Dynamic Regulation of Bicarbonate Permeability through CFTR Channel by WNK1

Abstract

Increase in relative HCO3− permeability (PHCO3/PCl) of cystic fibrosis transmembrane conductance regulator (CFTR) channel by[Cl−]i-sensitive kinases is essential for pancreatic HCO3− secretion. Defects in pancreatic HCO3− secretion caused by CFTR mutations are associated with cystic fibrosis and pancreatitis. However, the molecular mechanisms by which [Cl−]i-sensitive kinases regulate CFTR anion selectivity are unknown. We examined the mechanisms of CFTR PHCO3/PCl regulation by [Cl−]i-sensitive kinases using the electrophysiological and molecular approach, including whole-cell, outside-out, and inside-out patch clamp recordings and molecular dissection of WNK1 and CFTR proteins. Moreover, we analyzed the effects of pancreatitis-causing CFTR mutations on the WNK1-mediated regulation of CFTR. Although SPAK and/or OSR, which constitute a [Cl−]i-sensitive kinase cascade, wasn't expressed, the expression of WNK1 alone was sufficient to increase the CFTR PHCO3/PCl in patch clamp recordings. In molecular dissection of the WNK1 protein, the WNK1 kinase domain contributes CFTR PHCO3/PCl regulation by direct association with CFTR, while the surrounding N-terminal regions regulate the [Cl−]i-sensitivity of WNK1. Therefore, the inclusion of WNK1 N-terminal recombinant proteins in excised patch clamp recordings increased the CFTR PHCO3/PCl in a dose-dependent manner. The pancreatitis-causing R74Q and R75Q mutations in elbow helix 1 of CFTR hampered WNK1-CFTR physical associations and reduced WNK1-mediated CFTR PHCO3/PCl regulation. The CFTR PHCO3/PCl is regulated by [Cl−]i and a WNK1-dependent mechanism. In low [Cl−]i, physical associations between the N-terminal region of WNK1 and CFTR near p.R75 increase PHCO3/PCl. Defects in this process underlie the pathogenesis of some CFTR-related disorders.