Cystic Fibrosis (CF) and hypertension are life-shortening diseases of epithelial ion transport. In the CF airway, when the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is functionally absent, there is a relative and unbalanced increase in the activity of the Epithelial Sodium Channel (ENaC) that leads to Na+ absorption from and depletion of the airway surface liquid. In turn, this impairs mucociliary clearance and predisposes the CF airway to bacterial colonization/infection. ENaC can also play a key role in causing hypertension; abnormally increased function of ENaC in the distal tubule of the nephron leads to increased Na+ retention, increased blood volume and hypertension. The regulation of ENaC activity is therefore key in understanding and developing therapies for these diseases of epithelial ion transport. A critical determinant of ENaC's open probability (Po), and therefore its function, is the proteolytic cleavage of the luminal/extracellular loops of its ? and ? subunits; uncleaved channel has Po ~0, while fully cleaved channel has Po ~1. In fact, increased ENaC cleavage is reported in CF airway epithelial cells. Interestingly, ENaC can be delivered to the epithelial surface in either a cleaved or an uncleaved form, but the factor(s) that determine whether or not ENaC undergoes cleavage during biogenesis are not known. We have extensively studied Sodium 4-Phenylbutyrate (4PBA), the prototype small molecule corrector of the aberrant trafficking of the most common CF causing CFTR mutation, ?F508. Others demonstrated that 4PBA also increases the function of ENaC. We found that ERp29 (Endoplasmic Reticulum Protein of 29 kD), a novel, ubiquitously expressed endoplasmic reticulum (ER) luminal chaperone has increased expression in bronchiolar epithelial cells treated with 4PBA, and that ERp29 promotes the trafficking of both wild type and ?F508-CFTR. ERp29 was thus the first ER luminal component demonstrated to positively influence CFTR trafficking. We have also recently demonstrated that ERp29 promotes the function of ENaC. Interestingly, our data suggest that ERp29 does this by directing ENaC for cleavage in the Golgi during biogenesis, as depletion of ERp29 decreased ENaC function without altering expression of ENaC at the apical epithelial surface. The hypothesis of this proposal is that interaction of the luminal face of epithelial ion channels, such as CFTR and ENaC, with ERp29 during their biogenesis is required to direct these clients from the ER to the Golgi. This hypothesis will be tested with studies Specifically Aiming: 1) To test if ER-luminal facing mutations of CFTR that cause CF and are predicted to not interact with ERp29 have abnormal maturation. 2) To test the mechanism by which ERp29 directs ENaC to the Golgi during biogenesis. 3) To test the mechanism by which ENaC can bypass cleavage in the Golgi en route to the plasma membrane. These studies will yield key mechanistic insights that will be crucial in designing potential ERp29-directed therapeutic interventions for CFTR- and ENaC-opathies.

Public Health Relevance

Cystic Fibrosis (CF) and hypertension are life-shortening diseases of epithelial ion transport. The roles of proteins within the lumen of the endoplasmic reticulum (ER) in promoting proper biogenesis of the epithelial ion channels that underlie CF and hypertension are not well defined, and such knowledge is of vital importance. The data we propose to obtain here will delineate the mechanism by which one ER protein, ERp29, regulates such epithelial ion channels, and will define whether ERp29 is a viable therapeutic target in these disorders.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Lung Cellular, Molecular, and Immunobiology Study Section (LCMI)
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Lu, Jining
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Children's Hospital of Philadelphia
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Rubenstein, Ronald C (2018) Editorial Focus: CFTR-dependent bicarbonate secretion by Calu-3 cells. Physiol Rep 6:e13691