Backgraoud & Aims: Aberrant epithelial bicarbonate (HCO3 −) secretion caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is associated with several diseases including cystic fibrosis and pancreatitis. Dynamically regulated ion channel activity and anion selectivity of CFTR by kinases sensitive to intracellular chloride concentration ([Cl−]i) play an important role in epithelial HCO3 − secretion. However, the molecular mechanisms of how [Cl−]i-dependent mechanisms regulate CFTR are unknown. Methods: We examined the mechanisms of the CFTR HCO3 − channel regulation by [Cl−]i-sensitive kinases using an integrated electrophysiological, molecular, and computational approach including whole-cell, outside-out, and inside-out patch clamp recordings and molecular dissection of WNK1 and CFTR proteins. In addition, we analyzed the effects of pancreatitis-causing CFTR mutations on the WNK1-mediated regulation of CFTR. Results: Among the WNK1, SPAK, and OSR1 kinases that constitute a [Cl−]i-sensitive kinase cascade, the expression of WNK1 alone was sufficient to increase the CFTR bicarbonate permeability (PHCO3/PCl) and conductance (GHCO3) in patch clamp recordings. Molecular dissection of the WNK1 domains revealed that the WNK1 kinase domain is responsible for CFTR PHCO3/PCl regulation by direct association with CFTR, while the surrounding N-terminal regions mediate the [Cl−]i-sensitivity of WNK1. Furthermore, the pancreatitis-causing R74Q and R75Q mutations in the elbow helix 1 of CFTR hampered WNK1-CFTR physical associations and reduced WNK1-mediated CFTR PHCO3/PCl regulation. Conclusion: The CFTR HCO3 − channel activity is regulated by [Cl−]i and a WNK1-dependent mechanism. Our results provide new insights into the regulation of the ion selectivity of CFTR and the pathogenesis of CFTR-related disorders.
|Number of pages||25|
|Journal||Cellular and Molecular Gastroenterology and Hepatology|
|Publication status||Published - 2020|
Bibliographical noteFunding Information:
Funding This work was funded by grants 2013R1A3A2042197 (to Min Goo Lee) and 2015R1D1A1A01057618 (to Yonjung Kim) from the National Research Foundation of Korea, the Ministry of Science,ICT & Future Planning, Republic of Korea; and grant HI15C1543 (to Min Goo Lee) of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare, Republic of Korea. The work of Mary Hongying Cheng and Ivet Bahar was supported by National Institutes of Health grants P30DA035778 and P41GM103712.
© 2019 The Authors
All Science Journal Classification (ASJC) codes