The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Steven C Hebert - One of the best experts on this subject based on the ideXlab platform.
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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, Norma Vazquez, Pedro San Cristobal, Steven C Hebert, Richard P Lifton, Jesse Rinehart, Kristopher T. Kahle, David B. Mount, Norma A. Bobadilla, 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 modulates transport of cl in and out of cells implications for control of cell volume and neuronal excitability
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Kristopher T. Kahle, Paola De Los Heros, Norma Vazquez, Steven C Hebert, Angeliki Louvi, Ignacio Gimenez, Gerardo Gamba, Jesse Rinehart, Patricia Meade, Richard P LiftonAbstract:Abstract The regulation of Cl- transport into and out of cells plays a critical role in the maintenance of intracellular volume and the excitability of GABA responsive neurons. The molecular determinants of these seemingly diverse processes are related ion Cotransporters: Cl- influx is mediated by the Na-K-2Cl Cotransporter NKCC1 and Cl- efflux via K-Cl Cotransporters, KCC1 or KCC2. A Cl-/volume-sensitive kinase has been proposed to coordinately regulate these activities via altered phosphorylation of the transporters; phosphorylation activates NKCC1 while inhibiting KCCs, and dephosphorylation has the opposite effects. We show that WNK3, a member of the WNK family of serine-threonine kinases, colocalizes with NKCC1 and KCC1/2 in diverse Cl--transporting epithelia and in neurons expressing ionotropic GABAA receptors in the hippocampus, cerebellum, cerebral cortex, and reticular activating system. By expression studies in Xenopus oocytes, we show that kinase-active WNK3 increases Cl- influx via NKCC1, and that it inhibits Cl- exit through KCC1 and KCC2; kinase-inactive WNK3 has the opposite effects. WNK3's effects are imparted via altered phosphorylation and surface expression of its downstream targets and bypass the normal requirement of altered tonicity for activation of these transporters. Together, these data indicate that WNK3 can modulate the level of intracellular Cl- via opposing actions on entry and exit pathways. They suggest that WNK3 is part of the Cl-/volume-sensing mechanism necessary for the maintenance of cell volume during osmotic stress and the dynamic modulation of GABA neurotransmission. cell volume GABA ion transport protein serine-threonine kinases
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wnk3 modulates transport of cl in and out of cells implications for control of cell volume and neuronal excitability
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Kristopher T. Kahle, Norma Vazquez, Steven C Hebert, Angeliki Louvi, Ignacio Gimenez, Gerardo Gamba, Jesse Rinehart, Patricia Meade, Paola De Los Heros, Richard P LiftonAbstract:The regulation of Cl(-) transport into and out of cells plays a critical role in the maintenance of intracellular volume and the excitability of GABA responsive neurons. The molecular determinants of these seemingly diverse processes are related ion Cotransporters: Cl(-) influx is mediated by the Na-K-2Cl Cotransporter NKCC1 and Cl(-) efflux via K-Cl Cotransporters, KCC1 or KCC2. A Cl(-)/volume-sensitive kinase has been proposed to coordinately regulate these activities via altered phosphorylation of the transporters; phosphorylation activates NKCC1 while inhibiting KCCs, and dephosphorylation has the opposite effects. We show that WNK3, a member of the WNK family of serine-threonine kinases, colocalizes with NKCC1 and KCC1/2 in diverse Cl(-)-transporting epithelia and in neurons expressing ionotropic GABA(A) receptors in the hippocampus, cerebellum, cerebral cortex, and reticular activating system. By expression studies in Xenopus oocytes, we show that kinase-active WNK3 increases Cl(-) influx via NKCC1, and that it inhibits Cl(-) exit through KCC1 and KCC2; kinase-inactive WNK3 has the opposite effects. WNK3's effects are imparted via altered phosphorylation and surface expression of its downstream targets and bypass the normal requirement of altered tonicity for activation of these transporters. Together, these data indicate that WNK3 can modulate the level of intracellular Cl(-) via opposing actions on entry and exit pathways. They suggest that WNK3 is part of the Cl(-)/volume-sensing mechanism necessary for the maintenance of cell volume during osmotic stress and the dynamic modulation of GABA neurotransmission.
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n glycosylation at two sites critically alters thiazide binding and activity of the rat thiazide sensitive na cl Cotransporter
Journal of The American Society of Nephrology, 2003Co-Authors: Robert S Hoover, Norma Vazquez, Gerardo Gamba, Esteban Poch, Adriana Monroy, Toshiyuki Nishio, Steven C HebertAbstract:ABSTRACT. The rat thiazide-sensitive Na-Cl Cotransporter (rNCC) is expressed in the renal distal convoluted tubule and is the site of action of an important class of antihypertensive agents, the thiazide diuretics. The amino acid sequence contains two potential N -linked glycosylation consensus sites, N404 and N424. Either enzymatic deglycosylation or tunicamycin reduced the Cotransporter to its core molecular weight (113 kD). Glycosylation site single mutants expressed in oocytes ran as thick bands at 115 kD, consistent with the high-mannose glycoprotein. The double mutant produced the single thin 113-kD band seen in the deglycosylated Cotransporter. Functional expression of Cotransporters in Xenopus laevis oocytes revealed that the mutants displayed drastically decreased thiazide-sensitive 22 Na + uptake compared with wild-type NCC. Analysis of enhanced green fluorescence protein (EGFP)–tagged Cotransporters demonstrated that this decrease in function is predominantly secondary to decreased surface expression. The elimination of glycosylation in the double mutant increased thiazide sensitivity by more than two orders of magnitude and also increased Cl − affinity. Thus, we have demonstrated that rNCC is N -glycosylated in vivo at two sites, that glycosylation is essential for efficient function and surface expression of the Cotransporter, and that the elimination of glycosylation allows much greater access of thiazide diuretics to their binding site. E-mail: steven.hebert@yale.edu
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molecular cloning and chromosome localization of a putative basolateral na k 2cl Cotransporter from mouse inner medullary collecting duct mimcd 3 cells
Journal of Biological Chemistry, 1994Co-Authors: Eric Delpire, Steven C Hebert, M I Rauchman, David R Beier, Steven R GullansAbstract:Electroneutral Na(+)-K(+)-2Cl- Cotransporters represent one of the major routes for Cl- movement in epithelia. A secretory form of the Cotransporter has been described in the basolateral membrane of a variety of epithelia from fish to mammals. We isolated a putative bumetanide-sensitive Na(+)-K(+)-2Cl- Cotransporter cDNA, BSC2, from mIMCD-3 cells. Northern analysis indicates that in contrast to BSC1, the recently cloned renal-specific apical isoform of the Cotransporter, BSC2 is expressed in secretory epithelia and thus appears to represent the basolateral isoform. Furthermore, BSC2 is also expressed in non-polarized cells, such as red cells and myocytes. Sequence comparison and chromosome localization demonstrate that BSC2 and BSC1 are different genes that diverged before the evolution of vertebrates.
Gerardo Gamba - One of the best experts on this subject based on the ideXlab platform.
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SPAK and OSR1 play essential roles in potassium homeostasis through actions on the distal convoluted tubule.
The Journal of Physiology, 2016Co-Authors: Mohammed Zubaerul Ferdaus, Karl W. Barber, Karen I. López-cayuqueo, Andrew S. Terker, Eduardo R. Argaiz, Brandon M. Gassaway, Régine Chambrey, Gerardo Gamba, Jesse Rinehart, James A. MccormickAbstract:Key points STE20 (Sterile 20)/SPS-1 related proline/alanine-rich kinase (SPAK) and oxidative stress-response kinase-1 (OSR1) phosphorylate and activate the renal Na+–K+–2Cl− Cotransporter 2 (NKCC2) and Na+Cl− Cotransporter (NCC). Mouse models suggest that OSR1 mainly activates NKCC2-mediated sodium transport along the thick ascending limb, while SPAK mainly activates NCC along the distal convoluted tubule, but the kinases may compensate for each other. We hypothesized that disruption of both kinases would lead to polyuria and severe salt-wasting, and generated SPAK/OSR1 double knockout mice to test this. Despite a lack of SPAK and OSR1, phosphorylated NKCC2 abundance was still high, suggesting the existence of an alternative activating kinase. Compensatory changes in SPAK/OSR1-independent phosphorylation sites on both NKCC2 and NCC and changes in sodium transport along the collecting duct were also observed. Potassium restriction revealed that SPAK and OSR1 play essential roles in the emerging model that NCC activation is central to sensing changes in plasma [K+]. Abstract STE20 (Sterile 20)/SPS-1 related proline/alanine-rich kinase (SPAK) and oxidative stress-response kinase-1 (OSR1) activate the renal cation Cotransporters Na+–K+–2Cl− Cotransporter (NKCC2) and Na+–Cl− Cotransporter (NCC) via phosphorylation. Knockout mouse models suggest that OSR1 mainly activates NKCC2, while SPAK mainly activates NCC, with possible cross-compensation. We tested the hypothesis that disrupting both kinases causes severe polyuria and salt-wasting by generating SPAK/OSR1 double knockout (DKO) mice. DKO mice displayed lower systolic blood pressure compared with SPAK knockout (SPAK-KO) mice, but displayed no severe phenotype even after dietary salt restriction. Phosphorylation of NKCC2 at SPAK/OSR1-dependent sites was lower than in SPAK-KO mice, but still significantly greater than in wild type mice. In the renal medulla, there was significant phosphorylation of NKCC2 at SPAK/OSR1-dependent sites despite a complete absence of SPAK and OSR1, suggesting the existence of an alternative activating kinase. The distal convoluted tubule has been proposed to sense plasma [K+], with NCC activation serving as the primary effector pathway that modulates K+ secretion, by metering sodium delivery to the collecting duct. Abundance of phosphorylated NCC (pNCC) is dramatically lower in SPAK-KO mice than in wild type mice, and the additional disruption of OSR1 further reduced pNCC. SPAK-KO and kidney-specific OSR1 single knockout mice maintained plasma [K+] following dietary potassium restriction, but DKO mice developed severe hypokalaemia. Unlike mice lacking SPAK or OSR1 alone, DKO mice displayed an inability to phosphorylate NCC under these conditions. These data suggest that SPAK and OSR1 are essential components of the effector pathway that maintains plasma [K+].
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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, Norma Vazquez, Pedro San Cristobal, Steven C Hebert, Richard P Lifton, Jesse Rinehart, Kristopher T. Kahle, David B. Mount, Norma A. Bobadilla, 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 modulates transport of cl in and out of cells implications for control of cell volume and neuronal excitability
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Kristopher T. Kahle, Paola De Los Heros, Norma Vazquez, Steven C Hebert, Angeliki Louvi, Ignacio Gimenez, Gerardo Gamba, Jesse Rinehart, Patricia Meade, Richard P LiftonAbstract:Abstract The regulation of Cl- transport into and out of cells plays a critical role in the maintenance of intracellular volume and the excitability of GABA responsive neurons. The molecular determinants of these seemingly diverse processes are related ion Cotransporters: Cl- influx is mediated by the Na-K-2Cl Cotransporter NKCC1 and Cl- efflux via K-Cl Cotransporters, KCC1 or KCC2. A Cl-/volume-sensitive kinase has been proposed to coordinately regulate these activities via altered phosphorylation of the transporters; phosphorylation activates NKCC1 while inhibiting KCCs, and dephosphorylation has the opposite effects. We show that WNK3, a member of the WNK family of serine-threonine kinases, colocalizes with NKCC1 and KCC1/2 in diverse Cl--transporting epithelia and in neurons expressing ionotropic GABAA receptors in the hippocampus, cerebellum, cerebral cortex, and reticular activating system. By expression studies in Xenopus oocytes, we show that kinase-active WNK3 increases Cl- influx via NKCC1, and that it inhibits Cl- exit through KCC1 and KCC2; kinase-inactive WNK3 has the opposite effects. WNK3's effects are imparted via altered phosphorylation and surface expression of its downstream targets and bypass the normal requirement of altered tonicity for activation of these transporters. Together, these data indicate that WNK3 can modulate the level of intracellular Cl- via opposing actions on entry and exit pathways. They suggest that WNK3 is part of the Cl-/volume-sensing mechanism necessary for the maintenance of cell volume during osmotic stress and the dynamic modulation of GABA neurotransmission. cell volume GABA ion transport protein serine-threonine kinases
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wnk3 modulates transport of cl in and out of cells implications for control of cell volume and neuronal excitability
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Kristopher T. Kahle, Norma Vazquez, Steven C Hebert, Angeliki Louvi, Ignacio Gimenez, Gerardo Gamba, Jesse Rinehart, Patricia Meade, Paola De Los Heros, Richard P LiftonAbstract:The regulation of Cl(-) transport into and out of cells plays a critical role in the maintenance of intracellular volume and the excitability of GABA responsive neurons. The molecular determinants of these seemingly diverse processes are related ion Cotransporters: Cl(-) influx is mediated by the Na-K-2Cl Cotransporter NKCC1 and Cl(-) efflux via K-Cl Cotransporters, KCC1 or KCC2. A Cl(-)/volume-sensitive kinase has been proposed to coordinately regulate these activities via altered phosphorylation of the transporters; phosphorylation activates NKCC1 while inhibiting KCCs, and dephosphorylation has the opposite effects. We show that WNK3, a member of the WNK family of serine-threonine kinases, colocalizes with NKCC1 and KCC1/2 in diverse Cl(-)-transporting epithelia and in neurons expressing ionotropic GABA(A) receptors in the hippocampus, cerebellum, cerebral cortex, and reticular activating system. By expression studies in Xenopus oocytes, we show that kinase-active WNK3 increases Cl(-) influx via NKCC1, and that it inhibits Cl(-) exit through KCC1 and KCC2; kinase-inactive WNK3 has the opposite effects. WNK3's effects are imparted via altered phosphorylation and surface expression of its downstream targets and bypass the normal requirement of altered tonicity for activation of these transporters. Together, these data indicate that WNK3 can modulate the level of intracellular Cl(-) via opposing actions on entry and exit pathways. They suggest that WNK3 is part of the Cl(-)/volume-sensing mechanism necessary for the maintenance of cell volume during osmotic stress and the dynamic modulation of GABA neurotransmission.
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n glycosylation at two sites critically alters thiazide binding and activity of the rat thiazide sensitive na cl Cotransporter
Journal of The American Society of Nephrology, 2003Co-Authors: Robert S Hoover, Norma Vazquez, Gerardo Gamba, Esteban Poch, Adriana Monroy, Toshiyuki Nishio, Steven C HebertAbstract:ABSTRACT. The rat thiazide-sensitive Na-Cl Cotransporter (rNCC) is expressed in the renal distal convoluted tubule and is the site of action of an important class of antihypertensive agents, the thiazide diuretics. The amino acid sequence contains two potential N -linked glycosylation consensus sites, N404 and N424. Either enzymatic deglycosylation or tunicamycin reduced the Cotransporter to its core molecular weight (113 kD). Glycosylation site single mutants expressed in oocytes ran as thick bands at 115 kD, consistent with the high-mannose glycoprotein. The double mutant produced the single thin 113-kD band seen in the deglycosylated Cotransporter. Functional expression of Cotransporters in Xenopus laevis oocytes revealed that the mutants displayed drastically decreased thiazide-sensitive 22 Na + uptake compared with wild-type NCC. Analysis of enhanced green fluorescence protein (EGFP)–tagged Cotransporters demonstrated that this decrease in function is predominantly secondary to decreased surface expression. The elimination of glycosylation in the double mutant increased thiazide sensitivity by more than two orders of magnitude and also increased Cl − affinity. Thus, we have demonstrated that rNCC is N -glycosylated in vivo at two sites, that glycosylation is essential for efficient function and surface expression of the Cotransporter, and that the elimination of glycosylation allows much greater access of thiazide diuretics to their binding site. E-mail: steven.hebert@yale.edu
Kristopher T. Kahle - One of the best experts on this subject based on the ideXlab platform.
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regulated phosphorylation of the k cl Cotransporter kcc3 is a molecular switch of intracellular potassium content and cell volume homeostasis
Frontiers in Cellular Neuroscience, 2015Co-Authors: Norma C Adragna, Kristopher T. Kahle, Dandan Sun, Nagendra Babu Ravilla, Peter K Lauf, Gulnaz Begum, Arjun KhannaAbstract:The defense of cell volume against excessive shrinkage or swelling is a requirement for cell function and organismal survival. Cell swelling triggers a coordinated homeostatic response termed regulatory volume decrease (RVD), resulting in K+ and Cl– efflux via the activation of K+ channels, volume-regulated anion channels (VRACs), and the K+-Cl– Cotransporters, including KCC3. Here, we show genetic alanine (Ala) substitution at threonines (Thr) 991 and 1048 in the KCC3a isoform carboxyl-terminus, preventing inhibitory phosphorylation at these sites, not only significantly up-regulates KCC3a activity up to 25-fold in normally inhibitory isotonic conditions, but is also accompanied by reversal of activity of the related bumetanide-sensitive Na+-K+-2Cl– Cotransporter isoform 1 (NKCC1). This results in a rapid (90 %) reduction in intracellular K+ content (Ki) via both Cl-dependent (KCC3a + NKCC1) and Cl-independent (DCPIB [VRAC inhibitor]-sensitive) pathways, which collectively renders cells less prone to acute swelling in hypotonic osmotic stress. Together, these data demonstrate the phosphorylation state of Thr991/Thr1048 in the KCC3a encodes a potent switch of transporter activity, Ki homeostasis, and cell volume regulation, and reveal novel observations into the functional interaction among ion transport molecules involved in RVD.
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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, Norma Vazquez, Pedro San Cristobal, Steven C Hebert, Richard P Lifton, Jesse Rinehart, Kristopher T. Kahle, David B. Mount, Norma A. Bobadilla, 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 modulates transport of cl in and out of cells implications for control of cell volume and neuronal excitability
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Kristopher T. Kahle, Paola De Los Heros, Norma Vazquez, Steven C Hebert, Angeliki Louvi, Ignacio Gimenez, Gerardo Gamba, Jesse Rinehart, Patricia Meade, Richard P LiftonAbstract:Abstract The regulation of Cl- transport into and out of cells plays a critical role in the maintenance of intracellular volume and the excitability of GABA responsive neurons. The molecular determinants of these seemingly diverse processes are related ion Cotransporters: Cl- influx is mediated by the Na-K-2Cl Cotransporter NKCC1 and Cl- efflux via K-Cl Cotransporters, KCC1 or KCC2. A Cl-/volume-sensitive kinase has been proposed to coordinately regulate these activities via altered phosphorylation of the transporters; phosphorylation activates NKCC1 while inhibiting KCCs, and dephosphorylation has the opposite effects. We show that WNK3, a member of the WNK family of serine-threonine kinases, colocalizes with NKCC1 and KCC1/2 in diverse Cl--transporting epithelia and in neurons expressing ionotropic GABAA receptors in the hippocampus, cerebellum, cerebral cortex, and reticular activating system. By expression studies in Xenopus oocytes, we show that kinase-active WNK3 increases Cl- influx via NKCC1, and that it inhibits Cl- exit through KCC1 and KCC2; kinase-inactive WNK3 has the opposite effects. WNK3's effects are imparted via altered phosphorylation and surface expression of its downstream targets and bypass the normal requirement of altered tonicity for activation of these transporters. Together, these data indicate that WNK3 can modulate the level of intracellular Cl- via opposing actions on entry and exit pathways. They suggest that WNK3 is part of the Cl-/volume-sensing mechanism necessary for the maintenance of cell volume during osmotic stress and the dynamic modulation of GABA neurotransmission. cell volume GABA ion transport protein serine-threonine kinases
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wnk3 modulates transport of cl in and out of cells implications for control of cell volume and neuronal excitability
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Kristopher T. Kahle, Norma Vazquez, Steven C Hebert, Angeliki Louvi, Ignacio Gimenez, Gerardo Gamba, Jesse Rinehart, Patricia Meade, Paola De Los Heros, Richard P LiftonAbstract:The regulation of Cl(-) transport into and out of cells plays a critical role in the maintenance of intracellular volume and the excitability of GABA responsive neurons. The molecular determinants of these seemingly diverse processes are related ion Cotransporters: Cl(-) influx is mediated by the Na-K-2Cl Cotransporter NKCC1 and Cl(-) efflux via K-Cl Cotransporters, KCC1 or KCC2. A Cl(-)/volume-sensitive kinase has been proposed to coordinately regulate these activities via altered phosphorylation of the transporters; phosphorylation activates NKCC1 while inhibiting KCCs, and dephosphorylation has the opposite effects. We show that WNK3, a member of the WNK family of serine-threonine kinases, colocalizes with NKCC1 and KCC1/2 in diverse Cl(-)-transporting epithelia and in neurons expressing ionotropic GABA(A) receptors in the hippocampus, cerebellum, cerebral cortex, and reticular activating system. By expression studies in Xenopus oocytes, we show that kinase-active WNK3 increases Cl(-) influx via NKCC1, and that it inhibits Cl(-) exit through KCC1 and KCC2; kinase-inactive WNK3 has the opposite effects. WNK3's effects are imparted via altered phosphorylation and surface expression of its downstream targets and bypass the normal requirement of altered tonicity for activation of these transporters. Together, these data indicate that WNK3 can modulate the level of intracellular Cl(-) via opposing actions on entry and exit pathways. They suggest that WNK3 is part of the Cl(-)/volume-sensing mechanism necessary for the maintenance of cell volume during osmotic stress and the dynamic modulation of GABA neurotransmission.
Richard P Lifton - One of the best experts on this subject based on the ideXlab platform.
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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, Norma Vazquez, Pedro San Cristobal, Steven C Hebert, Richard P Lifton, Jesse Rinehart, Kristopher T. Kahle, David B. Mount, Norma A. Bobadilla, 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 modulates transport of cl in and out of cells implications for control of cell volume and neuronal excitability
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Kristopher T. Kahle, Paola De Los Heros, Norma Vazquez, Steven C Hebert, Angeliki Louvi, Ignacio Gimenez, Gerardo Gamba, Jesse Rinehart, Patricia Meade, Richard P LiftonAbstract:Abstract The regulation of Cl- transport into and out of cells plays a critical role in the maintenance of intracellular volume and the excitability of GABA responsive neurons. The molecular determinants of these seemingly diverse processes are related ion Cotransporters: Cl- influx is mediated by the Na-K-2Cl Cotransporter NKCC1 and Cl- efflux via K-Cl Cotransporters, KCC1 or KCC2. A Cl-/volume-sensitive kinase has been proposed to coordinately regulate these activities via altered phosphorylation of the transporters; phosphorylation activates NKCC1 while inhibiting KCCs, and dephosphorylation has the opposite effects. We show that WNK3, a member of the WNK family of serine-threonine kinases, colocalizes with NKCC1 and KCC1/2 in diverse Cl--transporting epithelia and in neurons expressing ionotropic GABAA receptors in the hippocampus, cerebellum, cerebral cortex, and reticular activating system. By expression studies in Xenopus oocytes, we show that kinase-active WNK3 increases Cl- influx via NKCC1, and that it inhibits Cl- exit through KCC1 and KCC2; kinase-inactive WNK3 has the opposite effects. WNK3's effects are imparted via altered phosphorylation and surface expression of its downstream targets and bypass the normal requirement of altered tonicity for activation of these transporters. Together, these data indicate that WNK3 can modulate the level of intracellular Cl- via opposing actions on entry and exit pathways. They suggest that WNK3 is part of the Cl-/volume-sensing mechanism necessary for the maintenance of cell volume during osmotic stress and the dynamic modulation of GABA neurotransmission. cell volume GABA ion transport protein serine-threonine kinases
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wnk3 modulates transport of cl in and out of cells implications for control of cell volume and neuronal excitability
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Kristopher T. Kahle, Norma Vazquez, Steven C Hebert, Angeliki Louvi, Ignacio Gimenez, Gerardo Gamba, Jesse Rinehart, Patricia Meade, Paola De Los Heros, Richard P LiftonAbstract:The regulation of Cl(-) transport into and out of cells plays a critical role in the maintenance of intracellular volume and the excitability of GABA responsive neurons. The molecular determinants of these seemingly diverse processes are related ion Cotransporters: Cl(-) influx is mediated by the Na-K-2Cl Cotransporter NKCC1 and Cl(-) efflux via K-Cl Cotransporters, KCC1 or KCC2. A Cl(-)/volume-sensitive kinase has been proposed to coordinately regulate these activities via altered phosphorylation of the transporters; phosphorylation activates NKCC1 while inhibiting KCCs, and dephosphorylation has the opposite effects. We show that WNK3, a member of the WNK family of serine-threonine kinases, colocalizes with NKCC1 and KCC1/2 in diverse Cl(-)-transporting epithelia and in neurons expressing ionotropic GABA(A) receptors in the hippocampus, cerebellum, cerebral cortex, and reticular activating system. By expression studies in Xenopus oocytes, we show that kinase-active WNK3 increases Cl(-) influx via NKCC1, and that it inhibits Cl(-) exit through KCC1 and KCC2; kinase-inactive WNK3 has the opposite effects. WNK3's effects are imparted via altered phosphorylation and surface expression of its downstream targets and bypass the normal requirement of altered tonicity for activation of these transporters. Together, these data indicate that WNK3 can modulate the level of intracellular Cl(-) via opposing actions on entry and exit pathways. They suggest that WNK3 is part of the Cl(-)/volume-sensing mechanism necessary for the maintenance of cell volume during osmotic stress and the dynamic modulation of GABA neurotransmission.
Norma Vazquez - One of the best experts on this subject based on the ideXlab platform.
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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, Norma Vazquez, Pedro San Cristobal, Steven C Hebert, Richard P Lifton, Jesse Rinehart, Kristopher T. Kahle, David B. Mount, Norma A. Bobadilla, 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 modulates transport of cl in and out of cells implications for control of cell volume and neuronal excitability
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Kristopher T. Kahle, Paola De Los Heros, Norma Vazquez, Steven C Hebert, Angeliki Louvi, Ignacio Gimenez, Gerardo Gamba, Jesse Rinehart, Patricia Meade, Richard P LiftonAbstract:Abstract The regulation of Cl- transport into and out of cells plays a critical role in the maintenance of intracellular volume and the excitability of GABA responsive neurons. The molecular determinants of these seemingly diverse processes are related ion Cotransporters: Cl- influx is mediated by the Na-K-2Cl Cotransporter NKCC1 and Cl- efflux via K-Cl Cotransporters, KCC1 or KCC2. A Cl-/volume-sensitive kinase has been proposed to coordinately regulate these activities via altered phosphorylation of the transporters; phosphorylation activates NKCC1 while inhibiting KCCs, and dephosphorylation has the opposite effects. We show that WNK3, a member of the WNK family of serine-threonine kinases, colocalizes with NKCC1 and KCC1/2 in diverse Cl--transporting epithelia and in neurons expressing ionotropic GABAA receptors in the hippocampus, cerebellum, cerebral cortex, and reticular activating system. By expression studies in Xenopus oocytes, we show that kinase-active WNK3 increases Cl- influx via NKCC1, and that it inhibits Cl- exit through KCC1 and KCC2; kinase-inactive WNK3 has the opposite effects. WNK3's effects are imparted via altered phosphorylation and surface expression of its downstream targets and bypass the normal requirement of altered tonicity for activation of these transporters. Together, these data indicate that WNK3 can modulate the level of intracellular Cl- via opposing actions on entry and exit pathways. They suggest that WNK3 is part of the Cl-/volume-sensing mechanism necessary for the maintenance of cell volume during osmotic stress and the dynamic modulation of GABA neurotransmission. cell volume GABA ion transport protein serine-threonine kinases
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wnk3 modulates transport of cl in and out of cells implications for control of cell volume and neuronal excitability
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Kristopher T. Kahle, Norma Vazquez, Steven C Hebert, Angeliki Louvi, Ignacio Gimenez, Gerardo Gamba, Jesse Rinehart, Patricia Meade, Paola De Los Heros, Richard P LiftonAbstract:The regulation of Cl(-) transport into and out of cells plays a critical role in the maintenance of intracellular volume and the excitability of GABA responsive neurons. The molecular determinants of these seemingly diverse processes are related ion Cotransporters: Cl(-) influx is mediated by the Na-K-2Cl Cotransporter NKCC1 and Cl(-) efflux via K-Cl Cotransporters, KCC1 or KCC2. A Cl(-)/volume-sensitive kinase has been proposed to coordinately regulate these activities via altered phosphorylation of the transporters; phosphorylation activates NKCC1 while inhibiting KCCs, and dephosphorylation has the opposite effects. We show that WNK3, a member of the WNK family of serine-threonine kinases, colocalizes with NKCC1 and KCC1/2 in diverse Cl(-)-transporting epithelia and in neurons expressing ionotropic GABA(A) receptors in the hippocampus, cerebellum, cerebral cortex, and reticular activating system. By expression studies in Xenopus oocytes, we show that kinase-active WNK3 increases Cl(-) influx via NKCC1, and that it inhibits Cl(-) exit through KCC1 and KCC2; kinase-inactive WNK3 has the opposite effects. WNK3's effects are imparted via altered phosphorylation and surface expression of its downstream targets and bypass the normal requirement of altered tonicity for activation of these transporters. Together, these data indicate that WNK3 can modulate the level of intracellular Cl(-) via opposing actions on entry and exit pathways. They suggest that WNK3 is part of the Cl(-)/volume-sensing mechanism necessary for the maintenance of cell volume during osmotic stress and the dynamic modulation of GABA neurotransmission.
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n glycosylation at two sites critically alters thiazide binding and activity of the rat thiazide sensitive na cl Cotransporter
Journal of The American Society of Nephrology, 2003Co-Authors: Robert S Hoover, Norma Vazquez, Gerardo Gamba, Esteban Poch, Adriana Monroy, Toshiyuki Nishio, Steven C HebertAbstract:ABSTRACT. The rat thiazide-sensitive Na-Cl Cotransporter (rNCC) is expressed in the renal distal convoluted tubule and is the site of action of an important class of antihypertensive agents, the thiazide diuretics. The amino acid sequence contains two potential N -linked glycosylation consensus sites, N404 and N424. Either enzymatic deglycosylation or tunicamycin reduced the Cotransporter to its core molecular weight (113 kD). Glycosylation site single mutants expressed in oocytes ran as thick bands at 115 kD, consistent with the high-mannose glycoprotein. The double mutant produced the single thin 113-kD band seen in the deglycosylated Cotransporter. Functional expression of Cotransporters in Xenopus laevis oocytes revealed that the mutants displayed drastically decreased thiazide-sensitive 22 Na + uptake compared with wild-type NCC. Analysis of enhanced green fluorescence protein (EGFP)–tagged Cotransporters demonstrated that this decrease in function is predominantly secondary to decreased surface expression. The elimination of glycosylation in the double mutant increased thiazide sensitivity by more than two orders of magnitude and also increased Cl − affinity. Thus, we have demonstrated that rNCC is N -glycosylated in vivo at two sites, that glycosylation is essential for efficient function and surface expression of the Cotransporter, and that the elimination of glycosylation allows much greater access of thiazide diuretics to their binding site. E-mail: steven.hebert@yale.edu
