Regulation of CFTR Bicarbonate Channel Activity by WNK1: Implications for Pancreatitis and CFTR-Related Disorders

Abstract

Backgraoud & AimsAberrant 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.MethodsWe 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.ResultsAmong 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.ConclusionThe 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.

The CFTR Mutation c.3453G > C (D1152H) Confers an Anion Selectivity Defect in Primary Airway Tissue that Can be Rescued by Ivacaftor

“…The bicarbonate ion is larger than chloride ion and according to molecular dynamic simulations, the D1152H substitution may introduce a barrier to the internal vestibule that specifically affects bicarbonate ion access. The relative permeability of the pore to bicarbonate ions is also dependent on intracellular chloride ion concentrations and WNK1-SPAK signaling [ 38 , 42 , 43 ]. It remains possible that the D1152H substitution alters the interaction of CFTR with this signaling pathway and this concept will be investigated in future studies.…”

NKCC1: Newly Found as a Human Disease-Causing Ion Transporter

“…This WNK1 effect was not dependent on SPAK and OSR1 and did not affect CFTR expression or trafficking, but affected the physical property of the CFTR channel. 87 Finally, Hoegger and colleagues demonstrated that pharmacologic inhibition of NKCC1 in pig airway epithelial cell led to the secretion of a viscid mucus that remained attached to the epithelium in the absence of bicarbonate. 88 These observations indicate that NKCC1 and CFTR dysfunction may share common underlining mechanisms that need to be further explored.…”

Airway Surface Liquid pH Regulation in Airway Epithelium Current Understandings and Gaps in Knowledge

“…Moreover, the amount of CFTR is rate-limiting for HCO 3 − transport in contrast to Cl − secretion, which nearly reaches the WT level in CFTR+/F508del pig epithelia [ 28 ]. In addition, a low intracellular Cl − concentration may switch CFTR from a predominant Cl − channel to a predominant HCO 3 − channel, by activating intracellular Cl − -sensitive kinases (with-no-lysine kinase WNK1, oxidative stress-responsive kinase 1, OSR1, and sterile 20/SPS1-related proline/alanine-rich kinase, SPAK) [ 108 , 109 ]. Although this has been shown in pancreatic cells, this may be a mechanism that is also in place in the lung, as WNK1 and OSR1 are largely present in the lung [ 110 ].…”

“…Although this has been shown in pancreatic cells, this may be a mechanism that is also in place in the lung, as WNK1 and OSR1 are largely present in the lung [ 110 ]. Interestingly some CFTR mutations located in transmembrane domains selectively decrease the transport of HCO 3 − by hindering the physical association between CFTR and WNK1 [ 109 , 111 , 112 ]. Third, HCO 3 − transport is often assessed indirectly, i.e., by measuring pH changes in experimental protocols.…”