The Experts below are selected from a list of 192 Experts worldwide ranked by ideXlab platform
Christoph Korbmacher - One of the best experts on this subject based on the ideXlab platform.
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Conservation of pH sensitivity in the epithelial sodium channel (ENaC) with Liddle's syndrome mutation.
Pflugers Archiv : European journal of physiology, 2000Co-Authors: Angelosaristeidis Konstas, D Mavrelos, Christoph KorbmacherAbstract:Gain-of-function mutations of the epithelial Na+ channel (ENaC) cause a rare form of Hereditary Hypertension, Liddle's syndrome. How these mutations lead to increased channel activity is not yet fully understood. Since wild-type ENaC (wt-ENaC) is highly pH-sensitive, we wondered whether an altered pH-sensitivity of ENaC might contribute to the hyperactivity of ENaC with Liddle's syndrome mutation (Liddle-ENaC). Using Xenopus laevis oocytes as an expression system, we compared the pH-sensitivity of wt-ENaC (alphabetagammarENaC) and Liddle-ENaC (alphabeta(R564stop)gammarENaC). Oocytes were assayed for an amiloride-sensitive (2 microM) inward current (deltaIami) at -60 mV holding potential and cytosolic pH was altered by changing the extracellular pH in the presence of 60 mM sodium acetate. Alternatively, cytosolic acidification was achieved by proton loading the cells using a proton-coupled oligopeptide transporter (PepT-1) co-expressed in the oocytes together with ENaC. Cytosolic but not extracellular acidification substantially reduced deltaIami while cytosolic alkalinisation had a stimulatory effect. This pH-sensitivity was largely preserved in oocytes expressing Liddle-ENaC. The inhibition of wt-ENaC and Liddle-ENaC by cytosolic acidification was independent of so-called sodium-feedback inhibition, since it was not associated with a concomitant increase in intracellular Na+ concentration estimated from the reversal potential of deltaIami. In addition C-terminal deletions in the alpha or gamma subunits or in all three subunits of ENaC did not abolish the inhibitory effect of cytosolic acidification. We conclude that ENaC's pH-sensitivity is not mediated by its cytoplasmic C-termini and that an altered pH-sensitivity of ENaC does not contribute to the pathophysiology of Liddle's syndrome.
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conservation of ph sensitivity in the epithelial sodium channel enac with liddle s syndrome mutation
Pflügers Archiv: European Journal of Physiology, 2000Co-Authors: Angelosaristeidis Konstas, D Mavrelos, Christoph KorbmacherAbstract:Gain-of-function mutations of the epithelial Na+ channel (ENaC) cause a rare form of Hereditary Hypertension, Liddle's syndrome. How these mutations lead to increased channel activity is not yet fully understood. Since wild-type ENaC (wt-ENaC) is highly pH-sensitive, we wondered whether an altered pH-sensitivity of ENaC might contribute to the hyperactivity of ENaC with Liddle's syndrome mutation (Liddle-ENaC). Using Xenopus laevis oocytes as an expression system, we compared the pH-sensitivity of wt-ENaC (αβγrENaC) and Liddle-ENaC (αβR564stopγrENaC). Oocytes were assayed for an amiloride-sensitive (2 µM) inward current (ΔI ami) at –60 mV holding potential and cytosolic pH was altered by changing the extracellular pH in the presence of 60 mM sodium acetate. Alternatively, cytosolic acidification was achieved by proton loading the cells using a proton-coupled oligopeptide transporter (PepT-1) co-expressed in the oocytes together with ENaC. Cytosolic but not extracellular acidification substantially reduced ΔI ami while cytosolic alkalinisation had a stimulatory effect. This pH-sensitivity was largely preserved in oocytes expressing Liddle-ENaC. The inhibition of wt-ENaC and Liddle-ENaC by cytosolic acidification was independent of so-called sodium-feedback inhibition, since it was not associated with a concomitant increase in intracellular Na+ concentration estimated from the reversal potential of ΔI ami. In addition C-terminal deletions in the α or γ subunits or in all three subunits of ENaC did not abolish the inhibitory effect of cytosolic acidification. We conclude that ENaC's pH-sensitivity is not mediated by its cytoplasmic C-termini and that an altered pH-sensitivity of ENaC does not contribute to the pathophysiology of Liddle's syndrome.
Sei Sasaki - One of the best experts on this subject based on the ideXlab platform.
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two sporadic cases of liddle s syndrome caused by de novo enac mutations
American Journal of Kidney Diseases, 2001Co-Authors: Yumi Yamashita, Shinichi Uchida, Masafumi Koga, Yasuhiro Takeda, Nobuyuki Enomoto, Kunihiko Hashimoto, Shigeru Yamano, Kazuhiro Dohi, Fumiaki Marumo, Sei SasakiAbstract:Abstract Liddle's syndrome is a rare form of Hereditary Hypertension caused by mutations of the epithelial sodium (Na + ) channel (ENaC). Analysis of the diseased pedigrees indicates an autosomal dominant inheritance, and the identified mutations are heterozygotes of gain-of-function mutations. However, sporadic cases of Liddle's syndrome have been reported in the literature, including one recently reported case caused by a de novo mutation of ENaC. We identified two patients with Liddle's syndrome who did not have family histories of Hypertension. Sequence analysis showed a mutation in each case (P616L in βENaC and W576X in γENaC), both confirmed to be de novo mutations. These data indicate that Liddle's syndrome should be considered even in patients without a family history of Hypertension.
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Two sporadic cases of liddle’s syndrome caused by de novo ENaC mutations
American Journal of Kidney Diseases, 2001Co-Authors: Yumi Yamashita, Shinichi Uchida, Masafumi Koga, Yasuhiro Takeda, Nobuyuki Enomoto, Kunihiko Hashimoto, Shigeru Yamano, Kazuhiro Dohi, Fumiaki Marumo, Sei SasakiAbstract:Abstract Liddle's syndrome is a rare form of Hereditary Hypertension caused by mutations of the epithelial sodium (Na + ) channel (ENaC). Analysis of the diseased pedigrees indicates an autosomal dominant inheritance, and the identified mutations are heterozygotes of gain-of-function mutations. However, sporadic cases of Liddle's syndrome have been reported in the literature, including one recently reported case caused by a de novo mutation of ENaC. We identified two patients with Liddle's syndrome who did not have family histories of Hypertension. Sequence analysis showed a mutation in each case (P616L in βENaC and W576X in γENaC), both confirmed to be de novo mutations. These data indicate that Liddle's syndrome should be considered even in patients without a family history of Hypertension.
Richard P Lifton - One of the best experts on this subject based on the ideXlab platform.
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wnk3 a kinase related to genes mutated in Hereditary Hypertension with hyperkalaemia regulates the k channel romk1 kir1 1
The Journal of Physiology, 2006Co-Authors: Qiang Leng, Richard P Lifton, Kristopher T Kahle, Jesse Rinehart, Gordon G Macgregor, Frederick H Wilson, Cecilia M Canessa, Steven C HebertAbstract:The serine–threonine kinase WNK3 modulates Cl− transport into and out of cells through its regulation of SLC12A cation–Cl− cotransporters, implicating it as (one of) the long-sought Cl−/volume-sensitive kinase(s). Integrators in homeostatic systems regulate structurally diverse but functionally coupled elements. For example, the related kinase WNK4 regulates the Na+–Cl− cotransporter (NCC), paracellular Cl− flux, and the K+ channel ROMK1 (Kir1.1) to maintain renal NaCl and K+ homeostasis; mutations in PRKWNK4, encoding WNK4, cause a Mendelian disease featuring Hypertension and hyperkalaemia. It is known that WNK3 is expressed in the nephron's distal convoluted tubule (DCT) and stimulates NCC activity. Here, we show that WNK3 is also expressed in cortical and outer medullary collecting duct principal cells. Accordingly, we tested WNK3's effect on the mediators of NaCl and K+ handling in these nephron segments – the epithelial sodium channel (ENaC), paracellular Cl− flux, and ROMK1 – using established model systems. WNK3 did not alter paracellular Cl− flux in tetracycline-responsive MDCK II cells, nor affect amiloride-sensitive currents when coexpressed with ENaC in Xenopus laevis oocytes. However, additional coexpression studies in oocytes revealed WNK3 inhibited the renal-specific K+ channel ROMK1 activity greater than 5.5-fold (P < 0.0001) by altering its plasmalemmal surface expression; WNK3 did not affect ROMK1's conductance or open/closed probability. In contrast, WNK3 had no effect on the activity of the cardiac long-QT syndrome K+ channel KCNQ1/KCNE1 when coexpressed in oocytes. Inhibition of ROMK1 is independent of WNK3's catalytic activity and is mediated by WNK3's carboxyl terminus – a mechanism distinct from its known kinase-dependent activation of NCC. A kinase-inactivating point mutation or a missense mutation homologous to one in WNK4 that causes disease produced a gain-of-function effect, enhancing WNK3's inhibition of ROMK1 greater than 2.5-fold relative to wild-type kinase (P < 0.0001). The magnitude and specificity of WNK3's effects at both NCC and ROMK1, its coexpression with its targets in the distal nephron, and the established in vivo effect of WNK4 at these same targets provide evidence that WNK3's action is physiologically relevant. WNK3 is probably a component of one of the mechanisms that determines the balance between renal NaCl reabsorption and K+ secretion.
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wnk3 bypasses the tonicity requirement for k cl cotransporter activation via a phosphatase dependent pathway
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Paola De Los Heros, Richard P Lifton, Kristopher T Kahle, Jesse Rinehart, Steven C Hebert, David B. Mount, Norma A Bobadilla, Norma Vazquez, Pedro San Cristobal, Gerardo GambaAbstract:Abstract SLC12A cation/Cl− cotransporters are mutated in human disease, are targets of diuretics, and are collectively involved in the regulation of cell volume, neuronal excitability, and blood pressure. This gene family has two major branches with different physiological functions and inverse regulation: K-Cl cotransporters (KCC1–KCC4) mediate cellular Cl− efflux, are inhibited by phosphorylation, and are activated by dephosphorylation; Na-(K)-Cl cotransporters (NCC and NKCC1/2) mediate cellular Cl− influx and are activated by phosphorylation. A single kinase/phosphatase pathway is thought to coordinate the activities of these cotransporters in a given cell; however, the mechanisms involved are as yet unknown. We previously demonstrated that WNK3, a paralog of serine-threonine kinases mutated in Hereditary Hypertension, is coexpressed with several cation/Cl− cotransporters and regulates their activity. Here, we show that WNK3 completely prevents the cell swelling-induced activation of KCC1–KCC4 in Xenopus oocytes. In contrast, catalytically inactive WNK3 abolishes the cell shrinkage-induced inhibition of KCC1–KCC4, resulting in a >100-fold stimulation of K-Cl cotransport during conditions in which transport is normally inactive. This activation is completely abolished by calyculin A and cyclosporine A, inhibitors of protein phosphatase 1 and 2B, respectively. Wild-type WNK3 activates Na-(K)-Cl cotransporters by increasing their phosphorylation, and catalytically inactive kinase inhibits Na-(K)-Cl cotransporters by decreasing their phosphorylation, such that our data suggest that WNK3 is a crucial component of the kinase/phosphatase signaling pathway that coordinately regulates the Cl− influx and efflux branches of the SLC12A cotransporter family. ion transport protein serine-threonine kinases Hypertension cell volume regulation
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WNK3, a kinase related to genes mutated in Hereditary Hypertension with hyperkalaemia, regulates the K+ channel ROMK1 (Kir1.1).
The Journal of physiology, 2005Co-Authors: Qiang Leng, Richard P Lifton, Kristopher T Kahle, Jesse Rinehart, Gordon G Macgregor, Frederick H Wilson, Cecilia M Canessa, Steven C HebertAbstract:The serine-threonine kinase WNK3 modulates Cl- transport into and out of cells through its regulation of SLC12A cation/Cl- cotransporters, implicating it as (one of) the long-sought Cl-/volume-sensitive kinase(s). Integrators in homeostatic systems regulate structurally diverse but functionally coupled elements. For example, the related kinase WNK4 regulates the Na-Cl co-transporter (NCC), paracellular Cl- flux, and the K+ channel ROMK1 (Kir1.1) to maintain renal NaCl and K+ homeostasis; mutations in PRKWNK4, encoding WNK4, cause a Mendelian disease featuring Hypertension and hyperkalemia. It is known that WNK3 is expressed in the nephron's distal convoluted tubule (DCT) and stimulates NCC activity. Here, we show that WNK3 is also expressed in cortical and outer medullary collecting duct principal cells. Accordingly, we tested WNK3's effect on the mediators of NaCl and K+ handling in these nephron segments--the epithelial sodium channel (ENaC), paracellular Cl- flux, and ROMK1--using established model systems. WNK3 did not alter paracellular Cl- flux in tetracycline-responsive MDCK II cells, nor affect amiloride-sensitive currents when co-expressed with ENaC in Xenopus laevis oocytes. However, additional co-expression studies in oocytes revealed WNK3 inhibited the renal-specific K+ channel ROMK1 activity greater than 5.5-fold (p < .0001) by altering its plasmalemmal surface expression; WNK3 did not affect ROMK1's conductance or open/closed probability. In contrast, WNK3 had no effect on the activity of the cardiac long-QT syndrome K+ channel KCNQ1/KCNE1 when co-expressed in oocytes. Inhibition of ROMK1 is independent of WNK3's catalytic activity and is mediated by WNK3's carboxyl terminus--a mechanism distinct from its known kinase-dependent activation of NCC. A kinase-inactivating point mutation, or a missense mutation homologous to one in WNK4 that causes disease produced a gain-of-function effect, enhancing WNK3's inhibition of ROMK1 greater than 2.5-fold relative to wild type kinase (p < .0001). The magnitude and specificity of WNK3's effects at both NCC and ROMK1, its co-expression with its targets in the distal nephron, and the established in vivo effect of WNK4 at these same targets provide evidence that WNK3's action is physiologically relevant. WNK3 is likely a component of one of the mechanisms that determines the balance between renal NaCl reabsorption and K+ secretion.
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Hereditary Hypertension caused by chimaeric gene duplications and ectopic expression of aldosterone synthase
Nature Genetics, 1992Co-Authors: Richard P Lifton, Robert G Dluhy, Michael Powers, Glenn M Rich, Michael Gutkin, Francesco Fallo, John R Gill, Leonard G Feld, Arunabha GangulyAbstract:Patients with glucocorticoid–remediable aldosteronism (GRA) from 12 kindreds possess chimaeric gene duplications arising from unequal crossing–over, fusing regulatory sequences of steroid 11β–hydroxylase to coding sequences of aldosterone synthase. These chimaeric genes are specific for GRA and explain the biochemistry, physiology and genetics of this form of Hypertension. Sites of crossing over range from intron 2 to intron 4. Most mutations have arisen independently from either sister or non–sister chromatid exchange between these genes, which are only 45 kilobases apart. The possibility of a susceptibility allele for GRA of Irish origin is suggested. These findings indicate the utility of a direct genetic test for this disorder.
Steven C Hebert - One of the best experts on this subject based on the ideXlab platform.
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wnk3 a kinase related to genes mutated in Hereditary Hypertension with hyperkalaemia regulates the k channel romk1 kir1 1
The Journal of Physiology, 2006Co-Authors: Qiang Leng, Richard P Lifton, Kristopher T Kahle, Jesse Rinehart, Gordon G Macgregor, Frederick H Wilson, Cecilia M Canessa, Steven C HebertAbstract:The serine–threonine kinase WNK3 modulates Cl− transport into and out of cells through its regulation of SLC12A cation–Cl− cotransporters, implicating it as (one of) the long-sought Cl−/volume-sensitive kinase(s). Integrators in homeostatic systems regulate structurally diverse but functionally coupled elements. For example, the related kinase WNK4 regulates the Na+–Cl− cotransporter (NCC), paracellular Cl− flux, and the K+ channel ROMK1 (Kir1.1) to maintain renal NaCl and K+ homeostasis; mutations in PRKWNK4, encoding WNK4, cause a Mendelian disease featuring Hypertension and hyperkalaemia. It is known that WNK3 is expressed in the nephron's distal convoluted tubule (DCT) and stimulates NCC activity. Here, we show that WNK3 is also expressed in cortical and outer medullary collecting duct principal cells. Accordingly, we tested WNK3's effect on the mediators of NaCl and K+ handling in these nephron segments – the epithelial sodium channel (ENaC), paracellular Cl− flux, and ROMK1 – using established model systems. WNK3 did not alter paracellular Cl− flux in tetracycline-responsive MDCK II cells, nor affect amiloride-sensitive currents when coexpressed with ENaC in Xenopus laevis oocytes. However, additional coexpression studies in oocytes revealed WNK3 inhibited the renal-specific K+ channel ROMK1 activity greater than 5.5-fold (P < 0.0001) by altering its plasmalemmal surface expression; WNK3 did not affect ROMK1's conductance or open/closed probability. In contrast, WNK3 had no effect on the activity of the cardiac long-QT syndrome K+ channel KCNQ1/KCNE1 when coexpressed in oocytes. Inhibition of ROMK1 is independent of WNK3's catalytic activity and is mediated by WNK3's carboxyl terminus – a mechanism distinct from its known kinase-dependent activation of NCC. A kinase-inactivating point mutation or a missense mutation homologous to one in WNK4 that causes disease produced a gain-of-function effect, enhancing WNK3's inhibition of ROMK1 greater than 2.5-fold relative to wild-type kinase (P < 0.0001). The magnitude and specificity of WNK3's effects at both NCC and ROMK1, its coexpression with its targets in the distal nephron, and the established in vivo effect of WNK4 at these same targets provide evidence that WNK3's action is physiologically relevant. WNK3 is probably a component of one of the mechanisms that determines the balance between renal NaCl reabsorption and K+ secretion.
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wnk3 bypasses the tonicity requirement for k cl cotransporter activation via a phosphatase dependent pathway
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Paola De Los Heros, Richard P Lifton, Kristopher T Kahle, Jesse Rinehart, Steven C Hebert, David B. Mount, Norma A Bobadilla, Norma Vazquez, Pedro San Cristobal, Gerardo GambaAbstract:Abstract SLC12A cation/Cl− cotransporters are mutated in human disease, are targets of diuretics, and are collectively involved in the regulation of cell volume, neuronal excitability, and blood pressure. This gene family has two major branches with different physiological functions and inverse regulation: K-Cl cotransporters (KCC1–KCC4) mediate cellular Cl− efflux, are inhibited by phosphorylation, and are activated by dephosphorylation; Na-(K)-Cl cotransporters (NCC and NKCC1/2) mediate cellular Cl− influx and are activated by phosphorylation. A single kinase/phosphatase pathway is thought to coordinate the activities of these cotransporters in a given cell; however, the mechanisms involved are as yet unknown. We previously demonstrated that WNK3, a paralog of serine-threonine kinases mutated in Hereditary Hypertension, is coexpressed with several cation/Cl− cotransporters and regulates their activity. Here, we show that WNK3 completely prevents the cell swelling-induced activation of KCC1–KCC4 in Xenopus oocytes. In contrast, catalytically inactive WNK3 abolishes the cell shrinkage-induced inhibition of KCC1–KCC4, resulting in a >100-fold stimulation of K-Cl cotransport during conditions in which transport is normally inactive. This activation is completely abolished by calyculin A and cyclosporine A, inhibitors of protein phosphatase 1 and 2B, respectively. Wild-type WNK3 activates Na-(K)-Cl cotransporters by increasing their phosphorylation, and catalytically inactive kinase inhibits Na-(K)-Cl cotransporters by decreasing their phosphorylation, such that our data suggest that WNK3 is a crucial component of the kinase/phosphatase signaling pathway that coordinately regulates the Cl− influx and efflux branches of the SLC12A cotransporter family. ion transport protein serine-threonine kinases Hypertension cell volume regulation
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WNK3, a kinase related to genes mutated in Hereditary Hypertension with hyperkalaemia, regulates the K+ channel ROMK1 (Kir1.1).
The Journal of physiology, 2005Co-Authors: Qiang Leng, Richard P Lifton, Kristopher T Kahle, Jesse Rinehart, Gordon G Macgregor, Frederick H Wilson, Cecilia M Canessa, Steven C HebertAbstract:The serine-threonine kinase WNK3 modulates Cl- transport into and out of cells through its regulation of SLC12A cation/Cl- cotransporters, implicating it as (one of) the long-sought Cl-/volume-sensitive kinase(s). Integrators in homeostatic systems regulate structurally diverse but functionally coupled elements. For example, the related kinase WNK4 regulates the Na-Cl co-transporter (NCC), paracellular Cl- flux, and the K+ channel ROMK1 (Kir1.1) to maintain renal NaCl and K+ homeostasis; mutations in PRKWNK4, encoding WNK4, cause a Mendelian disease featuring Hypertension and hyperkalemia. It is known that WNK3 is expressed in the nephron's distal convoluted tubule (DCT) and stimulates NCC activity. Here, we show that WNK3 is also expressed in cortical and outer medullary collecting duct principal cells. Accordingly, we tested WNK3's effect on the mediators of NaCl and K+ handling in these nephron segments--the epithelial sodium channel (ENaC), paracellular Cl- flux, and ROMK1--using established model systems. WNK3 did not alter paracellular Cl- flux in tetracycline-responsive MDCK II cells, nor affect amiloride-sensitive currents when co-expressed with ENaC in Xenopus laevis oocytes. However, additional co-expression studies in oocytes revealed WNK3 inhibited the renal-specific K+ channel ROMK1 activity greater than 5.5-fold (p < .0001) by altering its plasmalemmal surface expression; WNK3 did not affect ROMK1's conductance or open/closed probability. In contrast, WNK3 had no effect on the activity of the cardiac long-QT syndrome K+ channel KCNQ1/KCNE1 when co-expressed in oocytes. Inhibition of ROMK1 is independent of WNK3's catalytic activity and is mediated by WNK3's carboxyl terminus--a mechanism distinct from its known kinase-dependent activation of NCC. A kinase-inactivating point mutation, or a missense mutation homologous to one in WNK4 that causes disease produced a gain-of-function effect, enhancing WNK3's inhibition of ROMK1 greater than 2.5-fold relative to wild type kinase (p < .0001). The magnitude and specificity of WNK3's effects at both NCC and ROMK1, its co-expression with its targets in the distal nephron, and the established in vivo effect of WNK4 at these same targets provide evidence that WNK3's action is physiologically relevant. WNK3 is likely a component of one of the mechanisms that determines the balance between renal NaCl reabsorption and K+ secretion.
Angelosaristeidis Konstas - One of the best experts on this subject based on the ideXlab platform.
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Conservation of pH sensitivity in the epithelial sodium channel (ENaC) with Liddle's syndrome mutation.
Pflugers Archiv : European journal of physiology, 2000Co-Authors: Angelosaristeidis Konstas, D Mavrelos, Christoph KorbmacherAbstract:Gain-of-function mutations of the epithelial Na+ channel (ENaC) cause a rare form of Hereditary Hypertension, Liddle's syndrome. How these mutations lead to increased channel activity is not yet fully understood. Since wild-type ENaC (wt-ENaC) is highly pH-sensitive, we wondered whether an altered pH-sensitivity of ENaC might contribute to the hyperactivity of ENaC with Liddle's syndrome mutation (Liddle-ENaC). Using Xenopus laevis oocytes as an expression system, we compared the pH-sensitivity of wt-ENaC (alphabetagammarENaC) and Liddle-ENaC (alphabeta(R564stop)gammarENaC). Oocytes were assayed for an amiloride-sensitive (2 microM) inward current (deltaIami) at -60 mV holding potential and cytosolic pH was altered by changing the extracellular pH in the presence of 60 mM sodium acetate. Alternatively, cytosolic acidification was achieved by proton loading the cells using a proton-coupled oligopeptide transporter (PepT-1) co-expressed in the oocytes together with ENaC. Cytosolic but not extracellular acidification substantially reduced deltaIami while cytosolic alkalinisation had a stimulatory effect. This pH-sensitivity was largely preserved in oocytes expressing Liddle-ENaC. The inhibition of wt-ENaC and Liddle-ENaC by cytosolic acidification was independent of so-called sodium-feedback inhibition, since it was not associated with a concomitant increase in intracellular Na+ concentration estimated from the reversal potential of deltaIami. In addition C-terminal deletions in the alpha or gamma subunits or in all three subunits of ENaC did not abolish the inhibitory effect of cytosolic acidification. We conclude that ENaC's pH-sensitivity is not mediated by its cytoplasmic C-termini and that an altered pH-sensitivity of ENaC does not contribute to the pathophysiology of Liddle's syndrome.
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conservation of ph sensitivity in the epithelial sodium channel enac with liddle s syndrome mutation
Pflügers Archiv: European Journal of Physiology, 2000Co-Authors: Angelosaristeidis Konstas, D Mavrelos, Christoph KorbmacherAbstract:Gain-of-function mutations of the epithelial Na+ channel (ENaC) cause a rare form of Hereditary Hypertension, Liddle's syndrome. How these mutations lead to increased channel activity is not yet fully understood. Since wild-type ENaC (wt-ENaC) is highly pH-sensitive, we wondered whether an altered pH-sensitivity of ENaC might contribute to the hyperactivity of ENaC with Liddle's syndrome mutation (Liddle-ENaC). Using Xenopus laevis oocytes as an expression system, we compared the pH-sensitivity of wt-ENaC (αβγrENaC) and Liddle-ENaC (αβR564stopγrENaC). Oocytes were assayed for an amiloride-sensitive (2 µM) inward current (ΔI ami) at –60 mV holding potential and cytosolic pH was altered by changing the extracellular pH in the presence of 60 mM sodium acetate. Alternatively, cytosolic acidification was achieved by proton loading the cells using a proton-coupled oligopeptide transporter (PepT-1) co-expressed in the oocytes together with ENaC. Cytosolic but not extracellular acidification substantially reduced ΔI ami while cytosolic alkalinisation had a stimulatory effect. This pH-sensitivity was largely preserved in oocytes expressing Liddle-ENaC. The inhibition of wt-ENaC and Liddle-ENaC by cytosolic acidification was independent of so-called sodium-feedback inhibition, since it was not associated with a concomitant increase in intracellular Na+ concentration estimated from the reversal potential of ΔI ami. In addition C-terminal deletions in the α or γ subunits or in all three subunits of ENaC did not abolish the inhibitory effect of cytosolic acidification. We conclude that ENaC's pH-sensitivity is not mediated by its cytoplasmic C-termini and that an altered pH-sensitivity of ENaC does not contribute to the pathophysiology of Liddle's syndrome.