Lipid-driven CFTR clustering is impaired in cystic fibrosis and restored by corrector drugs.

Membrane proteins often cluster in nanoscale membrane domains (lipid rafts) that coalesce into ceramide-rich platforms during cell stress, however the clustering mechanisms remain uncertain. The cystic fibrosis transmembrane conductance regulator (CFTR), which is mutated in cystic fibrosis (CF), forms clusters that are cholesterol dependent and become incorporated into long-lived platforms during hormonal stimulation. We report here that clustering does not involve known tethering interactions of CFTR with PDZ domain proteins, filamin A or the actin cytoskeleton. It also does not require CFTR palmitoylation but is critically dependent on membrane lipid order and is induced by detergents that increase the phase separation of membrane lipids. Clustering and integration of CFTR into ceramide-rich platforms are abolished by the disease mutations F508del and S13F and rescued by the CFTR modulators elexacaftor plus tezacaftor. These results indicate CF therapeutics that correct mutant protein folding restore both trafficking and normal lipid interactions in the plasma membrane. This article has an associated First Person interview with the first author of the paper.

mRNA Delivery: Challenges and Advances through Polymeric Soft Nanoparticles.

Single-stranded messenger ribonucleic acid (mRNA) plays a pivotal role in transferring genetic information, and tremendous effort has been devoted over the years to utilize its transcription efficacy in therapeutic interventions for a variety of diseases with high morbidity and mortality. Lipid nanocarriers have been extensively investigated for mRNA delivery and enabled the rapid and successful development of mRNA vaccines against SARS-CoV-2. Some constraints of lipid nanocarriers have encouraged the development of alternative delivery systems, such as polymer-based soft nanoparticles, which offer a modular gene delivery platform. Such macromolecule-based nanocarriers can be synthetically articulated for tailored parameters including mRNA protection, loading efficacy, and targeted release. In this review, we highlight recent advances in the development of polymeric architectures for mRNA delivery, their limitations, and the challenges that still exist, with the aim of expediting further research and the clinical translation of such formulations.

Ionocyte-Specific Regulation of Cystic Fibrosis Transmembrane Conductance Regulator.

CFTR (cystic fibrosis transmembrane conductance regulator) is a tightly regulated anion channel that mediates chloride and bicarbonate conductance in many epithelia and in other tissues, but whether its regulation varies depending on the cell type has not been investigated. Epithelial CFTR expression is highest in rare cells called ionocytes. We studied CFTR regulation in control and ionocyte-enriched cultures by transducing bronchial basal cells with adenoviruses that encode only eGFP or FOXI1 (forkhead box I1) + eGFP as separate polypeptides. FOXI1 dramatically increased the number of transcripts for ionocyte markers ASCL3 (Achaete-Scute Family BHLH Transcription Factor 3), BSND, ATP6V1G3, ATP6V0D2, KCNMA1, and CFTR without altering those for secretory (SCGB1A1), basal (KRT5, KRT6, TP63), goblet (MUC5AC), or ciliated (FOXJ1) cells. The number of cells displaying strong FOXI1 expression was increased 7-fold, and there was no evidence for a broad increase in background immunofluorescence. Total CFTR mRNA and protein levels increased 10-fold and 2.5-fold, respectively. Ionocyte-enriched cultures displayed elevated basal current, increased adenylyl cyclase 5 expression, and tonic suppression of CFTR activity by the phosphodiesterase PDE1C, which has not been shown previously to regulate CFTR activity. The results indicate that CFTR regulation depends on cell type and identifies PDE1C as a potential target for therapeutics that aim to increase CFTR function specifically in ionocytes.

BAP31 interacts with Sec61 translocons and promotes retrotranslocation of CFTRDeltaF508 via the derlin-1 complex.

BAP31 is an endoplasmic reticulum protein-sorting factor that associates with newly synthesized integral membrane proteins and controls their fate (i.e., egress, retention, survival, or degradation). BAP31 is itself an integral membrane protein and a constituent of several large protein complexes. Here, we show that a part of the BAP31 population interacts with two components of the Sec61 preprotein translocon, Sec61beta and TRAM. BAP31 associates with the N terminus of one of its newly synthesized client proteins, the DeltaF508 mutant of CFTR, and promotes its retrotranslocation from the ER and degradation by the cytoplasmic 26S proteasome system. Depletion of BAP31 reduces the proteasomal degradation of DeltaF508 and permits a significant fraction of the surviving protein to reach the cell surface. Of note, BAP31 also associates physically and functionally with the Derlin-1 protein disclocation complex in the DeltaF508 degradation pathway. Thus, BAP31 operates at early steps to deliver newly synthesized CFTRDeltaF508 to its degradation pathway.

Chronic obstructive pulmonary disease and the modulation of CFTR by acute exposure to cigarette smoke.

Chronic obstructive pulmonary disease (COPD) is a leading cause of death and cigarette smoke is the main risk factor. Detecting its earliest stages and preventing a decline in lung function are key goals. The pathogenesis of COPD is complex but has some similarities to cystic fibrosis (CF), a disease caused by mutations in the cftr gene. CF leads to chronic inflammation, abnormal mucus, and cycles of infection. Cigarette smoke exposure also causes CFTR dysfunction, and it is probably not a coincidence that inflammation, mucus obstruction, and infections are also characteristics of COPD, although the exacerbations can be quite different. We review here the acute effects of cigarette smoke on CFTR function and its potential role in COPD. Understanding CFTR regulation by cigarette smoke may identify novel drug targets and facilitate the development of therapeutics that reduce the progression and severity of COPD.

Phosphodiesterase 8A Regulates CFTR Activity in Airway Epithelial Cells.

BACKGROUND/AIMS: Cystic fibrosis transmembrane conductance regulator (CFTR), the anion channel that is defective in cystic fibrosis (CF), is phosphorylated and activated by cAMP-dependent protein kinase (PKA). cAMP levels are downregulated by a large family of phosphodiesterases that have variable expression in different cell types. We have previously observed high levels of PDE8A expression in well-differentiated primary human bronchial epithelial (pHBE) cells and thus aimed to assess whether it played a role in cAMP-dependent regulation of CFTR activity. METHODS: We assessed the effect of the selective PDE8 inhibitor PF-04957325 (PF) on intracellular cAMP levels ([cAMP]i) in well differentiated pHBE cells from non-CF or CF donors and also in CFBE41o- cells that stably express wild-type CFTR (CFBE41o- WT) using ELISA and FRET-FLIM microscopy. CFTR channel function was also measured using electrophysiological recordings from pHBE and CFBE41o- WT cells mounted in Ussing Chambers. RESULTS: PDE8 inhibition elevated [cAMP]i in well-differentiated pHBE cells and stimulated wild-type CFTR-dependent ion transport under basal conditions or after cells had been pre-stimulated with physiological cAMP-elevating agents. The response to PDE8 inhibition was larger than to PDE3 or PDE5 inhibition but smaller and synergistic with that elicited by PDE4 inhibition. CRISPR Cas9-mediated knockdown of PDE8A enhanced CFTR gene and protein expression yet reduced the effect of PDE8 inhibition. Acute pharmacological inhibition PDE8 increased CFTR activity in CF pHBE cells (F508del/F508del and F508del/R117H-5T) treated with clinically-approved CFTR modulators. CONCLUSION: These results provide the first evidence that PDE8A regulates CFTR and identifies PDE8A as a potential target for adjunct therapies to treat CF.

The anion transporter SLC26A9 localizes to tight junctions and is degraded by the proteasome when co-expressed with F508del-CFTR.

Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) disrupt epithelial secretion and cause cystic fibrosis (CF). Available CFTR modulators provide only modest clinical benefits, so alternative therapeutic targets are being explored. The anion-conducting transporter solute carrier family 26 member 9 (SLC26A9) is a promising candidate, but its functional expression is drastically reduced in cells that express the most common CF-associated CFTR variant, F508del-CFTR, through mechanisms that remain incompletely understood. Here, we examined the metabolic stability and location of SLC26A9 and its relationship to CFTR. Compared with SLC26A9 levels in BHK cells expressing SLC26A9 alone or with WT-CFTR, co-expression of SLC26A9 with F508del-CFTR reduced total and plasma membrane levels of SLC26A9. Proteasome inhibitors increased SLC26A9 immunofluorescence in primary human bronchial epithelial cells (pHBEs) homozygous for F508del-CFTR but not in non-CF pHBEs, suggesting that F508del-CFTR enhances proteasomal SLC26A9 degradation. Apical SLC26A9 expression increased when F508del-CFTR trafficking was partially corrected by low temperature or with the CFTR modulator VX-809. The immature glycoforms of SLC26A9 and CFTR co-immunoprecipitated, consistent with their interaction in the endoplasmic reticulum (ER). Transfection with increasing amounts of WT-CFTR cDNA progressively increased SLC26A9 levels in F508del-CFTR-expressing cells, suggesting that WT-CFTR competes with F508del-CFTR for SLC26A9 binding. Immunofluorescence staining of endogenous SLC26A9 and transfection of a 3HA-tagged construct into well-differentiated cells revealed that SLC26A9 is mostly present at tight junctions. We conclude that SLC26A9 interacts with CFTR in both the ER and Golgi and that its interaction with F508del-CFTR increases proteasomal SLC26A9 degradation.

The dual phosphodiesterase 3/4 inhibitor RPL554 stimulates rare class III and IV CFTR mutants.

Over 2,000 mutations have been reported in the cystic fibrosis transmembrane conductance regulator (cftr) gene, many of which cause disease but are rare and have no effective treatment. Thus, there is an unmet need for new, mutation-agnostic therapies for cystic fibrosis (CF). Phosphodiesterase (PDE) inhibitors are one such class of therapeutics that have been shown to elevate intracellular cAMP levels and stimulate CFTR-dependent anion secretion in human airway epithelia; however, the number of people with CF that could be helped by PDE inhibitors remains to be determined. Here we used Fisher rat thyroid (FRT) cells stably transduced with rare human CFTR mutants and studied their responsiveness to the dual phosphodiesterase 3/4 inhibitor RPL554 (Verona Pharma). Through its inhibitory effect on PDE4D, we find that RPL554 can elevate intracellular cAMP leading to a potentiation of forskolin-stimulated current mediated by R334W, T338I, G551D, and S549R mutants of CFTR when used alone or in combination with CFTR modulators. We also were able to reproduce these effects of RPL554 on G551D-CFTR when it was expressed in primary human bronchial epithelial cells, indicating that RPL554 would have stimulatory effects on rare CFTR mutants in human airways and validating FRT cells as a model for PDE inhibitor studies. Furthermore, we provide biochemical evidence that VX-809 causes surprisingly robust correction of several class III and IV CFTR mutants. Together, our findings further support the therapeutic potential of RPL554 for patients with CF with class III/IV mutations and emphasize the potential of PDEs as potential drug targets that could benefit patients with CF.

The Phosphodiesterase Inhibitor Ensifentrine Reduces Production of Proinflammatory Mediators in Well Differentiated Bronchial Epithelial Cells by Inhibiting PDE4.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel that impair airway salt and fluid secretion. Excessive release of proinflammatory cytokines and chemokines by CF bronchial epithelium during airway infection leads to chronic inflammation and a slow decline in lung function; thus, there is much interest in finding safe and effective treatments that reduce inflammation in CF. We showed previously that the cyclic nucleotide phosphodiesterase (PDE) inhibitor ensifentrine (RPL554; Verona Pharma) stimulates the channel function of CFTR mutants with abnormal gating and also those with defective trafficking that are partially rescued using a clinically approved corrector drug. PDE inhibitors also have known anti-inflammatory effects; therefore, we examined whether ensifentrine alters the production of proinflammatory cytokines in CF bronchial epithelial cells. Ensifentrine reduced the production of monocyte chemoattractant protein-1 and granulocyte monocyte colony-stimulating factor (GM-CSF) during challenge with interleukin-1ß Comparing the effect of ensifentrine with milrinone and roflumilast, selective PDE3 and PDE4 inhibitors, respectively, demonstrated that the anti-inflammatory effect of ensifentrine was mainly due to inhibition of PDE4. Beneficial modulation of GM-CSF was further enhanced when ensifentrine was combined with low concentrations of the ß 2-adrenergic agonist isoproterenol or the corticosteroid dexamethasone. The results indicate that ensifentrine may have beneficial anti-inflammatory effects in CF airways particularly when used in combination with ß 2-adrenergic agonists or corticosteroids. SIGNIFICANCE STATEMENT: Airway inflammation that is disproportionate to the burden of chronic airway infection causes much of the pathology in the cystic fibrosis (CF) lung. We show here that ensifentrine beneficially modulates the release of proinflammatory factors in well differentiated CF bronchial epithelial cells that is further enhanced when combined with ß2-adrenergic agonists or low-concentration corticosteroids. The results encourage further clinical testing of ensifentrine, alone and in combination with ß2-adrenergic agonists or low-concentration corticosteroids, as a novel anti-inflammatory therapy for CF.

Pendrin Mediates Bicarbonate Secretion and Enhances Cystic Fibrosis Transmembrane Conductance Regulator Function in Airway Surface Epithelia.

Bicarbonate facilitates mucin unpacking and bacterial killing; however, its transport mechanisms in the airways are not well understood. cAMP stimulates anion efflux through the cystic fibrosis (CF) transmembrane conductance regulator (CFTR; ABCC7) anion channel, and this is defective in CF. The anion exchanger pendrin (SLC26A4) also mediates HCO3- efflux and is upregulated by proinflammatory cytokines. Here, we examined pendrin and CFTR expression and their contributions to HCO3- secretion by human nasal and bronchial epithelia. In native tissue, both proteins were most abundant at the apical pole of ciliated surface cells with little expression in submucosal glands. In well-differentiated primary nasal and bronchial cell cultures, IL-4 dramatically increased pendrin mRNA levels and apical immunostaining. Exposure to low-Cl- apical solution caused intracellular alkalinization (ΔpHi) that was enhanced fourfold by IL-4 pretreatment. ΔpHi was unaffected by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) or CFTR inhibitor CFTRinh-172, but was reduced by adenoviral shRNA targeting pendrin. Forskolin increased ΔpHi, and this stimulation was prevented by CFTRinh-172, implicating CFTR, yet forskolin only increased ΔpHi after pendrin expression had been induced by IL-4. The dependence of ΔpHi on pendrin suggests there is minimal electrical coupling between Cl- and HCO3- fluxes and that CFTR activation increases anion exchange-mediated HCO3- influx. Conversely, inducing pendrin expression increased forskolin-stimulated, CFTRinh-172-sensitive current by approximately twofold in epithelial and nonepithelial cells. We conclude that pendrin mediates most HCO3- secretion across airway surface epithelium during inflammation and enhances electrogenic Cl- secretion via CFTR, as described for other SLC26A transporters.

Velocity landscape correlation resolves multiple flowing protein populations from fluorescence image time series.

Molecular, vesicular and organellar flows are of fundamental importance for the delivery of nutrients and essential components used in cellular functions such as motility and division. With recent advances in fluorescence/super-resolution microscopy modalities we can resolve the movements of these objects at higher spatio-temporal resolutions and with better sensitivity. Previously, spatio-temporal image correlation spectroscopy has been applied to map molecular flows by correlation analysis of fluorescence fluctuations in image series. However, an underlying assumption of this approach is that the sampled time windows contain one dominant flowing component. Although this was true for most of the cases analyzed earlier, in some situations two or more different flowing populations can be present in the same spatio-temporal window. We introduce an approach, termed velocity landscape correlation (VLC), which detects and extracts multiple flow components present in a sampled image region via an extension of the correlation analysis of fluorescence intensity fluctuations. First we demonstrate theoretically how this approach works, test the performance of the method with a range of computer simulated image series with varying flow dynamics. Finally we apply VLC to study variable fluxing of STIM1 proteins on microtubules connected to the plasma membrane of Cystic Fibrosis Bronchial Epithelial (CFBE) cells.

Cigarette smoke activates CFTR through ROS-stimulated cAMP signaling in human bronchial epithelial cells.

Air pollution stimulates airway epithelial secretion through a cholinergic reflex that is unaffected in cystic fibrosis (CF), yet a strong correlation is observed between passive smoke exposure in the home and impaired lung function in CF children. Our aim was to study the effects of low smoke concentrations on cystic fibrosis transmembrane conductance regulator (CFTR) function in vitro. Cigarette smoke extract stimulated robust anion secretion that was transient, mediated by CFTR, and dependent on cAMP-dependent protein kinase activation. Secretion was initiated by reactive oxygen species (ROS) and mediated by at least two distinct pathways: autocrine activation of EP4 prostanoid receptors and stimulation of Ca2+ store-operated cAMP signaling. The response was absent in cells expressing the most common disease-causing mutant F508del-CFTR. In addition to the initial secretion, prolonged exposure of non-CF bronchial epithelial cells to low levels of smoke also caused a gradual decline in CFTR functional expression. F508del-CFTR channels that had been rescued by the CF drug combination VX-809 (lumacaftor) + VX-770 (ivacaftor) were more sensitive to this downregulation than wild-type CFTR. The results suggest that CFTR-mediated secretion during acute cigarette smoke exposure initially protects the airway epithelium while prolonged exposure reduces CFTR functional expression and reduces the efficacy of CF drugs.

Latonduine Analogs Restore F508del-Cystic Fibrosis Transmembrane Conductance Regulator Trafficking through the Modulation of Poly-ADP Ribose Polymerase 3 and Poly-ADP Ribose Polymerase 16 Activity.

Cystic fibrosis (CF) is a major lethal genetic disease caused by mutations in the CF transmembrane conductance regulator gene (CFTR). This encodes a chloride ion channel on the apical surface of epithelial cells. The most common mutation in CFTR (F508del-CFTR) generates a protein that is misfolded and retained in the endoplasmic reticulum. Identifying small molecules that correct this CFTR trafficking defect is a promising approach in CF therapy. However, to date only modest efficacy has been reported for correctors in clinical trials. We identified the marine sponge metabolite latonduine as a corrector. We have now developed a series of latonduine derivatives that are more potent F508del-CFTR correctors with one (MCG315 [2,3-dihydro-1H-2-benzazepin-1-one]) having 10-fold increased corrector activity and an EC50 of 72.25 nM. We show that the latonduine analogs inhibit poly-ADP ribose polymerase (PARP) isozymes 1, 3, and 16. Further our molecular modeling studies point to the latonduine analogs binding to the PARP nicotinamide-binding domain. We established the relationship between the ability of the latonduine analogs to inhibit PARP-16 and their ability to correct F508del-CFTR trafficking. We show that latonduine can inhibit both PARP-3 and -16 and that this is necessary for CFTR correction. We demonstrate that latonduine triggers correction by regulating the activity of the unfolded protein response activator inositol-requiring enzyme (IRE-1) via modulation of the level of its ribosylation by PARP-16. These results establish latonduines novel site of action as well as its proteostatic mechanism of action.

The dual phosphodiesterase 3 and 4 inhibitor RPL554 stimulates CFTR and ciliary beating in primary cultures of bronchial epithelia.

Cystic fibrosis (CF), a genetic disease caused by mutations in the CFTR gene, is a life-limiting disease characterized by chronic bacterial airway infection and severe inflammation. Some CFTR mutants have reduced responsiveness to cAMP/PKA signaling; hence, pharmacological agents that elevate intracellular cAMP are potentially useful for the treatment of CF. By inhibiting cAMP breakdown, phosphodiesterase (PDE) inhibitors stimulate CFTR in vitro and in vivo. Here, we demonstrate that PDE inhibition by RPL554, a drug that has been shown to cause bronchodilation in asthma and chronic obstructive pulmonary disease (COPD) patients, stimulates CFTR-dependent ion secretion across bronchial epithelial cells isolated from patients carrying the R117H/F508del CF genotype. RPL554-induced CFTR activity was further increased by the potentiator VX-770, suggesting an additional benefit by the drug combination. RPL554 also increased cilia beat frequency in primary human bronchial epithelial cells. The results indicate RPL554 may increase mucociliary clearance through stimulation of CFTR and increasing ciliary beat frequency and thus could provide a novel therapeutic option for CF.

ß2-Adrenergic receptor agonists activate CFTR in intestinal organoids and subjects with cystic fibrosis.

We hypothesized that people with cystic fibrosis (CF) who express CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations associated with residual function may benefit from G-protein coupled receptor (GPCR)-targeting drugs that can activate and enhance CFTR function.We used intestinal organoids to screen a GPCR-modulating compound library and identified ß2-adrenergic receptor agonists as the most potent inducers of CFTR function.ß2-Agonist-induced organoid swelling correlated with the CFTR genotype, and could be induced in homozygous CFTR-F508del organoids and highly differentiated primary CF airway epithelial cells after rescue of CFTR trafficking by small molecules. The in vivo response to treatment with an oral or inhaled ß2-agonist (salbutamol) in CF patients with residual CFTR function was evaluated in a pilot study. 10 subjects with a R117H or A455E mutation were included and showed changes in the nasal potential difference measurement after treatment with oral salbutamol, including a significant improvement of the baseline potential difference of the nasal mucosa (+6.35 mV, p<0.05), suggesting that this treatment might be effective in vivo Furthermore, plasma that was collected after oral salbutamol treatment induced CFTR activation when administered ex vivo to organoids.This proof-of-concept study suggests that organoids can be used to identify drugs that activate CFTR function in vivo and to select route of administration.

Regulation of the cystic fibrosis transmembrane conductance regulator anion channel by tyrosine phosphorylation.

The cystic fibrosis transmembrane conductance regulator (CFTR) channel is activated by PKA phosphorylation of a regulatory domain that interacts dynamically with multiple CFTR domains and with other proteins. The large number of consensus sequences for phosphorylation by PKA has naturally focused most attention on regulation by this kinase. We report here that human CFTR is also phosphorylated by the tyrosine kinases p60c-Src (proto-oncogene tyrosine-protein kinase) and the proline-rich tyrosine kinase 2 (Pyk2), and they can also cause robust activation of quiescent CFTR channels. In excised patch-clamp experiments, CFTR activity during exposure to Src or Pyk2 reached ∼80% of that stimulated by PKA. Exposure to PKA after Src or Pyk2 caused a further increase to the level induced by PKA alone, implying a common limiting step. Channels became spontaneously active when v-Src or the catalytic domain of Pyk2 was coexpressed with CFTR and were further stimulated by the tyrosine phosphatase inhibitor dephostatin. Exogenous Src also activated 15SA-CFTR, a variant that lacks 15 potential PKA sites and has little response to PKA. PKA-independent activation by tyrosine phosphorylation has implications for the mechanism of regulation by the R domain and for the physiologic functions of CFTR.

Cholesterol modulates CFTR confinement in the plasma membrane of primary epithelial cells.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma-membrane anion channel that, when mutated, causes the disease cystic fibrosis. Although CFTR has been detected in a detergent-resistant membrane fraction prepared from airway epithelial cells, suggesting that it may partition into cholesterol-rich membrane microdomains (lipid rafts), its compartmentalization has not been demonstrated in intact cells and the influence of microdomains on CFTR lateral mobility is unknown. We used live-cell imaging, spatial image correlation spectroscopy, and k-space image correlation spectroscopy to examine the aggregation state of CFTR and its dynamics both within and outside microdomains in the plasma membrane of primary human bronchial epithelial cells. These studies were also performed during treatments that augment or deplete membrane cholesterol. We found two populations of CFTR molecules that were distinguishable based on their dynamics at the cell surface. One population showed confinement and had slow dynamics that were highly cholesterol dependent. The other, more abundant population was less confined and diffused more rapidly. Treatments that deplete the membrane of cholesterol caused the confined fraction and average number of CFTR molecules per cluster to decrease. Elevating cholesterol had the opposite effect, increasing channel aggregation and the fraction of channels displaying confinement, consistent with CFTR recruitment into cholesterol-rich microdomains with dimensions below the optical resolution limit. Viral infection caused the nanoscale microdomains to fuse into large platforms and reduced CFTR mobility. To our knowledge, these results provide the first biophysical evidence for multiple CFTR populations and have implications for regulation of their surface expression and channel function.

Polymer assisted entrapment of netilmicin in PLGA nanoparticles for sustained antibacterial activity.

This study was aimed to develop poly(dl-lactide-co-glycolide) (PLGA) nanoparticle of highly water soluble antibiotic drug, netilmicin sulfate (NS) with improved entrapment efficiency (EE) and antibacterial activity. Dextran sulfate was introduced as helper polymer to form electrostatic complex with NS. Nanoparticles were prepared by double emulsification method and optimized using 2(5-1) fractional factorial design. EE was mainly influenced by dextran sulfate: NS charge ratio and PLGA concentration, whereas particle size (PS) was affected by all factors examined. The optimized NS-loaded-NPs had EE and PS of 93.23 ± 2.7% and 140.83 ± 2.4 nm respectively. NS-loaded-NPs effectively inhibited bacterial growth compared to free NS. Sustained release protected its inactivation and reduced the decline in its killing activity over time even in presence of bronchial cells. A MIC value of 18 µg/mL was observed for NPs on P. aeruginosa. Therefore, NPs with sustained bactericidal efficiency against P. aeruginosa may provide therapeutic benefit in chronic pulmonary infection, like cystic fibrosis.

Role of tyrosine phosphorylation in the muscarinic activation of the cystic fibrosis transmembrane conductance regulator (CFTR).

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride (Cl(-)) channel, which plays an important role in physiological anion and fluid secretion, and is defective in several diseases. Although its activation by PKA and PKC has been studied extensively, its regulation by receptors is less well understood. To study signaling involved in CFTR activation, we measured whole-cell Cl(-) currents in BHK cells cotransfected with GPCRs and CFTR. In cells expressing the M3 muscarinic acetylcholine receptor, the agonist carbachol (Cch) caused strong activation of CFTR through two pathways; the canonical PKA-dependent mechanism and a second mechanism that involves tyrosine phosphorylation. The role of PKA was suggested by partial inhibition of cholinergic stimulation by the specific PKA inhibitor Rp-cAMPS. The role of tyrosine kinases was suggested by Cch stimulation of 15SA-CFTR and 9CA-CFTR, mutants that lack 15 PKA or 9 PKC consensus sequences and are unresponsive to PKA or PKC stimulation, respectively. Moreover the residual Cch response was sensitive to inhibitors of the Pyk2 and Src tyrosine kinase family. Our results suggest that tyrosine phosphorylation acts on CFTR directly and through inhibition of the phosphatase PP2A. Results suggest that PKA and tyrosine kinases contribute to CFTR regulation by GPCRs that are expressed at the apical membrane of intestinal and airway epithelia.

CRISPR/Cas9 bioluminescence-based assay for monitoring CFTR trafficking to the plasma membrane.

CFTR is a membrane protein that functions as an ion channel. Mutations that disrupt its biosynthesis, trafficking or function cause cystic fibrosis (CF). Here, we present a novel in vitro model system prepared using CRISPR/Cas9 genome editing with endogenously expressed WT-CFTR tagged with a HiBiT peptide. To enable the detection of CFTR in the plasma membrane of live cells, we inserted the HiBiT tag in the fourth extracellular loop of WT-CFTR. The 11-amino acid HiBiT tag binds with high affinity to a large inactive subunit (LgBiT), generating a reporter luciferase with bright luminescence. Nine homozygous clones with the HiBiT knock-in were identified from the 182 screened clones; two were genetically and functionally validated. In summary, this work describes the preparation and validation of a novel reporter cell line with the potential to be used as an ultimate building block for developing unique cellular CF models by CRISPR-mediated insertion of CF-causing mutations.