IRBIT governs epithelial secretion in mice by antagonizing the WNK/SPAK kinase pathway
Dongki Yang, Qin Li, Insuk So, Chou-Long Huang, Hideaki Ando, Akihiro Mizutani, George Seki, Katsuhiko Mikoshiba, Philip J. Thomas, Shmuel Muallem
Dongki Yang, Qin Li, Insuk So, Chou-Long Huang, Hideaki Ando, Akihiro Mizutani, George Seki, Katsuhiko Mikoshiba, Philip J. Thomas, Shmuel Muallem
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Research Article

IRBIT governs epithelial secretion in mice by antagonizing the WNK/SPAK kinase pathway

Abstract

Fluid and HCO3– secretion are fundamental functions of epithelia and determine bodily fluid volume and ionic composition, among other things. Secretion of ductal fluid and HCO3– in secretory glands is fueled by Na+/HCO3– cotransport mediated by basolateral solute carrier family 4 member 4 (NBCe1-B) and by Cl–/HCO3– exchange mediated by luminal solute carrier family 26, member 6 (Slc26a6) and CFTR. However, the mechanisms governing ductal secretion are not known. Here, we have shown that pancreatic ductal secretion in mice is suppressed by silencing of the NBCe1-B/CFTR activator inositol-1,4,5-trisphosphate (IP3) receptor–binding protein released with IP3 (IRBIT) and by inhibition of protein phosphatase 1 (PP1). In contrast, silencing the with-no-lysine (WNK) kinases and Ste20-related proline/alanine-rich kinase (SPAK) increased secretion. Molecular analysis revealed that the WNK kinases acted as scaffolds to recruit SPAK, which phosphorylated CFTR and NBCe1-B, reducing their cell surface expression. IRBIT opposed the effects of WNKs and SPAK by recruiting PP1 to the complex to dephosphorylate CFTR and NBCe1-B, restoring their cell surface expression, in addition to stimulating their activities. Silencing of SPAK and IRBIT in the same ducts rescued ductal secretion due to silencing of IRBIT alone. These findings stress the pivotal role of IRBIT in epithelial fluid and HCO3– secretion and provide a molecular mechanism by which IRBIT coordinates these processes. They also have implications for WNK/SPAK kinase–regulated processes involved in systemic fluid homeostasis, hypertension, and cystic fibrosis.

Authors

Dongki Yang, Qin Li, Insuk So, Chou-Long Huang, Hideaki Ando, Akihiro Mizutani, George Seki, Katsuhiko Mikoshiba, Philip J. Thomas, Shmuel Muallem

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Figure 6

Physiological relevance of regulation of the WNK/SPAK pathway by the IRBIT/PP1 pathway.

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Physiological relevance of regulation of the WNK/SPAK pathway by the IRB...
(A) Sealed pancreatic ducts were treated with 10 nM scrambled siRNA (black, red, and green traces), 5 nM IRBIT siRNA (red and blue traces), 2.5 nM IRBIT siRNA (green and purple traces), and 10 nM SPAK siRNA (blue and purple traces) and were used to measure stimulated ductal fluid secretion. (B) Sealed pancreatic ducts were treated with 10 nM scrambled (black trace) or SPAK siRNA (red trace). Ten minutes before and during the experiments, the ducts were treated with the indicated concentration of tautomycin, and fluid secretion was measured by video microscopy for 40 minutes. Results in A and B are mean ± SEM of 3–6 experiments.