, reflective of a polarized effect of increased [Ca(2+)]i on ENaC and what underlying mechanism is responsible. We began by performing patch clamp experiments in which ENaC activity was measured during apical or basolateral application of ionomycin to increase [Ca(2+)]i near the apical or basolateral membrane, respectively. We found that ENaC does indeed respond to increased [Ca(2+)]i in a polarized fashion, with apical increases being inhibitory and basolateral increases stimulating channel activity. In other epithelial cell types, mitochondria sequester [Ca(2+)]i, creating [Ca(2+)]i signaling microdomains within the cell that are dependent on mitochondrial localization. We found that mitochondria localize in bands just beneath the apical and basolateral membranes in two different cortical collecting duct principal cell lines and in cortical collecting duct principal cells in mouse kidney tissue. We found that inhibiting mitochondrial [Ca(2+)]i uptake destroyed the polarized response of ENaC to [Ca(2+)]i. Overall, our data suggest that ENaC is regulated by [Ca(2+)]i in a polarized fashion and that this polarization is maintained by mitochondrial [Ca(2+)]i sequestration.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Canais Epiteliais de Sódio/metabolismo , Túbulos Renais Coletores/metabolismo , Mitocôndrias/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Camundongos , Xenopus laevisRESUMO
Many hormonal pathways contribute to the regulation of renal epithelial sodium channel (ENaC) function, a key process for maintaining blood volume and controlling blood pressure. In the present study, we examined whether the peptide hormone prolactin (PRL) regulates ENaC function in renal epithelial cells (A6). Basolateral application of several different concentrations of PRL dramatically stimulated the transepithelial current in A6 cells, increasing both amiloride-sensitive (ENaC) and amiloride-insensitive currents. Using cell-attached patch clamp, we determined that PRL increased both the number (N) and open probability (Po) of ENaC present in the apical membrane. Inhibition of PKA with H-89 abolished the effect of PRL on amiloride-sensitive and insensitive transepithelial currents and eliminated the increase in ENaC NPo with PRL exposure. PRL also increased cAMP in A6 cells, consistent with signaling through the cAMP-dependent PKA pathway. We also identified that PRL induced activity of a 2-pS anion channel with outward rectification, electrophysiological properties consistent with ClC4 or ClC5. RT-PCR only detected ClC4, but not ClC5 transcripts. Here, we show for the first time that PRL activates sodium and chloride transport in renal epithelial cells via ENaC and ClC4.
Assuntos
Canais de Cloreto/metabolismo , Células Epiteliais/efeitos dos fármacos , Canais Epiteliais de Sódio/metabolismo , Prolactina/farmacologia , Sódio/metabolismo , Amilorida/farmacologia , Animais , Linhagem Celular , AMP Cíclico/metabolismo , Células Epiteliais/metabolismo , Camundongos , Técnicas de Patch-Clamp/métodosRESUMO
The polarized nature of epithelial cells allows for different responses to luminal or serosal stimuli. In kidney tubules, ATP is produced luminally in response to changes in luminal flow. Luminal increases in ATP have been previously shown to inhibit the renal epithelial Na⺠channel (ENaC). On the other hand, ATP is increased basolaterally in renal epithelia in response to aldosterone. We tested the hypothesis that basolateral ATP can stimulate ENaC function through activation of the P2X4receptor/channel. Using single channel cell-attached patch-clamp techniques, we demonstrated the existence of a basolaterally expressed channel stimulated by the P2X4agonist 2-methylthio-ATP (meSATP) in Xenopus A6 cells, a renal collecting duct principal cell line. This channel had a similar reversal potential and conductance to that of P2X4channels. Cell surface biotinylation of the basolateral side of these cells confirmed the basolateral presence of the P2X4 receptor. Basolateral addition of meSATP enhanced the activity of ENaC in single channel patch-clamp experiments, an effect that was absent in cells transfected with a dominant negative P2X4receptor construct, indicating that activation of P2X4channels stimulates ENaC activity in these cells. The effect of meSATP on ENaC activity was reduced after chelation of basolateral Ca²âº with EGTA or inhibition of phosphatidylinositol 3-kinase with LY-294002. Overall, our results show that ENaC is stimulated by P2X4receptor activation and that the stimulation is dependent on increases in intracellular Ca²âº and phosphatidylinositol 3-kinase activation.
Assuntos
Cálcio/metabolismo , Canais Epiteliais de Sódio/metabolismo , Túbulos Renais Coletores/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptores Purinérgicos P2X4/metabolismo , Animais , Linhagem Celular , XenopusRESUMO
Epithelial Na(+) channel (ENaC) activity, which determines the rate of renal Na(+) reabsorption, can be regulated by G protein-coupled receptors. Regulation of ENaC by Gα-mediated downstream effectors has been studied extensively, but the effect of Gßγ dimers on ENaC is unclear. A6 cells endogenously contain high levels of Gß1 but low levels of Gß3, Gß4, and Gß5 were detected by Q-PCR. We tested Gγ2 combined individually with Gß1 through Gß5 expressed in A6 cells, after which we recorded single-channel ENaC activity. Among the five ß and γ2 combinations, ß1γ2 strongly inhibits ENaC activity by reducing both ENaC channel number (N) and open probability (Po) compared with control cells. In contrast, the other four ß-isoforms combined with γ2 have no significant effect on ENaC activity. By using various inhibitors to probe Gß1γ2 effects on ENaC regulation, we found that Gß1γ2-mediated ENaC inhibition involved activation of phospholipase C-ß and its enzymatic products that induce protein kinase C and ERK1/2 signaling pathways.
Assuntos
Canais Epiteliais de Sódio/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/fisiologia , Subunidades gama da Proteína de Ligação ao GTP/fisiologia , Rim/metabolismo , Sódio/metabolismo , Animais , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Ativação do Canal Iônico/fisiologia , Técnicas de Patch-Clamp , Fosfoinositídeo Fosfolipase C/metabolismo , Isoformas de Proteínas/fisiologia , Transdução de Sinais/fisiologia , Xenopus laevisRESUMO
A serine-threonine protein kinase, WNK4, reduces Na⺠reabsorption and K⺠secretion in the distal convoluted tubule by reducing trafficking of the thiazide-sensitive Na-Cl cotransporter to and enhancing renal outer medullary potassium channel retrieval from the apical membrane. Epithelial sodium channels (ENaC) in the distal nephron also play a role in regulating Na⺠reabsorption and are also regulated by WNK4, but the mechanism is unclear. In A6 distal nephron cells, transepithelial current measurement and single channel recording show that WNK4 inhibits ENaC activity. Analysis of the number of channel per patch shows that WNK4 reduces channel number but has no effect on channel open probability. Western blots of apical and total ENaC provide additional evidence that WNK4 reduces apical as well as total ENaC expression. WNK4 enhances ENaC internalization independent of Nedd4-2-mediated ENaC ubiquitination. WNK4 also reduced the amount of ENaC available for recycling but has no effect on the rate of transepithelial current increase to forskolin. In contrast, Nedd4-2 not only reduced ENaC in the recycling pool but also decreased the rate of increase of current after forskolin. WNK4 associates with wild-type as well as Liddle's mutated ENaC, and WNK4 reduces both wild-type and mutated ENaC expressed in HEK293 cells.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Canais Epiteliais de Sódio/metabolismo , Néfrons/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Western Blotting , Colforsina/farmacologia , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Canais Epiteliais de Sódio/efeitos dos fármacos , Canais Epiteliais de Sódio/genética , Células HEK293 , Humanos , Potenciais da Membrana , Mutação , Ubiquitina-Proteína Ligases Nedd4 , Néfrons/efeitos dos fármacos , Técnicas de Patch-Clamp , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Fatores de Tempo , Transfecção , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
The epithelial sodium channel (ENaC) plays an important role in regulating sodium balance, extracellular volume, and blood pressure. Evidence suggests the α and γ subunits of ENaC are cleaved during assembly before they are inserted into the apical membranes of epithelial cells, and maximal activity of ENaC depends on cleavage of the extracellular loops of α and γ subunits. Here, we report that Xenopus 2F3 cells apically express the cysteine protease cathepsin B, as indicated by two-dimensional gel electrophoresis and mass spectrometry analysis. Recombinant GST ENaC α, ß, and γ subunit fusion proteins were expressed in Escherichia coli and then purified and recovered from bacterial inclusion bodies. In vitro cleavage studies revealed the full-length ENaC α subunit fusion protein was cleaved by active cathepsin B but not the full-length ß or γ subunit fusion proteins. Both single channel patch clamp studies and short circuit current experiments show ENaC activity decreases with the application of a cathepsin B inhibitor directly onto the apical side of 2F3 cells. We suggest a role for the proteolytic cleavage of ENaC by cathepsin B, and we suggest two possible mechanisms by which cathepsin B could regulate ENaC. Cathepsin B may cleave ENaC extracellularly after being secreted or intracellularly, while ENaC is present in the Golgi or in recycling endosomes.
Assuntos
Catepsina B/metabolismo , Endossomos/metabolismo , Canais Epiteliais de Sódio/metabolismo , Complexo de Golgi/metabolismo , Proteólise , Animais , Catepsina B/genética , Endossomos/genética , Canais Epiteliais de Sódio/genética , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Complexo de Golgi/genética , Humanos , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Xenopus laevisRESUMO
Phosphatidylinositol phosphates (PIPs) are known to regulate epithelial sodium channels (ENaC). Lipid binding assays and coimmunoprecipitation showed that the amino-terminal domain of the ß- and γ-subunits of Xenopus ENaC can directly bind to phosphatidylinositol 4,5-bisphosphate (PIP(2)), phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), and phosphatidic acid (PA). Similar assays demonstrated various PIPs can bind strongly to a native myristoylated alanine-rich C-kinase substrate (MARCKS), but weakly or not at all to a mutant form of MARCKS. Confocal microscopy demonstrated colocalization between MARCKS and PIP(2). Confocal microscopy also showed that MARCKS redistributes from the apical membrane to the cytoplasm after PMA-induced MARCKS phosphorylation or ionomycin-induced intracellular calcium increases. Fluorescence resonance energy transfer studies revealed ENaC and MARCKS in close proximity in 2F3 cells when PKC activity and intracellular calcium concentrations are low. Transepithelial current measurements from Xenopus 2F3 cells treated with PMA and single-channel patch-clamp studies of Xenopus 2F3 cells treated with a PKC inhibitor altered Xenopus ENaC activity, which suggest an essential role for MARCKS in the regulation of Xenopus ENaC activity.
Assuntos
Canais Epiteliais de Sódio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Xenopus laevis/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Cálcio/análise , Cálcio/fisiologia , Ionóforos de Cálcio/farmacologia , Transferência Ressonante de Energia de Fluorescência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Ionomicina/farmacologia , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutação , Substrato Quinase C Rico em Alanina Miristoilada , Fosforilação , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/química , Proteína Quinase C/metabolismo , Análise de Sequência de DNA , Acetato de Tetradecanoilforbol/farmacologia , Xenopus laevis/genéticaRESUMO
Altering the splice variant composition of large-conductance Ca(2+)-activated potassium (BK) channels can alter their activity and apparent sensitivity to Ca(2+) and other regulators of activity. We hypothesized that differences in the responsiveness to arachidonic acid of GH3 and GH4 cells was due to a difference in two splice variants, one present in GH3 cells and the other in GH4 cells. The sequences of the two splice variants differ from one another in several ways, but the largest difference is the presence or absence of 27 amino acids in the COOH terminus of the BK alpha-subunit. Open probability of the variant containing the 27 amino acids is significantly increased by arachidonic acid, while the variant lacking the 27 amino acids is insensitive to arachidonic acid. In addition, sensitivity of BK channels to arachidonic acid depends on cytosolic phospholipase A(2) (cPLA(2)). Here we used the Mammalian Matchmaker two-hybrid assay and two BK alpha-subunit constructs with [rSlo(27)] and without [rSlo(0)] the 27-amino acid motif to determine whether cPLA(2) associates with one construct [rSlo(27)] and not the other. We hypothesized that differential association of cPLA(2) might explain the differing responsiveness of the two constructs and GH3 and GH4 cells to arachidonic acid. We found that cPLA(2) is strongly associated with the COOH terminus of rSlo(27) and only very weakly associated with rSlo(0). We also found that arachidonic acid has a lower affinity for rSlo(0) than for rSlo(27). We conclude that the lack of response of BK channels in GH4 cells to arachidonic acid can be explained, in part, by the poor binding of cPLA(2) to the COOH terminus of the rSlo(0) alpha-subunit, which is very similar to the splice variant found in the arachidonic acid-insensitive GH4 cells.
Assuntos
Fosfolipases A2 do Grupo IV/metabolismo , Ativação do Canal Iônico , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Sequência de Aminoácidos , Animais , Ácido Araquidônico/metabolismo , Células CHO , Cricetinae , Cricetulus , Fosfolipases A2 do Grupo IV/química , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Potenciais da Membrana , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Isoformas de Proteínas , Estrutura Terciária de Proteína , Ratos , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
Previous studies from this laboratory have demonstrated a critical role of cytosolic phospholipase A2 (cPLA2) and arachidonic acid in angiotensin II (Ang II) AT2 receptor-mediated signal transduction in renal epithelium. In primary proximal tubular epithelial cells exposed to hydrogen peroxide (H2O2), both the selective cPLA2 inhibitors and the cPLA2 antisense oligonucleotides significantly attenuated H2O2-induced arachidonic acid liberation and activation of p38(SAPK), ERK1/2, and Akt1. This H2O2-induced kinase activation was significantly attenuated by a Src kinase inhibitor PP2, or by transient transfection of carboxyl-terminal Src kinase (CSK) that maintained Src in the dormant form. Under basal conditions, Src coimmunoprecipitated with epidermal growth factor receptor (EGFR), while H2O2 increased EGFR phosphorylation in the complex. We observed that inhibition of EGFR kinase activity with AG1478 significantly attenuated H2O2-induced p38(SAPK) and ERK1/2 activation, but did not inhibit Akt1 activation. Furthermore, it seems that p38(SAPK) is upstream of ERK1/2 and Akt1, since a p38(SAPK) inhibitor SB203580 significantly blocked H2O2-induced activation of ERK1/2 and Akt1. Interestingly, overexpression of the dominant-negative p38(SAPK) isoform alpha inhibited ERK1/2 but not Akt1 activation. Our observations demonstrate that in these nontransformed cells, activation of cPLA2 is a converging point for oxidative stress and Ang II, which share common downstream signaling mechanisms including Src and EGFR. In addition, p38(SAPK) provides a positive input to both growth and antiapoptotic signaling pathways induced by acute oxidative stress.
Assuntos
Citosol/enzimologia , Rim/metabolismo , Estresse Oxidativo , Fosfolipases A/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Sequência de Bases , Primers do DNA , DNA Complementar , Epitélio/metabolismo , Fosfolipases A2RESUMO
Whole cell voltage clamp experiments were performed in a mouse cortical collecting duct principal cell line using patch pipettes back-filled with a solution containing phosphatidylinositol 3,4,5-trisphosphate (PIP(3)). PIP(3) significantly increased amiloridesensitive current in control cells but not in the cells prestimulated by aldosterone. Additionally, aldosterone stimulated amiloridesensitive current in control cells, but not in the cells that expressed a PIP(3)-binding protein (Grp1-PH), which sequestered intracellular PIP(3). 12 amino acids from the N-terminal tail (APGEKIKAKIKK) of gamma-epithelial sodium channel (gamma-ENaC) were truncated by PCRbased mutagenesis (gammaT-ENaC). Whole cell and confocal microscopy experiments were conducted in Madin-Darby canine kidney cells co-expressing alpha- and beta-ENaC only or with either gamma-ENaC or gamma(T)-ENaC. The data demonstrated that the N-terminal tail truncation significantly decreased amiloride-sensitive current and that both the N-terminal tail truncation and LY-294002 (a PI3K inhibitor) prevented ENaC translocation to the plasmamembrane. These data suggest that PIP(3) mediates aldosterone-induced ENaC activity and trafficking and that the N-terminal tail of gamma-ENaC is necessary for channel trafficking, probably channel gating as well. Additionally, we demonstrated a novel interaction between gamma-ENaC and PIP(3).
Assuntos
Aldosterona/farmacologia , Fosfatos de Fosfatidilinositol/farmacologia , Canais de Sódio/fisiologia , Amilorida/farmacologia , Sequência de Aminoácidos , Animais , Transporte Biológico , Linhagem Celular , Membrana Celular/metabolismo , Cães , Interações Medicamentosas , Condutividade Elétrica , Canais Epiteliais de Sódio , Expressão Gênica , Rim , Camundongos , Microscopia Confocal , Mutagênese , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , Proteínas Recombinantes de Fusão , Canais de Sódio/química , Canais de Sódio/genética , Relação Estrutura-Atividade , TransfecçãoRESUMO
Previous studies using whole-cell recording methods suggest that human B lymphocytes express an amiloride-sensitive, sodium-permeable channel. The present studies aim to determine whether this channel has biophysical properties and a molecular structure related to the alpha, beta, and gamma subunits of the epithelial sodium channel (ENaC). Reverse transcriptase polymerase chain reaction and Northern blots showed that human B lymphocytes express messages for both alpha- and beta- but not gamma-ENaC. Western blots showed that both alpha- and beta- but not gamma-ENaC proteins are expressed and strongly reduced by antisense oligonucleotides. Patch clamp experiments demonstrated that lymphocyte sodium channels are not active in cell-attached patches. However, membrane stretch can activate a 21-pS nonselective cation channel. The frequency of observance of this channel was significantly reduced by antisense oligonucleotide against alpha-ENaC but not by antisense oligonucleotide against beta-ENaC, indicating that only the alpha subunit of ENaC is necessary to form stretch-activated cation channels. Aldosterone (1.5 microm) reduced the frequency of observance of 21-pS alpha-ENaC channels and simultaneously induced the appearance of spontaneously active 10-pS channels. Antisense oligonucleotide experiments showed that this 10-pS channel is formed from alpha- and beta-ENaC. After expression of exogenous gamma-ENaC, aldosterone again reduced the frequency of observance of the 21-pS alpha-ENaC channel but induced the appearance of a 5-pS channel, presumably a alphabetagamma-ENaC channel. In the absence of aldosterone, the alpha subunit forms an alpha-cryptic channel that is activated by stretch, and in the presence of aldosterone, beta and alpha subunits together form an active channel that is modulated by aldosterone.