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Wenhui Wang - One of the best experts on this subject based on the ideXlab platform.
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angiotensin ii type 2 receptor regulates ROMK like k channel activity in the renal cortical collecting duct during high dietary k adaptation
American Journal of Physiology-renal Physiology, 2014Co-Authors: Yi Liao, Wenhui Wang, Beth Zavilowitz, Pokman Chan, Rajeev Rohatgi, Genevieve Estilo, Edwin K Jackson, Lisa M SatlinAbstract:The kidney adjusts K+ excretion to match intake in part by regulation of the activity of apical K+ secretory channels, including renal outer medullary K+ (ROMK)-like K+ channels, in the cortical collecting duct (CCD). ANG II inhibits ROMK channels via the ANG II type 1 receptor (AT1R) during dietary K+ restriction. Because AT1Rs and ANG II type 2 receptors (AT2Rs) generally function in an antagonistic manner, we sought to characterize the regulation of ROMK channels by the AT2R. Patch-clamp experiments revealed that ANG II increased ROMK channel activity in CCDs isolated from high-K+ (HK)-fed but not normal K+ (NK)-fed rats. This response was blocked by PD-123319, an AT2R antagonist, but not by losartan, an AT1R antagonist, and was mimicked by the AT2R agonist CGP-42112. Nitric oxide (NO) synthase is present in CCD cells that express ROMK channels. Blockade of NO synthase with N-nitro-l-arginine methyl ester and free NO with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt completely abolished ANG II-stimulated ROMK channel activity. NO enhances the synthesis of cGMP, which inhibits phosphodiesterases (PDEs) that normally degrade cAMP; cAMP increases ROMK channel activity. Pretreatment of CCDs with IBMX, a broad-spectrum PDE inhibitor, or cilostamide, a PDE3 inhibitor, abolished the stimulatory effect of ANG II on ROMK channels. Furthermore, PKA inhibitor peptide, but not an activator of the exchange protein directly activated by cAMP (Epac), also prevented the stimulatory effect of ANG II. We conclude that ANG II acts at the AT2R to stimulate ROMK channel activity in CCDs from HK-fed rats, a response opposite to that mediated by the AT1R in dietary K+-restricted animals, via a NO/cGMP pathway linked to a cAMP-PKA pathway.
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microrna 802 stimulates ROMK channels by suppressing caveolin 1
Journal of The American Society of Nephrology, 2011Co-Authors: Wenhui WangAbstract:Dietary potassium stimulates the surface expression of ROMK channels in the aldosterone-sensitive distal nephron, but the mechanism by which this occurs is incompletely understood. Here, a highpotassium diet increased the transcription of microRNA (miR) 802 in the cortical collecting duct in mice. In addition, high-potassium intake decreased the expression of caveolin-1, whose 3 untranslated region contains the seed sequence of miR-802. In vitro, expression of miR-802 suppressed the expression of caveolin-1, and conversely, downregulation of endogenous miR-802 increased the expression of caveolin-1. Sucrose-gradient centrifugation suggested that caveolin-1 closely associated with ROMK channels, and immunoprecipitation showed that caveolin-1 interacted with the N terminus of ROMK. Expression of caveolin-1 varied inversely with the expression of ROMK1 in the plasma membrane, and caveolin-1 inhibited ROMK1 channel activity. Removal of the clathrin-dependent endocytosis motif from ROMK1 failed to abolish the effect of caveolin-1 on ROMK1 channel activity. Last, expression of miR-802 increased ROMK1 channel activity, an effect blocked by coexpression of caveolin-1. Taken together, miR-802 mediates the stimulatory effect of a high-potassium diet on ROMK channel activity by suppressing caveolin-1 expression, which leads to increased surface expression of ROMK channels in the distal nephron.
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src family protein tyrosine kinase ptk modulates the effect of sgk1 and wnk4 on ROMK channels
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Qiang Leng, Gerhard Giebisch, Richard P Lifton, Wenhui WangAbstract:WNK4 (with no lysine kinase 4) inhibits ROMK channel activity in the distal nephron by stimulating clathrin-dependent endocytosis, an effect attenuated by SGK1 (serum-glucocorticoids-induced kinase)-mediated phosphorylation. It has been suggested that increased ROMK activity because of SGK1-mediated inhibition of WNK4 plays a role in promoting renal K secretion in response to elevated serum K or high K (HK) intake. In contrast, intravascular volume depletion also increases SGK1 activity but fails to stimulate ROMK channels and K secretion. Because HK intake decreases Src family protein tyrosine kinase (PTK) activity an inhibitor of ROMK channels, it is possible that Src family PTK may modulate the effects of SGK1 on WNK4. Here, we show that c-Src prevents SGK1 from attenuating WNK4's inhibition of ROMK activity. This effect of c-Src was WNK4-dependent because c-Src had no effect on ROMK harboring mutation at the site of c-Src phosphorylation (R1Y337A) in the absence of WNK4. Moreover, expression c-Src diminished the SGK1-mediated increase in serine phosphorylation of WNK4, suggesting that c-Src enhances WNK4-mediated inhibition of ROMK channels by suppressing the SGK1-induced phosphorylation. This notion is also supported by the observation that c-Src was not able to modulate the interaction between SGK1 and WNK4 mutants (WNK4S1169A or WNK4S1169D) in which an SGK1-phosphorylation site (serine 1169) was mutated by alanine or aspartate. We conclude that c-Src inhibits SGK1-mediated phosphorylation hereby restoring the WNK4-mediated inhibition of ROMK channels thus suppressing K secretion.
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expression of tetraspan protein cd63 activates protein tyrosine kinase ptk and enhances the ptk induced inhibition of ROMK channels
Journal of Biological Chemistry, 2008Co-Authors: Erik Jan Kamsteeg, Hyacinth Sterling, Yan Zhang, Marcel Roos, Amy S Duffield, Joanna Spencer, Michael S Caplan, Wenhui WangAbstract:Abstract In the present study, we tested the role of CD63 in regulating ROMK1 channels by protein-tyrosine kinase (PTK). Immunocytochemical staining shows that CD63 and receptor-linked tyrosine phosphatase α (RPTPα) are expressed in the cortical collecting duct and outer medulla collecting duct. Immunoprecipitation of tissue lysates from renal cortex and outer medulla or 293T cells transfected with CD63 reveals that CD63 was associated with RPTPα both in situ and in transfected cells. Expression of CD63 in 293T cells stimulated the phosphorylation of tyrosine residue 416 of c-Src but decreased the phosphorylation of tyrosine residue 527, indicating that expression of CD63 stimulates the activity of c-Src. Furthermore, c-Src was coimmunoprecipitated with RPTPα and CD63 both in 293T cells transfected with CD63 and in lysates prepared from native rat kidney. Potassium restriction had no effect on the expression of RPTPα, but it increased the association between c-Src and RPTPα in the renal cortex and outer medulla. We also used two-electrode voltage clamp to study the effect of CD63 on ROMK channels in Xenopus oocytes. Expression of CD63 had no significant effect on potassium currents in oocytes injected with ROMK1; however, it significantly enhanced the c-Src-induced inhibition of ROMK channels in oocytes injected with ROMK1+c-Src. The effect of CD63 on the c-Src-induced inhibition was not due to a decreased expression of ROMK1 channels, because blocking PTK with herbimycin A abolished the inhibitory effect of c-Src on ROMK channels in oocytes injected with ROMK1+c-Src+CD63. Furthermore, coexpression of CD63 enhanced tyrosine phosphorylation of ROMK1. We conclude that CD63 plays a role in the regulation of ROMK channels through its association with RPTPα, which in turn interacts with and activates Src family PTK, thus reducing ROMK activity.
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role of gp91phox containing nadph oxidase in mediating the effect of k restriction on ROMK channels and renal k excretion
Journal of The American Society of Nephrology, 2007Co-Authors: Elisa Babilonia, Yan Zhang, Wenhui WangAbstract:Previous study has demonstrated that superoxide and the related products are involved in mediating the effect of low K intake on renal K secretion and ROMK channel activity in the cortical collecting duct (CCD). This study investigated the role of gp91 phox -containing NADPH oxidase (NOXII) in mediating the effect of low K intake on renal K excretion and ROMK channel activity in gp91(−/−) mice. K depletion increased superoxide levels, phosphorylation of c-Jun, expression of c-Src, and tyrosine phosphorylation of ROMK in renal cortex and outer medulla in wild-type (WT) mice. In contrast, tempol treatment in WT mice abolished whereas deletion of gp91 significantly attenuated the effect of low K intake on superoxide production, c-Jun phosphorylation, c-Src expression, and tyrosine phosphorylation of ROMK. Patch-clamp experiments demonstrated that low K intake decreased mean product of channel number (N) and open probability (P) (NP o ) of ROMK channels from 1.1 to 0.4 in the CCD. However, the effect of low K intake on ROMK channel activity was significantly attenuated in the CCD from gp91(−/−) mice and completely abolished by tempol treatment. Immunocytochemical staining also was used to examine the ROMK distribution in WT, gp91(−/−), and WT mice with tempol treatment in response to K restriction. K restriction decreased apical staining of ROMK in WT mice. In contrast, a sharp apical ROMK staining was observed in the tempol-treated WT or gp91(−/−) mice. Metabolic cage study further showed that urinary K loss is significantly higher in gp91(−/−) mice than in WT mice. It is concluded that superoxide anions play a key role in suppressing K secretion during K restriction and that NOXII is involved in mediating the effect of low K intake on renal K secretion and ROMK channel activity.
Steven C Hebert - One of the best experts on this subject based on the ideXlab platform.
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phosphorylation regulated endoplasmic reticulum retention signal in the renal outer medullary k channel ROMK
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Anthony D Oconnell, Gordon G Macgregor, Ke Dong, Qiang Leng, Gerhard Giebisch, Steven C HebertAbstract:The renal outer-medullary K + channel (ROMK; Kir1.1) mediates K + secretion in the renal mammalian nephron that is critical to both sodium and potassium homeostasis. The posttranscriptional expression of ROMK in the plasma membrane of cells is regulated by delivery of protein from endoplasmic reticulum (ER) to the cell surface and by retrieval by dynamin-dependent endocytic mechanisms in clathrin-coated pits. The S44 in the NH 2 terminus of ROMK1 can be phosphorylated by PKA and serum- and glucocorticoid-inducible kinase-1, and this process increases surface expression of functional channels. We present evidence that phosphorylation of S44 modulates channel expression by increasing its cell surface delivery consequent to suppression of a COOH-terminal ER retention signal. This phosphorylation switch of the ER retention signal could provide a pool of mature and properly folded channels for rapid delivery to the plasma membrane. The x-ray crystal structures of inward rectifier K + channels have shown a close apposition of the NH 2 terminus with the distal COOH terminus of the adjacent subunit in the channel homotetramer, which is important to channel gating. Thus, NH 2 -terminal phosphorylation modifying a COOH-terminal ER retention signal in ROMK1 could serve as a checkpoint for proper subunit folding critical to channel gating.
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regulation by glucocorticoids and osmolality of expression of ROMK kir 1 1 the apical k channel of thick ascending limb
American Journal of Physiology-renal Physiology, 2003Co-Authors: Morgan Gallazzini, Steven C Hebert, Amel Attmaneelakeb, David B Mount, Maurice BicharaAbstract:Mechanisms of regulation of ROMK channel mRNA and protein expression in medullary thick ascending limb (MTAL) were assessed in rat MTAL fragments incubated for 7 h. ROMK mRNA was quantified by quantitative RT-PCR and ROMK protein by immunoblotting analysis of crude membranes. Medium hyperosmolality (450 mosmol/kgH2O; NaCl plus urea added to isoosmotic medium) increased ROMK mRNA (P < 0.04) and protein (P < 0.006), and 10 nM dexamethasone also increased ROMK mRNA (P < 0.02). Hyperosmolality and dexamethasone had no additive effects on ROMK mRNA. NaCl alone, but not urea or mannitol, reproduced the hyperosmolality effect on ROMK mRNA. 1-Deamino-(8-d-arginine) vasopressin (1 nM) or 0.5 mM 8-bromo-cAMP had no effect per se on ROMK mRNA and protein. However, 8-bromo-cAMP abolished the stimulatory effect of dexamethasone on ROMK mRNA in the isoosmotic but not in the hyperosmotic medium (P < 0.004). In in vivo studies, the abundance of ROMK protein and mRNA increased in adrenalectomized (ADX) rats infused with d...
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absence of small conductance k channel sk activity in apical membranes of thick ascending limb and cortical collecting duct in ROMK bartter s knockout mice
Journal of Biological Chemistry, 2002Co-Authors: Ming Lu, G Giebisch, Wenhui Wang, Ke Dong, Tong Wang, Xinbo Yang, Mark A Knepper, Gary E Shull, Steven C HebertAbstract:Abstract The ROMK (Kir1.1; Kcnj1) gene is believed to encode the apical small conductance K+channels (SK) of the thick ascending limb (TAL) and cortical collecting duct (CCD). Loss-of-function mutations in the human ROMK gene cause Bartter's syndrome with renal Na+ wasting, consistent with the role of this channel in apical K+ recycling in the TAL that is crucial for NaCl reabsorption. However, the mechanism of renal K+ wasting and hypokalemia that develop in individuals with ROMK Bartter's syndrome is not apparent given the proposed loss of the collecting duct SK channel. Thus, we generated a colony of ROMK null mice with ∼25% survival to adulthood that provides a good model for ROMK Bartter's syndrome. The remaining 75% of null mice die in less than 14 days after birth. The surviving ROMK null mice have normal gross renal morphology with no evidence of significant hydronephrosis, whereas non-surviving null mice exhibit marked hydronephrosis. ROMK protein expression was absent in TAL and CCD from null mice but exhibited normal abundance and localization in wild-type littermates. ROMK null mice were polyuric and natriuretic with an elevated hematocrit consistent with mild extracellular volume depletion. SK channel activity in TAL and CCD was assessed by patch clamp analysis in ROMK wild-type ROMK(+/+), heterozygous ROMK(+/−), and null ROMK(−/−) mice. In 313 patches with successful seals from the three ROMK genotypes, SK channel activity in ROMK (+/+ and +/−) exhibited normal single channel kinetics. The expression frequencies are as follows: 67 (TAL) and 58% (CCD) in ROMK(+/+); about half that of the wild-type in ROMK(+/−), being 38 (TAL) and 25% (CCD); absent in both TAL or CCD in ROMK(−/−) between 2 and 5 weeks in 15 mice (61 and 66 patches, respectively). The absence of SK channel activity in ROMK null mice demonstrates that ROMK is essential for functional expression of SK channels in both TAL and CCD. Despite loss of ROMK expression, the normokalemic null mice exhibited significantly increased kaliuresis, indicating alternative mechanisms for K+absorption/secretion in the nephron.
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ph dependent modulation of the cloned renal k channel ROMK
American Journal of Physiology-renal Physiology, 1998Co-Authors: Carmel M Mcnicholas, Gordon G Macgregor, Leon D Islas, Yinhai Yang, Steven C Hebert, G GiebischAbstract:pH is an important modulator of the low-conductance ATP-sensitive K+ channel of the distal nephron. To examine the mechanism of interaction of protons with the channel-forming protein, we expressed the cloned renal K channel, ROMK (Kir1.x), in Xenopus oocytes and examined the response to varied concentrations of protons both in the presence and in the absence of ATP. Initial experiments were performed on inside-out patches in the absence of ATP in Mg2+-free solution, which prevents channel rundown. A steep sigmoidal relationship was shown between bath pH and ROMK1 or ROMK2 channel function with intracellular acidification reducing channel activity. We calculated values for pK = 7.18 and 7.04 and Hill coefficients = 3.1 and 3.3, for ROMK1 and ROMK2, respectively. Intracellular acidification (pH 7.2) also increased the Mg-ATP binding affinity of ROMK2, resulting in a leftward shift of the relationship between ATP concentration and the reduction in channel activity. TheK 1/2 for Mg-ATP decreased from 2.4 mM ...
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localization of the ROMK protein on apical membranes of rat kidney nephron segments
American Journal of Physiology-renal Physiology, 1997Co-Authors: Jason Z Xu, Amy E Hall, L N Peterson, Michael J Bienkowski, Thomas E Eessalu, Steven C HebertAbstract:The ATP-sensitive, inwardly rectifying K+ channel, ROMK, has been suggested to be the low-conductance ATP-sensitive K+ channel identified in apical membranes of mammalian renal thick ascending limb (TAL) and cortical collecting duct (CCD). Mutations in the human ROMK gene (KIR1.2) have been identified in kindreds with neonatal Bartter’s syndrome. In the present study, we generated polyclonal antibodies raised against both a COOH-terminal (amino acids 252–391) ROMK-maltose binding protein (MBP) fusion protein and an NH2-terminal (amino acids 34–49) ROMK peptide. Affinity-purified anti-ROMK COOH-terminal antibody detected the 45-kDa ROMK protein in kidney tissues and HEK-293 cells transfected with ROMK1 cDNA. The antibody also recognized 85- to 90-kDa proteins in kidney tissue; these higher molecular weight proteins were abolished by immunoabsorption with ROMK-MBP fusion protein and were also detected on Western blots using anti-ROMK NH2-terminal antibody. Immunofluorescence studies using anti-ROMK COOH-ter...
Christian Derst - One of the best experts on this subject based on the ideXlab platform.
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classification and rescue of ROMK mutations underlying hyperprostaglandin e syndrome antenatal bartter syndrome
Kidney International, 2003Co-Authors: Melanie Peters, Christian Derst, Hannsjorg W Seyberth, Nikola Jeck, Stefanie Weber, Saskia Ermert, Ulla Pechmann, Karl P Schlingmann, Siegfried Waldegger, Martin KonradAbstract:Classification and rescue of ROMK mutations underlying hyperprostaglandin E syndrome/antenatal Bartter syndrome. Background Mutations in the renal K + channel ROMK (Kir 1.1) cause hyperprostaglandin E syndrome/antenatal Bartter syndrome (HPS/aBS), a severe tubular disorder leading to renal salt and water wasting. Several studies confirmed the predominance of alterations of current properties in ROMK mutants. However, in most of these studies, analysis was restricted to nonmammalian cells and electrophysiologic methods. Therefore, for the majority of ROMK mutations, disturbances in protein trafficking remained unclear. The aim of the present study was the evaluation of different pathogenic mechanisms of 20 naturally occurring ROMK mutations with consecutive classification into mutational classes and identification of distinct rescue mechanisms according to the underlying defect. Methods Mutated ROMK potassium channels were expressed in Xenopus oocytes and a human kidney cell line and analyzed by two electrode voltage clamp analysis, immunofluorescence, and Western blot analysis. Results We identified 14 out of 20 ROMK mutations that did not reach the cell surface, indicating defective membrane trafficking. High expression levels rescued six out of 14 ROMK mutants, leading to significant K + currents. In addition, two early inframe stop mutations could be rescued by aminoglycosides, resulting in full-length ROMK and correct trafficking to the plasma membrane in a subset of transfected cells. Conclusion In contrast to previous reports, most of the investigated ROMK mutations displayed a trafficking defect that might be rescued by pharmacologic agents acting as molecular chaperones. The evaluation of different disease-causing mechanisms will be essential for establishing new and more specific therapeutic strategies for HPS/aBS patients.
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Classification and rescue of ROMK mutations underlying hyperprostaglandin E syndrome/antenatal Bartter syndrome.
Kidney International, 2003Co-Authors: Melanie Peters, Christian Derst, Hannsjorg W Seyberth, Nikola Jeck, Stefanie Weber, Saskia Ermert, Ulla Pechmann, Karl P Schlingmann, Siegfried Waldegger, Martin KonradAbstract:Classification and rescue of ROMK mutations underlying hyperprostaglandin E syndrome/antenatal Bartter syndrome. Background Mutations in the renal K + channel ROMK (Kir 1.1) cause hyperprostaglandin E syndrome/antenatal Bartter syndrome (HPS/aBS), a severe tubular disorder leading to renal salt and water wasting. Several studies confirmed the predominance of alterations of current properties in ROMK mutants. However, in most of these studies, analysis was restricted to nonmammalian cells and electrophysiologic methods. Therefore, for the majority of ROMK mutations, disturbances in protein trafficking remained unclear. The aim of the present study was the evaluation of different pathogenic mechanisms of 20 naturally occurring ROMK mutations with consecutive classification into mutational classes and identification of distinct rescue mechanisms according to the underlying defect. Methods Mutated ROMK potassium channels were expressed in Xenopus oocytes and a human kidney cell line and analyzed by two electrode voltage clamp analysis, immunofluorescence, and Western blot analysis. Results We identified 14 out of 20 ROMK mutations that did not reach the cell surface, indicating defective membrane trafficking. High expression levels rescued six out of 14 ROMK mutants, leading to significant K + currents. In addition, two early inframe stop mutations could be rescued by aminoglycosides, resulting in full-length ROMK and correct trafficking to the plasma membrane in a subset of transfected cells. Conclusion In contrast to previous reports, most of the investigated ROMK mutations displayed a trafficking defect that might be rescued by pharmacologic agents acting as molecular chaperones. The evaluation of different disease-causing mechanisms will be essential for establishing new and more specific therapeutic strategies for HPS/aBS patients.
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functional heterogeneity of ROMK mutations linked to hyperprostaglandin e syndrome
Kidney International, 2001Co-Authors: Christian Derst, Nikola Jeck, Erhard Wischmeyer, Stefanie Weber, Christoph RudinAbstract:Functional heterogeneity of ROMK mutations linked to hyperprostaglandin E syndrome. Background The renal K + channel ROMK (Kir1.1) controls salt reabsorption in the kidney. Loss-of-function mutations in this channel cause hyperprostaglandin E syndrome/antenatal Bartter syndrome (HPS/aBS), which is characterized by severe renal salt and fluid wasting. Methods We investigated 10 HPS/aBS patients for mutations in the ROMK gene by single-strand conformation polymorphism analysis (SSCA) and direct sequencing. To assess the functional consequences, Ba 2+ -sensitive K + currents were measured in five mutants of the core region as well as one mutant with truncated C-terminus, using the two-electrode voltage-clamp technique after an injection of mutant cRNA into Xenopus oocytes. Results Three novel ROMK mutations were identified together with six mutations described previously. The mutations were categorized into three groups: ( 1 ) amino acid exchanges in the core region (M1-H5-M2), ( 2 ) truncation at the cytosolic C-terminus, and ( 3 ) deletions of putative promoter elements. While the core mutations W99C, N124K, and I142T led to significantly reduced macroscopic K + currents (1 to 8% of wild-type currents), the A103V and P110L variants retained substantial K + conductivity (23 and 35% of wild-type currents, respectively). Coexpression of A103V and P110L, resembling the compound heterozygous state of the affected individual, further reduced macroscopic currents to 9% of the wild-type currents. All mutants in the core region exerted a dominant-negative effect on wild-type ROMK1. The C-terminal frameshift (fs) mutation (H354fs) did not change current amplitudes compared with ROMK1 wild type, suggesting that a mechanism other than alteration of the electrophysiological properties may responsible for loss of channel activity. Conclusions Analysis of ROMK mutants linked to HPS/aBS revealed a spectrum of mechanisms accounting for loss of channel function. Further characterization of the molecular defects might be helpful for the development of new therapeutic approaches.
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mutations in the ROMK gene in antenatal bartter syndrome are associated with impaired k channel function
Biochemical and Biophysical Research Communications, 1997Co-Authors: Christian Derst, Martin Konrad, Arnold Kockerling, Lothar Karolyi, Georges Deschenes, Jurgen Daut, Andreas Karschin, Hannsjorg W SeyberthAbstract:Abstract Children with the antenatal variant of Bartter syndrome present the typical pattern of impaired salt reabsorption in the thick ascending limb of Henle's loop (TALH) resulting in marked ante- and postnatal salt wasting. In some of these patients mutations in the renal potassium channel ROMK (KCNJ1) have been found. We analyzed the electrophysiological function of five recently described ROMK channel mutations (V72E, D108H, P110L, A198T and V315G). In whole cell patch clamp recordings wildtype rat ROMK1 exhibited K+currents of >1 nA at a membrane potential of 100 mV when transfected into COS-7 kidney cells. These currents were sensitive to external Ba2+and internal Mg2+, which are typical features of the inwardly rectifying KIRchannel. In contrast mutated ROMK1 cDNAs expressed either no or only infrequently small currents (
Hannsjorg W Seyberth - One of the best experts on this subject based on the ideXlab platform.
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Expression of the potassium channel ROMK in adult and fetal human kidney
Histochemistry and Cell Biology, 2005Co-Authors: Rolf M. Nüsing, Hannsjorg W Seyberth, Fiore Pantalone, Hermann-josef Gröne, Markus WegmannAbstract:The renal potassium channel ROMK is a crucial element of K^+ recycling and secretion in the distal tubule and the collecting duct system. Mutations in the ROMK gene ( KCNJ1 ) lead to hyperprostaglandin E syndrome/antenatal Bartter syndrome, a life-threatening hypokalemic disorder of the newborn. The localization of ROMK channel protein, however, remains unknown in humans. We generated an affinity-purified specific polyclonal anti-ROMK antibody raised against a C-terminal peptide of human ROMK. Immunoblotting revealed a 45 kDa protein band in both rat and human kidney tissue. In human kidney sections, the antibody showed intense staining of epithelial cells in the cortical and medullary thick ascending limb (TAL), the connecting tubule, and the collecting duct. Moreover, a strong expression of ROMK protein was detected in cells of the macula densa. In epithelial cells of the TAL expression of ROMK protein was mainly restricted to the apical membrane. In human fetal kidney expression of ROMK protein was detected mainly in distal tubules of mature nephrons but not or only marginally in the collecting system. No expression was found in early developmental stages such as comma or S shapes, indicating a differentiation-dependent expression of ROMK protein. In summary, these findings support the proposed role of ROMK channels in potassium recycling and in the regulation of K^+ secretion and present a rationale for the phenotype observed in patients with ROMK deficiency.
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classification and rescue of ROMK mutations underlying hyperprostaglandin e syndrome antenatal bartter syndrome
Kidney International, 2003Co-Authors: Melanie Peters, Christian Derst, Hannsjorg W Seyberth, Nikola Jeck, Stefanie Weber, Saskia Ermert, Ulla Pechmann, Karl P Schlingmann, Siegfried Waldegger, Martin KonradAbstract:Classification and rescue of ROMK mutations underlying hyperprostaglandin E syndrome/antenatal Bartter syndrome. Background Mutations in the renal K + channel ROMK (Kir 1.1) cause hyperprostaglandin E syndrome/antenatal Bartter syndrome (HPS/aBS), a severe tubular disorder leading to renal salt and water wasting. Several studies confirmed the predominance of alterations of current properties in ROMK mutants. However, in most of these studies, analysis was restricted to nonmammalian cells and electrophysiologic methods. Therefore, for the majority of ROMK mutations, disturbances in protein trafficking remained unclear. The aim of the present study was the evaluation of different pathogenic mechanisms of 20 naturally occurring ROMK mutations with consecutive classification into mutational classes and identification of distinct rescue mechanisms according to the underlying defect. Methods Mutated ROMK potassium channels were expressed in Xenopus oocytes and a human kidney cell line and analyzed by two electrode voltage clamp analysis, immunofluorescence, and Western blot analysis. Results We identified 14 out of 20 ROMK mutations that did not reach the cell surface, indicating defective membrane trafficking. High expression levels rescued six out of 14 ROMK mutants, leading to significant K + currents. In addition, two early inframe stop mutations could be rescued by aminoglycosides, resulting in full-length ROMK and correct trafficking to the plasma membrane in a subset of transfected cells. Conclusion In contrast to previous reports, most of the investigated ROMK mutations displayed a trafficking defect that might be rescued by pharmacologic agents acting as molecular chaperones. The evaluation of different disease-causing mechanisms will be essential for establishing new and more specific therapeutic strategies for HPS/aBS patients.
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Classification and rescue of ROMK mutations underlying hyperprostaglandin E syndrome/antenatal Bartter syndrome.
Kidney International, 2003Co-Authors: Melanie Peters, Christian Derst, Hannsjorg W Seyberth, Nikola Jeck, Stefanie Weber, Saskia Ermert, Ulla Pechmann, Karl P Schlingmann, Siegfried Waldegger, Martin KonradAbstract:Classification and rescue of ROMK mutations underlying hyperprostaglandin E syndrome/antenatal Bartter syndrome. Background Mutations in the renal K + channel ROMK (Kir 1.1) cause hyperprostaglandin E syndrome/antenatal Bartter syndrome (HPS/aBS), a severe tubular disorder leading to renal salt and water wasting. Several studies confirmed the predominance of alterations of current properties in ROMK mutants. However, in most of these studies, analysis was restricted to nonmammalian cells and electrophysiologic methods. Therefore, for the majority of ROMK mutations, disturbances in protein trafficking remained unclear. The aim of the present study was the evaluation of different pathogenic mechanisms of 20 naturally occurring ROMK mutations with consecutive classification into mutational classes and identification of distinct rescue mechanisms according to the underlying defect. Methods Mutated ROMK potassium channels were expressed in Xenopus oocytes and a human kidney cell line and analyzed by two electrode voltage clamp analysis, immunofluorescence, and Western blot analysis. Results We identified 14 out of 20 ROMK mutations that did not reach the cell surface, indicating defective membrane trafficking. High expression levels rescued six out of 14 ROMK mutants, leading to significant K + currents. In addition, two early inframe stop mutations could be rescued by aminoglycosides, resulting in full-length ROMK and correct trafficking to the plasma membrane in a subset of transfected cells. Conclusion In contrast to previous reports, most of the investigated ROMK mutations displayed a trafficking defect that might be rescued by pharmacologic agents acting as molecular chaperones. The evaluation of different disease-causing mechanisms will be essential for establishing new and more specific therapeutic strategies for HPS/aBS patients.
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mutations in the ROMK gene in antenatal bartter syndrome are associated with impaired k channel function
Biochemical and Biophysical Research Communications, 1997Co-Authors: Christian Derst, Martin Konrad, Arnold Kockerling, Lothar Karolyi, Georges Deschenes, Jurgen Daut, Andreas Karschin, Hannsjorg W SeyberthAbstract:Abstract Children with the antenatal variant of Bartter syndrome present the typical pattern of impaired salt reabsorption in the thick ascending limb of Henle's loop (TALH) resulting in marked ante- and postnatal salt wasting. In some of these patients mutations in the renal potassium channel ROMK (KCNJ1) have been found. We analyzed the electrophysiological function of five recently described ROMK channel mutations (V72E, D108H, P110L, A198T and V315G). In whole cell patch clamp recordings wildtype rat ROMK1 exhibited K+currents of >1 nA at a membrane potential of 100 mV when transfected into COS-7 kidney cells. These currents were sensitive to external Ba2+and internal Mg2+, which are typical features of the inwardly rectifying KIRchannel. In contrast mutated ROMK1 cDNAs expressed either no or only infrequently small currents (
Chou-long Huang - One of the best experts on this subject based on the ideXlab platform.
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activation of pi3 kinase stimulates endocytosis of ROMK via akt1 sgk1 dependent phosphorylation of wnk1
Journal of The American Society of Nephrology, 2011Co-Authors: Chih-jen Cheng, Chou-long HuangAbstract:WNK kinases stimulate endocytosis of ROMK channels to regulate renal K+ handling. Phosphatidylinositol 3-kinase (PI3K)-activating hormones, such as insulin and IGF 1, phosphorylate WNK1, but how this affects the regulation of ROMK abundance is unknown. Here, serum starvation of ROMK-transfected HEK cells led to an increase of ROMK current density; subsequent addition of insulin or IGF1 inhibited ROMK currents in a PI3K-dependent manner. Serum and insulin also increased phosphorylation of the downstream kinases Akt1 and SGK1 as well as WNK1. A biotinylation assay suggested that insulin and IGF1 inhibit ROMK by enhancing its endocytosis, a process that WNK1 may mediate. Knockdown of WNK1 with siRNA or expression of a phospho-deficient WNK1 mutant (T58A) both prevented insulin-induced inhibition of ROMK currents, suggesting that phosphorylation at Threonine-58 of WNK1 is important to mediate the inhibition of ROMK by PI3K-activating hormones or growth factors. In vitro and in vivo kinase assays supported the notion that Akt1 and SGK1 can phosphorylate WNK1 at this site, and we established that Akt1 and SGK1 synergistically inhibit ROMK through WNK1. We used dominant-negative intersectin and dynamin constructs to show that SGK1-mediated phosphorylation of WNK1 inhibits ROMK by promoting its endocytosis. Taken together, these results suggest that PI3K-activating hormones inhibit ROMK by enhancing its endocytosis via a mechanism that involves phosphorylation of WNK1 by Akt1 and SGK1.
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Activation of PI3-Kinase Stimulates Endocytosis of ROMK via Akt1/SGK1-Dependent Phosphorylation of WNK1
Journal of The American Society of Nephrology, 2011Co-Authors: Chih-jen Cheng, Chou-long HuangAbstract:WNK kinases stimulate endocytosis of ROMK channels to regulate renal K+ handling. Phosphatidylinositol 3-kinase (PI3K)-activating hormones, such as insulin and IGF 1, phosphorylate WNK1, but how this affects the regulation of ROMK abundance is unknown. Here, serum starvation of ROMK-transfected HEK cells led to an increase of ROMK current density; subsequent addition of insulin or IGF1 inhibited ROMK currents in a PI3K-dependent manner. Serum and insulin also increased phosphorylation of the downstream kinases Akt1 and SGK1 as well as WNK1. A biotinylation assay suggested that insulin and IGF1 inhibit ROMK by enhancing its endocytosis, a process that WNK1 may mediate. Knockdown of WNK1 with siRNA or expression of a phospho-deficient WNK1 mutant (T58A) both prevented insulin-induced inhibition of ROMK currents, suggesting that phosphorylation at Threonine-58 of WNK1 is important to mediate the inhibition of ROMK by PI3K-activating hormones or growth factors. In vitro and in vivo kinase assays supported the notion that Akt1 and SGK1 can phosphorylate WNK1 at this site, and we established that Akt1 and SGK1 synergistically inhibit ROMK through WNK1. We used dominant-negative intersectin and dynamin constructs to show that SGK1-mediated phosphorylation of WNK1 inhibits ROMK by promoting its endocytosis. Taken together, these results suggest that PI3K-activating hormones inhibit ROMK by enhancing its endocytosis via a mechanism that involves phosphorylation of WNK1 by Akt1 and SGK1.
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regulation of ROMK channel and k homeostasis by kidney specific wnk1 kinase
Journal of Biological Chemistry, 2009Co-Authors: Hao Ran Wang, Chou-long HuangAbstract:WNK kinases are serine-threonine kinases with an atypical placement of the catalytic lysine. WNK1, the first member discovered, has multiple alternatively spliced isoforms, including a ubiquitously expressed full-length long form (L-WNK1) and a kidney-specific form (KS-WNK1) predominantly expressed in the kidney. Intronic deletions of WNK1 that increase WNK1 transcript cause pseudohypoaldosteronism type 2, an autosomal-dominant disease characterized by hypertension and hyperkalemia. L-WNK1 inhibits renal K+ channel ROMK, likely contributing to hyperkalemia in PHAII. Previously, we reported that KS-WNK1 by itself has no effect on ROMK1 but antagonizes L-WNK1-mediated inhibition of ROMK1. Amino acids 1–253 of KS-WNK1 (KS-WNK1(1–253)) are sufficient for reversing the inhibition of ROMK1 caused by L-WNK1(1–491). Here, we further investigated the mechanisms by which KS-WNK1 counteracts L-WNK1 regulation of ROMK1. We reported that two regions of KS-WNK1(1–253) are involved in the antagonism of L-WNK1; one includes the first 30 amino acids unique for KS-WNK1 encoded by the alternatively spliced initiating exon 4A, and the other is equivalent to the autoinhibitory domain (AID) of L-WNK1. Mutations of two phenylalanine residues known to be critical for autoinhibitory function of AID abolish the ability of the AID region of KS-WNK1 to antagonize L-WNK1. To examine the physiological role of KS-WNK1 in the regulation of renal K+ secretion, we generated transgenic mice that overexpress amino acids 1–253 of KS-WNK1 under the control of a kidney-specific promoter. Transgenic mice have lower serum K+ levels and higher urinary fractional excretion of K+ compared with wild type littermates despite the same amount of daily urinary K+ excretion. Moreover, transgenic mice (compared with wild type littermates) displayed a higher abundance of ROMK on the apical membrane of distal nephron. Thus, KS-WNK1 is an important physiological regulator of renal K+ excretion, likely through its effects on the ROMK1 channel.
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domains of wnk1 kinase in the regulation of ROMK1
American Journal of Physiology-renal Physiology, 2008Co-Authors: Hao Ran Wang, Chou-long HuangAbstract:WNK1 kinase belongs to a family of serine-threonine protein kinases with an atypical placement of the catalytic lysine. Increased expression of WNK1 causes hypertension and hyperkalemia in humans. WNK1 inhibits renal potassium channel ROMK1 by enhancing its endocytosis, likely contributing to hyperkalemia in affected patients. The domains of WNK1 involved in inhibition of ROMK1 have not been completely elucidated. Here, we reported that an NH2-terminal proline-rich domain (N-PRD; amino acids 1-119) is necessary and sufficient for WNK1 inhibition of ROMK1. A region (named “NL” for N-linker; amino acids 120-220) located between N-PRD and the kinase domain of WNK1 (amino acids 220-491) antagonized the inhibition of ROMK1 caused by N-PRD. The WNK1 kinase domain reversed the antagonism of NL on N-PRD. Mutagenesis studies revealed that charge-charge interactions between two conserved catalytic residues (Lys-233 and Asp-368) within the kinase domain (not the kinase activity) are critical for kinase domain to reverse the antagonism of NL domain. The WNK1 autoinhibitory domain (AID; amino acids 491-555) also affected ROMK, presumably by modulating the kinase domain conformation. Mutations of two conserved phenylalanine abolished the ability of AID to modulate ROMK1. Finally, the first coiled-coil domain (CC1; amino acids 555-640) of WNK1 alleviated the effect of AID domain toward kinase domain. Thus, multiple intra- and/or intermolecular interactions of WNK1 domains are at play for regulation of ROMK1 by WNK1.
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antagonistic regulation of ROMK by long and kidney specific wnk1 isoforms
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Ahmed Lazrak, Chou-long HuangAbstract:WNK kinases are serine-threonine kinases with an atypical placement of the catalytic lysine. Intronic deletions with increased expression of a ubiquitous long WNK1 transcript cause pseudohypoaldosteronism type 2 (PHA II), characterized by hypertension and hyperkalemia. Here, we report that long WNK1 inhibited ROMK1 by stimulating its endocytosis. Inhibition of ROMK by long WNK1 was synergistic with, but not dependent on, WNK4. A smaller transcript of WNK1 lacking the N-terminal 1-437 amino acids is expressed highly in the kidney. Whether expression of the KS-WNK1 (kidney-specific, KS) is altered in PHA II is not known. We found that KS-WNK1 did not inhibit ROMK1 but reversed the inhibition of ROMK1 caused by long WNK1. Consistent with the lack of inhibition by KS-WNK1, we found that amino acids 1-491 of the long WNK1 were sufficient for inhibiting ROMK. Dietary K+ restriction decreases ROMK abundance in the renal cortical-collecting ducts by stimulating endocytosis, an adaptative response important for conservation of K+ during K+ deficiency. We found that K+ restriction in rats increased whole-kidney transcript of long WNK1 while decreasing that of KS-WNK1. Thus, KS-WNK1 is a physiological antagonist of long WNK1. Hyperkalemia in PHA II patients with PHA II mutations may be caused, at least partially, by increased expression of long WNK1 with or without decreased expression of KS-WNK1.
