Cystic Fibrosis Transmembrane Conductance Regulator Activity Research Articles

G551D mutation impairs PKA-dependent activation of CFTR channel that can be restored by novel GOF mutations.

G551D is a major disease-associated gating mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an ATP- and phosphorylation-dependent chloride channel. G551D causes severe cystic fibrosis (CF) disease by disrupting ATP-dependent channel opening; however, whether G551D affects phosphorylation-dependent channel activation is unclear. Here, we use macropatch recording and Ussing chamber approaches to demonstrate that G551D impacts on phosphorylation-dependent activation of CFTR, and PKA-mediated phosphorylation regulates the interaction between the x-loop in nucleotide-binding domain 2 (NBD2) and cytosolic loop (CL) 1. We show that G551D not only disrupts ATP-dependent channel opening but also impairs phosphorylation-dependent channel activation by largely reducing PKA sensitivity consistent with the reciprocal relationship between channel opening/gating, ligand binding, and phosphorylation. Furthermore, we identified two novel GOF mutations: D1341R in the x-loop near the ATP-binding cassette signature motif in NBD2 and D173R in CL1, each of which strongly increased PKA sensitivity both in the wild-type (WT) background and when introduced into G551D-CFTR. When D1341R was combined with a second GOF mutation (e.g., K978C in CL3), we find that the double GOF mutation maximally increased G551D channel activity such that VX-770 had no further effect. We further show that a double charge-reversal mutation of D1341R/D173R-CFTR exhibited similar PKA sensitivity when compared with WT-CFTR. Together, our results suggest that charge repulsion between D173 and D1341 of WT-CFTR normally inhibits channel activation at low PKA activity by reducing PKA sensitivity, and negative allostery by the G551D is coupled to reduced PKA sensitivity of CFTR that can be restored by second GOF mutations.

Correction of Airway Stem Cells: Genome Editing Approaches for the Treatment of Cystic Fibrosis.

Cystic fibrosis (CF) is an autosomal recessive disease caused by variations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although CF affects multiple organs, the primary cause of mortality is respiratory failure resulting from poor clearance of hyperviscous secretions and subsequent airway infection. Recently developed CFTR modulators provide significant therapeutic benefit to the majority of CF individuals. However, treatments directed at the underlying cause are needed for the ∼7% of CF patients who are not expected to be responsive to these modulators. Genome editing can restore the native CFTR genetic sequence and function to mutant cells, representing an approach to establish durable physiologic CFTR correction. Although editing the CFTR gene in various airway cell types may transiently restore CFTR activity, effort is focused on editing airway basal stem/progenitor cells, since their correction would allow appropriate and durable expression of CFTR in stem cell-derived epithelial cell types. Substantial progress has been made to directly correct airway basal cells in vitro, theoretically enabling transplantation of autologous corrected cells to regenerate an airway with CFTR functional cells. Another approach to create autologous, gene-edited airway basal cells is derivation of CF donor-specific induced pluripotent stem cells, correction of the CFTR gene, and subsequent directed differentiation to airway basal cells. Further work is needed to translate these advances by developing effective transplantation methods. Alternatively, gene editing in vivo may enable CFTR correction. However, this approach will require robust delivery methods ensuring that basal cells are efficiently targeted and corrected. Recent advances in gene editing-based therapies provide hope that the genetic underpinning of CF can be durably corrected in airway epithelial stem cells, thereby preventing or treating lung disease in all people with CF.

Novel Therapy of Bicarbonate, Glutathione, and Ascorbic Acid Improves Cystic Fibrosis Mucus Transport.

Defective airway mucus clearance is a defining characteristic of cystic fibrosis lung disease, and improvements to current mucolytic strategies are needed. Novel approaches targeting a range of contributing mechanisms are in various stages of preclinical and clinical development. ARINA-1 is a new nebulized product comprised of ascorbic acid, glutathione, and bicarbonate. Using microoptical coherence tomography, we tested the effect of ARINA-1 on central features of mucociliary clearance in F508del/F508del primary human bronchial epithelial cells to assess its potential as a mucoactive therapy in cystic fibrosis. We found that ARINA-1 significantly augmented mucociliary transport rates, both alone and with CFTR (cystic fibrosis transmembrane conductance regulator) modulator therapy, whereas airway hydration and ciliary beating were largely unchanged compared with PBS vehicle control. Analysis of mucus reflectivity and particle-tracking microrheology indicated that ARINA-1 restores mucus clearance by principally reducing mucus layer viscosity. The combination of bicarbonate and glutathione elicited increases in mucociliary transport rate comparable to those seen with ARINA-1, indicating the importance of this interaction to the impact of ARINA-1 on mucus transport; this effect was not recapitulated with bicarbonate alone or bicarbonate combined with ascorbic acid. Assessment of CFTR chloride transport revealed an increase in CFTR-mediated chloride secretion in response to ARINA-1 in CFBE41o- cells expressing wild-type CFTR, driven by CFTR activity stimulation by ascorbate. This response was absent in CFBE41o- F508del cells treated with VX-809 and primary human bronchial epithelial cells, implicating CFTR-independent mechanisms for the effect of ARINA-1 on cystic fibrosis mucus. Together, these studies indicate that ARINA-1 is a novel potential therapy for the treatment of impaired mucus clearance in cystic fibrosis.

Pendrin stimulates a chloride absorption pathway to increase CFTR-mediated chloride secretion from Cystic Fibrosis airway epithelia.

Cystic Fibrosis (CF), an inherited multi‐system disease, is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) that disrupt its ability to secrete anions from epithelia. Recovery of functional anion secretion may be curative for CF, so different components of the ion transport machinery have become attractive therapeutic targets. Several members of the SLC26 ion transporter family have been linked to epithelial ion flux, some through putative functional interactions with CFTR. Using a small‐scale qPCR screen, we confirmed that the anion transporter SLC26A4 (pendrin) is downregulated in CF. Upregulation of pendrin using interleukins IL‐4 or IL‐13 increased Cl− secretion through CFTR in human bronchial epithelial cell (HBEC) derived epithelia differentiated in vitro and measured in the Ussing Chamber. Inhibition or knockdown of pendrin prevented this increased secretion. Increased CFTR activity was not driven by increases in CFTR protein or upstream regulatory pathway components. When basolateral Cl− absorption through NKCC1 was inhibited, a pendrin‐dependent Cl− absorption pathway allowing CFTR to continue secreting Cl− from the epithelium was revealed. Although CFTR is often considered the bottleneck in the transepithelial Cl− transport pathway, these studies indicate that basolateral Cl− permeability becomes limiting as CFTR activity increases. Therefore, an increase of epithelial Cl− absorption via pendrin might have additional therapeutic benefit in combination with CFTR modulators.

ENaC inhibition in cystic fibrosis: potential role in the new era of CFTR modulator therapies.

ENaC inhibition with BI 1265162 is a promising strategy to optimise outcomes in patients with CF either eligible, or ineligible, for CFTR modulator therapy. Phase II clinical trials of BI 1265162 must now show this translates into clinical benefit. https://bit.ly/2OQ1IUI

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in Human Lung Microvascular Endothelial Cells Controls Oxidative Stress, Reactive Oxygen-Mediated Cell Signaling and Inflammatory Responses.

BackgroundPerturbation of endothelial function in people with cystic fibrosis (CF) has been reported, which may be associated with endothelial cell expression of the cystic fibrosis transmembrane conductance regulator (CFTR). Previous reports indicate that CFTR activity upregulates endothelial barrier function, endothelial nitric oxide synthase (eNOS) expression and NO release, while limiting interleukin-8 (IL-8) release, in human umbilical vein endothelial cells (HUVECs) in cell culture. In view of reported microvascular dysfunction in people with CF we investigated the role of CFTR expression and activity in the regulation of oxidative stress, cell signaling and inflammation in human lung microvascular endothelial cells (HLMVECs) in cell culture.MethodsHLMVECs were cultured in the absence and presence of the CFTR inhibitor GlyH-101 and CFTR siRNA. CFTR expression was analyzed using qRT-PCR, immunocytochemistry (IHC) and western blot, and function by membrane potential assay. IL-8 expression was analyzed using qRT-PCR and ELISA. Nrf2 expression, and NF-κB and AP-1 activation were determined using IHC and western blot. The role of the epidermal growth factor receptor (EGFR) in CFTR signaling was investigated using the EGFR tyrosine kinase inhibitor AG1478. Oxidative stress was measured as intracellular ROS and hydrogen peroxide (H2O2) concentration. VEGF and SOD-2 were measured in culture supernatants by ELISA.ResultsHLMVECs express low levels of CFTR that increase following inhibition of CFTR activity. Inhibition of CFTR, significantly increased intracellular ROS and H2O2 levels over 30 min and significantly decreased Nrf2 expression by 70% while increasing SOD-2 expression over 24 h. CFTR siRNA significantly increased constitutive expression of IL-8 by HLMVECs. CFTR inhibition activated the AP-1 pathway and increased IL-8 expression, without effect on NF-κB activity. Conversely, TNF-α activated the NF-κB pathway and increased IL-8 expression. The effects of TNF-α and GlyH-101 on IL-8 expression were additive and inhibited by AG1478. Inhibition of both CFTR and EGFR in HLMVECs significantly increased VEGF expression. The antioxidant N-acetyl cysteine significantly reduced ROS production and the increase in IL-8 and VEGF expression following CFTR inhibition.ConclusionFunctional endothelial CFTR limits oxidative stress and contributes to the normal anti-inflammatory state of HLMVECs. Therapeutic strategies to restore endothelial CFTR function in CF are warranted.

Ivacaftor Reverses Airway Mucus Abnormalities in a Rat Model Harboring a Humanized G551D-CFTR.

Rationale: Animal models have been highly informative for understanding the characteristics, onset, and progression of cystic fibrosis (CF) lung disease. In particular, the CFTR-/- rat has revealed insights into the airway mucus defect characteristic of CF but does not replicate a human-relevant CFTR (cystic fibrosis transmembrane conductance regulator) variant.Objectives: We hypothesized that a rat expressing a humanized version of CFTR and harboring the ivacaftor-sensitive variant G551D could be used to test the impact of CFTR modulators on pathophysiologic development and correction.Methods: In this study, we describe a humanized-CFTR rat expressing the G551D variant obtained by zinc finger nuclease editing of a human complementary DNA superexon, spanning exon 2-27, with a 5' insertion site into the rat gene just beyond intron 1. This targeted insertion takes advantage of the endogenous rat promoter, resulting in appropriate expression compared with wild-type animals.Measurements and Main Results: The bioelectric phenotype of the epithelia recapitulates the expected absence of CFTR activity, which was restored with ivacaftor. Large airway defects, including depleted airway surface liquid and periciliary layers, delayed mucus transport rates, and increased mucus viscosity, were normalized after the administration of ivacaftor.Conclusions: This model is useful to understand the mechanisms of disease and the extent of pathology reversal with CFTR modulators.

Cystic fibrosis transmembrane conductance regulator-dependent bicarbonate entry controls rat cardiomyocyte ATP release via pannexin1 through mitochondrial signalling and caspase activation.

Cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in the heart, but its function there is unclear. CFTR regulates an ATP release pore in many tissues, but the identity and regulatory mechanism of the pore are unknown. We investigated the role of CFTR in ATP release from primary cardiomyocytes and ventricular wall in vivo. Proteins involved in the signalling pathway for ATP release during simulated ischaemia (lactic acid treatment) were investigated using inhibitors and siRNA; colocalization was identified by coimmunofluorescence and proximity ligation assays; changes in near-membrane pH and calcium were identified with total internal reflection microscopy; in vivo ATP release was investigated using interstitial microdialysis of rat heart. Lactic acid-induced CFTR-dependent ATP release from cultured cardiomyocytes and left ventricle in vivo. Lactic acid entry elevated near-membrane calcium, which involved Na/H- and Na/Ca-exchangers colocalized with CFTR. Calcium entry-induced CFTR activation, which involved cAMP, protein kinase A, FAK, Pyk2 and Src. Removal of extracellular bicarbonate abolished cardiomyocyte ATP release induced by lactic acid or CFTR activators. Bicarbonate stimulated cytochrome c expression, cytochrome c release and ATP release from isolated cardiomyocyte mitochondria. Pannexin 1 (Panx1) colocalized with CFTR. Lactic acid increased cardiomyocyte caspase activity: caspase inhibitors or Panx1 siRNA abolished cardiomyocyte ATP release, while pannexin inhibition abolished cardiac ATP release in vivo. During simulated ischaemia, CFTR-dependent bicarbonate entry stimulated ATP and cytochrome c release from mitochondria; in the cytoplasm, cytochrome c-activated caspase 3, which in turn activated Panx1, and ATP was released through the opened Panx1 channel.

Defective Ion Channel in Cystic Fibrosis: Current Development in Treatment of Cystic Fibrosis

Cystic fibrosis is an inherited disorder that causes severe damage to the lungs, digestive system and other organs in the body. Cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the production of mucus, sweat and digestive juices. These secreted fluids are normally thin and slippery. But in people with cystic fibrosis, a defective gene in CFTR causes the secretions to become sticky and thick. Instead of acting as a lubricant, the secretions plug up tubes, ducts and passage ways, especially in the lungs and pancreas. This mucus leads to the formation of bacterial microenvironments known as biofilms (a niche that harbors bacteria; Staphylococcus aureus, Haemophilus influenzae, and Pseudomonas aeruginosa ) that are difficult for immune cells and antibiotics to penetrate. Viscous secretions and persistent respiratory infections repeatedly damage the lung by gradually remodeling the airways, which makes infection even more difficult to eradicate. CFTR, a Cl– selective ion channel, is a prototypic member of the ATP-binding cassette transporter super family that is expressed in several organs. Understanding how these complexes regulate the intracellular trafficking and activity of CFTR provides a unique insight into the aetiology of cystic fibrosis and other diseases associated to it. Cystic fibrosis patients exhibit lung disease consistent with a failure of innate airway defense mechanisms. The link between abnormal ion transport, disease initiation and progression is not fully understood, but airway mucus dehydration seems paramount in the initiation of CF lung disease. New therapies are currently in development that target the ion transport defects in CF with the intention of rehydrating airway surfaces.

The Effect of Sodium Bicarbonate, a Beneficial Adjuvant Molecule in Cystic Fibrosis, on Bronchial Epithelial Cells Expressing a Wild-Type or Mutant CFTR Channel.

Clinical and experimental results with inhaled sodium bicarbonate as an adjuvant therapy in cystic fibrosis (CF) are promising due to its mucolytic and bacteriostatic properties, but its direct effect has not been studied on respiratory epithelial cells. Our aim was to establish and characterize co-culture models of human CF bronchial epithelial (CFBE) cell lines expressing a wild-type (WT) or mutant (deltaF508) CF transmembrane conductance regulator (CFTR) channel with human vascular endothelial cells and investigate the effects of bicarbonate. Vascular endothelial cells induced better barrier properties in CFBE cells as reflected by the higher resistance and lower permeability values. Activation of CFTR by cAMP decreased the electrical resistance in WT but not in mutant CFBE cell layers confirming the presence and absence of functional channels, respectively. Sodium bicarbonate (100 mM) was well-tolerated by CFBE cells: it slightly reduced the impedance of WT but not that of the mutant CFBE cells. Sodium bicarbonate significantly decreased the more-alkaline intracellular pH of the mutant CFBE cells, while the barrier properties of the models were only minimally changed. These observations indicate that sodium bicarbonate is beneficial to deltaF508-CFTR expressing CFBE cells. Thus, sodium bicarbonate may have a direct therapeutic effect on the bronchial epithelium.

Human Nasal Epithelial Organoids for Therapeutic Development in Cystic Fibrosis.

We describe a human nasal epithelial (HNE) organoid model derived directly from patient samples that is well-differentiated and recapitulates the airway epithelium, including the expression of cilia, mucins, tight junctions, the cystic fibrosis transmembrane conductance regulator (CFTR), and ionocytes. This model requires few cells compared to airway epithelial monolayer cultures, with multiple outcome measurements depending on the application. A novel feature of the model is the predictive capacity of lumen formation, a marker of baseline CFTR function that correlates with short-circuit current activation of CFTR in monolayers and discriminates the cystic fibrosis (CF) phenotype from non-CF. Our HNE organoid model is amenable to automated measurements of forskolin-induced swelling (FIS), which distinguishes levels of CFTR activity. While the apical side is not easily accessible, RNA- and DNA-based therapies intended for systemic administration could be evaluated in vitro, or it could be used as an ex vivo biomarker of successful repair of a mutant gene. In conclusion, this highly differentiated airway epithelial model could serve as a surrogate biomarker to assess correction of the mutant gene in CF or other diseases, recapitulating the phenotypic and genotypic diversity of the population.

Immunomodulation in Cystic Fibrosis: Why and How?

Cystic fibrosis (CF) lung disease is characterized by unconventional mechanisms of inflammation, implicating a chronic immune response dominated by innate immune cells. Historically, therapeutic development has focused on the mutated cystic fibrosis transmembrane conductance regulator (CFTR), leading to the discovery of small molecules aiming at modulating and potentiating the presence and activity of CFTR at the plasma membrane. However, treatment burden sustained by CF patients, side effects of current medications, and recent advances in other therapeutic areas have highlighted the need to develop novel disease targeting of the inflammatory component driving CF lung damage. Furthermore, current issues with standard treatment emphasize the need for directed lung therapies that could minimize systemic side effects. Here, we summarize current treatment used to target immune cells in the lungs, and highlight potential benefits and caveats of novel therapeutic strategies.

An organoid model to assay the role of CFTR in the human epididymis epithelium.

Organoid cultures derived from primary human tissues facilitate the study of disease processes and the development of new therapeutics. Most men with cystic fibrosis (CF) are infertile due to defects in the epididymis and vas deferens; however, the causative mechanisms are still unclear. We used human epididymis epithelial cell (HEE) organoids and polarized HEE cell cultures to assay the CF transmembrane conductance regulator (CFTR) in the human epididymis. 3D HEE organoids and polarized 2D HEE cell cultures on membrane inserts were established from human caput epididymis. Single-cell RNA sequencing (scRNA-seq) was performed to map cell type-specific gene expression in the organoids. Using forskolin (FSK) to activate CFTR and inhibitor CFTRinh172 to block its activity, we assessed how CFTR contributes to organoid swelling and epithelial barrier function. The scRNA-seq data showed key caput epididymis cell types present in HEE organoid cultures. FSK at 10μM induced HEE organoid swelling by 20% at 16h, while 5 and 10μM CFTRinh172 treatment significantly reduced HEE organoid size. In transepithelial resistance (TER) measurements, FSK reduced TER, while inhibition of CFTR increased TER; also, depletion of CFTR with specific siRNAs significantly increased TER. FSK treatment significantly increased the flux of 4-kDa but not 70-kDa dextran, suggesting activation of CFTR mainly enhances transcellular diffusion. We have demonstrated that CFTR contributes to the maintenance of HEE cell TER and that cultured HEE organoids are a useful model to investigate human epididymis function. These results facilitate progress in elucidating how CFTR-dependent cellular processes impair fertility in CF.

Negative chronotropic and inotropic effects of lubiprostone on iPS cell-derived cardiomyocytes via activation of CFTR

BackgroundLubiprostone (LBP) is a novel chloride channel opener that has been reported to activate chloride channel protein 2 (ClC-2) and cystic fibrosis transmembrane conductance regulator (CFTR). LBP facilitates fluid secretion by activating CFTR in the intestine and is used as a drug for treating chronic constipation. While ClC-2 and CFTR expression has been confirmed in cardiomyocytes (CMs), the effect of LBP on CMs has not yet been investigated. Thus, the present study aimed to investigate the effect of LBP on CMs using mouse-induced pluripotent stem (iPS) cell-derived CMs (iPS-CMs).MethodsWe induced mouse iPS cells into CMs through embryoid body (EB) formation. We compared the differentiated cells to CMs isolated from adult and fetal mice using gene expression, spontaneous beating rate, and contraction ratio analyses.ResultsGene expression analysis revealed that, in the iPS-CMs, the mRNA expression of the undifferentiated cell markers Rex1 and Nanog decreased, whereas the expression of the unique cardiomyocyte markers cardiac troponin I (cTnI) and cardiac troponin T (cTNT), increased. Immunostaining showed that the localization of cTnI and connexin-43 in the iPS-CMs was similar to that in the primary fetal CMs (FCMs) and adult CMs (ACMs). LBP decreased the spontaneous beating rate of the iPS-CMs and FCMs, and decreased the contraction ratio of the iPS-CMs and ACMs. The reduction in the beating rate and contraction ratio caused by LBP was inhibited by glycine hydrazide (GlyH), which is a CFTR inhibitor.ConclusionThese results suggest that LBP stimulates CFTR in CMs and that LBP has negative chronotropic and inotropic effects on CMs. LBP may be useful for treating cardiac diseases such as heart failure, ischemia, and arrhythmia.

IPSC-Derived Intestinal Organoids from Cystic Fibrosis Patients Acquire CFTR Activity upon TALEN-Mediated Repair of the p.F508del Mutation

Cystic fibrosis (CF) is the main genetic cause of death among the Caucasian population. The disease is characterized by abnormal fluid and electrolyte mobility across secretory epithelia. The first manifestations occur within hours of birth (meconium ileus), later extending to other organs, generally affecting the respiratory tract. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR encodes a cyclic adenosine monophosphate (cAMP)-dependent, phosphorylation-regulated chloride channel required for transport of chloride and other ions through cell membranes. There are more than 2,000 mutations described in the CFTR gene, but one of them, phenylalanine residue at amino acid position 508 (p.F508del), a recessive allele, is responsible for the vast majority of CF cases worldwide. Here, we present the results of the application of genome-editing techniques to the restoration of CFTR activity in p.F508del patient-derived induced pluripotent stem cells (iPSCs). Gene-edited iPSCs were subsequently used to produce intestinal organoids on which the physiological activity of the restored gene was tested in forskolin-induced swelling tests. The seamless restoration of the p.F508del mutation resulted in normal expression of the mature CFTR glycoprotein, full recovery of CFTR activity, and a normal response of the repaired organoids to treatment with two approved CF therapies: VX-770 and VX-809.

Role of cystic fibrosis transmembrane conductance regulator in FHC colon epithelial cell dysfunction and colon cancer

ObjectiveTo determine the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in colon cancer development, with a focus on cell migration, EGFR signaling, and inflammation using model FHC colon epithelial cells.BackgroundOur clinical observations indicate that individuals with cystic fibrosis (CF) over the age of 40 are 4–8 times more likely to get colon cancer than the general population. Moreover, the likelihood of getting colon cancer increases to over 30 times the general population’s risk in CF patients who have had an organ transplant. The mechanism(s) leading to increased colon cancer in CF patients is not fully understood, but dysfunctional Cl− and Na+ transport are highly implicated. Chronic inflammation has also been shown to play an important role in the development and progression of many cancers. We hypothesize that dysregulated ion transport in CF patients leads to chronic inflammation and increased incidence of colon cancer via altered epidermal growth factor receptor (EGFR) signaling.MethodsWe performed scratch assays on cultured human colon epithelial cells (FHC) and measured the rate of cell migration every 24 hours for 5 days in the presence of either CFTR activating or inhibiting compounds. EGFR signaling was analyzed using standard immunohistochemical labeling techniques and commercially available reagents in similarly treated FHC cells.ResultsCells treated with 500 μM IBMX and 5 μM forskolin (CFTR activators) had significantly decreased rates of cell migration (26 μm2/24 hours) compared to the control group (51 μm2/24 hours) n=3; p<0.05. Control and colon cells treated with 500 μM IBMX and 5 μM forskolin showed low levels of EGFR labelling (restricted to cell periphery), whereas cells treated with 5 mM CFTR(inh)‐172 (CFTR inhibitor) showed more robust EGFR signal throughout the cytoplasm in 3 independent observations.ConclusionOur data indicates that suppressed CFTR activity impacts colon epithelial cell migration and EGFR localization in FHC cells. Additional research on CFTR and EGFR signaling is needed to better understand and improve treatment options for both colon cancer and CF patients.Support or Funding InformationFunding: NIH R01 HL137033‐01 awarded to MNH.

Protein kinase A phosphorylation potentiates cystic fibrosis transmembrane conductance regulator gating by relieving autoinhibition on the stimulatory C terminus of the regulatory domain

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel activated by protein kinase A (PKA) phosphorylation on the regulatory (R) domain. Phosphorylation at several R domain residues stimulates ATP-dependent channel openings and closings, termed channel gating. To explore the protein segment responsible for channel potentiation and PKA-dependent activation, deletion mutations were constructed by removing one to three protein segments of the R domain including residues 708-759 (ΔR708-759), R760-783, and R784-835, each of which contains one or two PKA phosphorylation sites. Deletion of R708-759 or R760-783 had little effect on CFTR gating, whereas all mutations lacking R784-835 reduced CFTR activity by decreasing the mean burst duration and increasing the interburst interval (IBI). The data suggest that R784-835 plays a major role in stimulating CFTR gating. For ATP-associated regulation, ΔR784-835 had minor impact on gating potentiation by 2'dATP, CaATP, and pyrophosphate. Interestingly, introducing a phosphorylated peptide matching R809-835 shortened the IBI of ΔR708-835-CFTR. Consistently, ΔR815-835, but not ΔR784-814, enhanced IBI, whereas both reduced mean burst duration. These data suggest that the entirety of R784-835 is required for stabilizing the open state of CFTR; however, R815-835, through interactions with the channel, is dominant for enhancing the opening rate. Of note, PKA markedly decreased the IBI of ΔR708-783-CFTR. Conversely, the IBI of ΔR708-814-CFTR was short and PKA-independent. These data reveal that for stimulating CFTR gating, PKA phosphorylation may relieve R784-814-mediated autoinhibition that prevents IBI shortening by R815-835 This mechanism may elucidate how the R domain potentiates channel gating and may unveil CFTR stimulation by other protein kinases.

Amplifiers co-translationally enhance CFTR biosynthesis via PCBP1-mediated regulation of CFTR mRNA

BackgroundCystic fibrosis (CF) is a recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We previously described a first-in-class CFTR modulator that functions as an amplifier to selectively increase CFTR expression and function. The amplifier mechanism is distinct from and complementary to corrector and potentiator classes of CFTR modulators. Here we characterize the mechanism by which amplifiers increase CFTR mRNA, protein, and activity. MethodsBiochemical studies elucidated the action of amplifiers on CFTR mRNA abundance and translation and defined the role of an amplifier-binding protein that was identified using chemical proteomics. ResultsAmplifiers stabilize CFTR mRNA through a process that requires only the translated sequence of CFTR and involves translational elongation. Amplifiers enrich ER-associated CFTR mRNA and increase its translational efficiency through increasing the fraction of CFTR mRNA associated with polysomes. Pulldowns identified the poly(rC)-binding protein 1 (PCBP1) as directly binding to amplifier. A PCBP1 consensus element was identified within the CFTR open reading frame that binds PCBP1. This sequence proved necessary for amplifier responsiveness. ConclusionsSmall molecule amplifiers co-translationally increase CFTR mRNA stability. They enhance translation through addressing the inherently inefficient membrane targeting of CFTR mRNA. Amplifiers bind directly to PCBP1, show enhanced affinity in the presence of bound RNA, and require a PCBP1 consensus element within CFTR mRNA to elicit translational effects. These modulators represent a promising new and mechanistically novel class of CFTR therapeutic. They may be useful as a monotherapy or in combination with other CFTR modulators.

Neuropeptide regulation of secretion and inflammation in human airway gland serous cells.

Airway submucosal gland serous cells are sites of expression of the cystic fibrosis transmembrane conductance regulator (CFTR) and are important for fluid secretion in conducting airways. To elucidate how neuropeptides regulate serous cells, we tested if human nasal turbinate serous cells secrete bicarbonate (HCO3 -), important for mucus polymerisation and antimicrobial peptide function, during stimulation with cAMP-elevating vasoactive intestinal peptide (VIP) and if this requires CFTR. Serous cells stimulated with VIP exhibited a ∼15-20% cAMP-dependent decrease in cell volume and a ∼0.15 unit decrease in intracellular pH (pHi), reflecting activation of Cl- and HCO3 - secretion, respectively. HCO3 - secretion was directly dependent on CFTR and was absent in cells from CF patients. In contrast, neuropeptide Y (NPY) reduced VIP-evoked cAMP increases, CFTR activation, and Cl-/HCO3 - secretion. Culture of primary serous cells in a model that maintained a serous phenotype confirmed the activating and inhibiting effects of VIP and NPY, respectively, on fluid and HCO3 - secretion. Moreover, VIP enhanced antimicrobial peptide secretion and antimicrobial efficacy of secretions while NPY reduced antimicrobial efficacy. In contrast, NPY enhanced cytokine release while VIP reduced cytokine release through a mechanism requiring CFTR. As levels of VIP and NPY are up-regulated in diseases like allergy, asthma, and chronic rhinosinusitis, the balance of these two peptides in the airway may control mucus rheology and inflammatory responses in serous cells. Furthermore, the loss of CFTR conductance in serous cells may contribute to CF pathophysiology by increasing serous cells inflammatory responses in addition to directly impairing Cl- and HCO3 - secretion.

Determining the Lipid Environment and Interactions of CFTR

The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is the ABC-Transporter family's only chloride channel, whose deficiency results in Cystic Fibrosis (CF). Currently, the major cause of death for CF patients is lung failure, and thus focusing on the structure and function of CFTR in its pulmonary epithelial cell environment is of utmost relevance. While several high-resolution structures of CFTR have been solved, none of the structures in the activated state included an open pore enabling chloride conduction. Importantly, these structures were solved in a detergent environment, although some copurified lipids are resolved in some structures. Inspired by data indicating that CFTR's function is highly dependent on the lipid environment, our lab has set out to determine more specifically the lipid environment CFTR encounters and the effects of these lipids on CFTR activity. Thus, our hypothesis is that determining these lipid interactions will provide the information necessary to solve an open-pore CFTR structure and perform more accurate functional analyses. We are approaching this question using detergent-purified CFTR as well as styrene maleic acid lipid particle (SMALP)-purified CFTR. Detergent purification of CFTR would leave only lipids that are tightly bound to the protein, indicating their importance in structure and function. Expanding from these tightly bound lipids, SMALPs require no detergent, but rather “hole-punch” the cell membrane, granting insight into the native lipid neighborhood surrounding CFTR. After determining these lipids, we will conduct functional ATPase and Planar Lipid Bilayer reconstitution studies to determine the specific effects of the lipids on CFTR activity. Alongside these experiments, we are conducting experiments to determine the change in ceramide composition of cultured pulmonary epithelial cells from non-CF and CF subjects, to understand the role of lipid-mediated signaling on CFTR function. [CFF MCCART17G0, MCCART18G0; NIH F31-HL143863-01].