Intracellular chloride and scaffold protein mo25 cooperatively regulate transepithelial ion transport through WNK signaling in the malpighian tubule

Qifei Sun, Yipin Wu, Sima Jonusaite, John M. Pleinis, John M. Humphreys, Haixia He, Jeffrey N. Schellinger, Radha Akella, Drew Stenesen, Helmut Krämer, Elizabeth J. Goldsmith, Aylin R. Rodan

Research output: Contribution to journalArticle

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Abstract

BackgroundWith No Lysine kinase (WNK) signaling regulates mammalian renal epithelial ion transport to maintain electrolyte and BP homeostasis. Our previous studies showed a conserved role for WNK in the regulation of transepithelial ion transport in the Drosophila Malpighian tubule. Methods Using in vitro assays and transgenic Drosophila lines, we examined two potential WNK regulators, chloride ion and the scaffold protein mouse protein 25 (Mo25), in the stimulation of transepithelial ion flux. Results In vitro, autophosphorylation of purified Drosophila WNK decreased as chloride concentration increased. In conditions inwhich tubule intracellular chloride concentration decreased from30 to 15mMas measured using a transgenic sensor, DrosophilaWNK activity acutely increased. DrosophilaWNK activity in tubules also increased or decreased when bath potassium concentration decreased or increased, respectively. However, a mutation that reduces chloride sensitivity of Drosophila WNK failed to alter transepithelial ion transport in 30 mM chloride. We, therefore, examined a role for Mo25. In in vitro kinase assays, Drosophila Mo25 enhanced the activity of the Drosophila WNK downstream kinase Fray, the fly homolog of mammalian Ste20-related proline/alanine-rich kinase (SPAK), and oxidative stress-responsive 1 protein (OSR1). Knockdown of Drosophila Mo25 in the Malpighian tubule decreased transepithelial ion flux under stimulated but not basal conditions. Finally, whereas overexpression of wild-type Drosophila WNK, with or without Drosophila Mo25, did not affect transepithelial ion transport, Drosophila Mo25 overexpressed with chloride-insensitive Drosophila WNK increased ion flux. Conclusions Cooperative interactions between chloride andMo25 regulateWNK signaling in a transporting renal epithelium.

Original languageEnglish (US)
Pages (from-to)1449-1461
Number of pages13
JournalJournal of the American Society of Nephrology
Volume29
Issue number5
DOIs
StatePublished - May 1 2018

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Malpighian Tubules
Ion Transport
Drosophila
Chlorides
Drosophila Proteins
Proteins
Ions
Phosphotransferases
Kidney
Baths
Diptera
Electrolytes
Lysine
Potassium
Oxidative Stress
Homeostasis
Epithelium
Mutation

ASJC Scopus subject areas

  • Nephrology

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Intracellular chloride and scaffold protein mo25 cooperatively regulate transepithelial ion transport through WNK signaling in the malpighian tubule. / Sun, Qifei; Wu, Yipin; Jonusaite, Sima; Pleinis, John M.; Humphreys, John M.; He, Haixia; Schellinger, Jeffrey N.; Akella, Radha; Stenesen, Drew; Krämer, Helmut; Goldsmith, Elizabeth J.; Rodan, Aylin R.

In: Journal of the American Society of Nephrology, Vol. 29, No. 5, 01.05.2018, p. 1449-1461.

Research output: Contribution to journalArticle

Sun, Qifei ; Wu, Yipin ; Jonusaite, Sima ; Pleinis, John M. ; Humphreys, John M. ; He, Haixia ; Schellinger, Jeffrey N. ; Akella, Radha ; Stenesen, Drew ; Krämer, Helmut ; Goldsmith, Elizabeth J. ; Rodan, Aylin R. / Intracellular chloride and scaffold protein mo25 cooperatively regulate transepithelial ion transport through WNK signaling in the malpighian tubule. In: Journal of the American Society of Nephrology. 2018 ; Vol. 29, No. 5. pp. 1449-1461.
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abstract = "BackgroundWith No Lysine kinase (WNK) signaling regulates mammalian renal epithelial ion transport to maintain electrolyte and BP homeostasis. Our previous studies showed a conserved role for WNK in the regulation of transepithelial ion transport in the Drosophila Malpighian tubule. Methods Using in vitro assays and transgenic Drosophila lines, we examined two potential WNK regulators, chloride ion and the scaffold protein mouse protein 25 (Mo25), in the stimulation of transepithelial ion flux. Results In vitro, autophosphorylation of purified Drosophila WNK decreased as chloride concentration increased. In conditions inwhich tubule intracellular chloride concentration decreased from30 to 15mMas measured using a transgenic sensor, DrosophilaWNK activity acutely increased. DrosophilaWNK activity in tubules also increased or decreased when bath potassium concentration decreased or increased, respectively. However, a mutation that reduces chloride sensitivity of Drosophila WNK failed to alter transepithelial ion transport in 30 mM chloride. We, therefore, examined a role for Mo25. In in vitro kinase assays, Drosophila Mo25 enhanced the activity of the Drosophila WNK downstream kinase Fray, the fly homolog of mammalian Ste20-related proline/alanine-rich kinase (SPAK), and oxidative stress-responsive 1 protein (OSR1). Knockdown of Drosophila Mo25 in the Malpighian tubule decreased transepithelial ion flux under stimulated but not basal conditions. Finally, whereas overexpression of wild-type Drosophila WNK, with or without Drosophila Mo25, did not affect transepithelial ion transport, Drosophila Mo25 overexpressed with chloride-insensitive Drosophila WNK increased ion flux. Conclusions Cooperative interactions between chloride andMo25 regulateWNK signaling in a transporting renal epithelium.",
author = "Qifei Sun and Yipin Wu and Sima Jonusaite and Pleinis, {John M.} and Humphreys, {John M.} and Haixia He and Schellinger, {Jeffrey N.} and Radha Akella and Drew Stenesen and Helmut Kr{\"a}mer and Goldsmith, {Elizabeth J.} and Rodan, {Aylin R.}",
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T1 - Intracellular chloride and scaffold protein mo25 cooperatively regulate transepithelial ion transport through WNK signaling in the malpighian tubule

AU - Sun, Qifei

AU - Wu, Yipin

AU - Jonusaite, Sima

AU - Pleinis, John M.

AU - Humphreys, John M.

AU - He, Haixia

AU - Schellinger, Jeffrey N.

AU - Akella, Radha

AU - Stenesen, Drew

AU - Krämer, Helmut

AU - Goldsmith, Elizabeth J.

AU - Rodan, Aylin R.

PY - 2018/5/1

Y1 - 2018/5/1

N2 - BackgroundWith No Lysine kinase (WNK) signaling regulates mammalian renal epithelial ion transport to maintain electrolyte and BP homeostasis. Our previous studies showed a conserved role for WNK in the regulation of transepithelial ion transport in the Drosophila Malpighian tubule. Methods Using in vitro assays and transgenic Drosophila lines, we examined two potential WNK regulators, chloride ion and the scaffold protein mouse protein 25 (Mo25), in the stimulation of transepithelial ion flux. Results In vitro, autophosphorylation of purified Drosophila WNK decreased as chloride concentration increased. In conditions inwhich tubule intracellular chloride concentration decreased from30 to 15mMas measured using a transgenic sensor, DrosophilaWNK activity acutely increased. DrosophilaWNK activity in tubules also increased or decreased when bath potassium concentration decreased or increased, respectively. However, a mutation that reduces chloride sensitivity of Drosophila WNK failed to alter transepithelial ion transport in 30 mM chloride. We, therefore, examined a role for Mo25. In in vitro kinase assays, Drosophila Mo25 enhanced the activity of the Drosophila WNK downstream kinase Fray, the fly homolog of mammalian Ste20-related proline/alanine-rich kinase (SPAK), and oxidative stress-responsive 1 protein (OSR1). Knockdown of Drosophila Mo25 in the Malpighian tubule decreased transepithelial ion flux under stimulated but not basal conditions. Finally, whereas overexpression of wild-type Drosophila WNK, with or without Drosophila Mo25, did not affect transepithelial ion transport, Drosophila Mo25 overexpressed with chloride-insensitive Drosophila WNK increased ion flux. Conclusions Cooperative interactions between chloride andMo25 regulateWNK signaling in a transporting renal epithelium.

AB - BackgroundWith No Lysine kinase (WNK) signaling regulates mammalian renal epithelial ion transport to maintain electrolyte and BP homeostasis. Our previous studies showed a conserved role for WNK in the regulation of transepithelial ion transport in the Drosophila Malpighian tubule. Methods Using in vitro assays and transgenic Drosophila lines, we examined two potential WNK regulators, chloride ion and the scaffold protein mouse protein 25 (Mo25), in the stimulation of transepithelial ion flux. Results In vitro, autophosphorylation of purified Drosophila WNK decreased as chloride concentration increased. In conditions inwhich tubule intracellular chloride concentration decreased from30 to 15mMas measured using a transgenic sensor, DrosophilaWNK activity acutely increased. DrosophilaWNK activity in tubules also increased or decreased when bath potassium concentration decreased or increased, respectively. However, a mutation that reduces chloride sensitivity of Drosophila WNK failed to alter transepithelial ion transport in 30 mM chloride. We, therefore, examined a role for Mo25. In in vitro kinase assays, Drosophila Mo25 enhanced the activity of the Drosophila WNK downstream kinase Fray, the fly homolog of mammalian Ste20-related proline/alanine-rich kinase (SPAK), and oxidative stress-responsive 1 protein (OSR1). Knockdown of Drosophila Mo25 in the Malpighian tubule decreased transepithelial ion flux under stimulated but not basal conditions. Finally, whereas overexpression of wild-type Drosophila WNK, with or without Drosophila Mo25, did not affect transepithelial ion transport, Drosophila Mo25 overexpressed with chloride-insensitive Drosophila WNK increased ion flux. Conclusions Cooperative interactions between chloride andMo25 regulateWNK signaling in a transporting renal epithelium.

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JO - Journal of the American Society of Nephrology

JF - Journal of the American Society of Nephrology

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