Conformational Changes of CFTR upon Phosphorylation and ATP Binding.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel evolved from an ATP-binding cassette transporter. CFTR channel gating is strictly coupled to phosphorylation and ATP hydrolysis. Previously, we reported essentially identical structures of zebrafish and human CFTR in the dephosphorylated, ATP-free form. Here, we present the structure of zebrafish CFTR in the phosphorylated, ATP-bound conformation, determined by cryoelectron microscopy to 3.4 Å resolution. Comparison of the two conformations shows major structural rearrangements leading to channel opening. The phosphorylated regulatory domain is disengaged from its inhibitory position; the nucleotide-binding domains (NBDs) form a "head-to-tail" dimer upon binding ATP; and the cytoplasmic pathway, found closed off in other ATP-binding cassette transporters, is cracked open, consistent with CFTR's unique channel function. Unexpectedly, the extracellular mouth of the ion pore remains closed, indicating that local movements of the transmembrane helices can control ion access to the pore even in the NBD-dimerized conformation.

Acute polymicrobial airway infections: analysis in cystic fibrosis mice.

Cystic fibrosis (CF) is a genetic disorder affecting epithelial ion transport, which among other impacts results in defective mucociliary clearance and innate defenses in the respiratory tract. Consequently, people with CF experience lifelong infections of the respiratory mucosa that are chronic and polymicrobial in nature. Young children with CF are initially colonized by opportunists like nontypeable Haemophilus influenzae (NTHi), which normally resides within the microbiome of the nasopharynx and upper airways and can also cause infections of the respiratory mucosa that include bronchitis and otitis media. NTHi is typically supplanted by other microbes as patients age; for example, people with CF are often chronically infected with mucoid strains of Pseudomonas aeruginosa, which prior work in our laboratory has shown to promote colonization and persistence by other opportunists that include Stenotrophomonas maltophilia. Our previous work has shown that polymicrobial infection impacts host colonization and persistence of incoming microbes via diverse mechanisms that include priming of host immunity that can promote microbial clearance, and cooperativity within polymicrobial biofilms, which can promote persistence. In infection studies with BALB/c Cftrtm1UNC mice, results showed, as previously observed for WT BALB/c mice, preceding infection with NTHi decreased colonization and persistence by P. aeruginosa. Likewise, polymicrobial infection of BALB/c Cftrtm1UNC and C57BL/6 Cftrtm1UncTg(FABPhCFTR)1Jaw/J mice showed correlation between S. maltophilia and P. aeruginosa, with increased bacterial colonization and lung pathology. Based on these results, we conclude that our previous observations regarding polymicrobial infections with CF opportunists in WT mice are also validated using CF mice.

The cAMP-phosphodiesterase 4 (PDE4) controls ß-adrenoceptor- and CFTR-dependent saliva secretion in mice.

Saliva, while often taken for granted, is indispensable for oral health and overall well-being, as inferred from the significant impairments suffered by patients with salivary gland dysfunction. Here, we show that treatment with several structurally distinct PAN-PDE4 inhibitors, but not a PDE3 inhibitor, induces saliva secretion in mice, indicating it is a class-effect of PDE4 inhibitors. In anesthetized mice, while neuronal regulations are suppressed, PDE4 inhibition potentiates a ß-adrenoceptor-induced salivation, that is ablated by the ß-blocker Propranolol and is absent from homozygous ΔF508-CFTR mice lacking functional CFTR. These data suggest that PDE4 acts within salivary glands to gate saliva secretion that is contingent upon the cAMP/PKA-dependent activation of CFTR. Indeed, PDE4 contributes the majority of total cAMP-hydrolytic capacity in submandibular-, sublingual-, and parotid glands, the three major salivary glands of the mouse. In awake mice, PDE4 inhibitor-induced salivation is reduced by CFTR deficiency or ß-blockers, but also by the muscarinic blocker Atropine, suggesting an additional, central/neuronal mechanism of PDE4 inhibitor action. The PDE4 family comprises four subtypes, PDE4A-D. Ablation of PDE4D, but not PDE4A-C, produced a minor effect on saliva secretion, implying that while PDE4D may play a predominant role, PDE4 inhibitor-induced salivation results from the concurrent inactivation of multiple (at least two) PDE4 subtypes. Taken together, our data reveal a critical role for PDE4/PDE4D in controlling CFTR function in an in vivo model and in inducing salivation, hinting at a therapeutic potential of PDE4 inhibition for cystic fibrosis and conditions associated with xerostomia.

Precise Targeting of miRNA Sites Restores CFTR Activity in CF Bronchial Epithelial Cells.

MicroRNAs that are overexpressed in cystic fibrosis (CF) bronchial epithelial cells (BEC) negatively regulate CFTR and nullify the beneficial effects of CFTR modulators. We hypothesized that it is possible to reverse microRNA-mediated inhibition of CFTR using CFTR-specific target site blockers (TSBs) and to develop a drug-device combination inhalation therapy for CF. Lead microRNA expression was quantified in a series of human CF and non-CF samples and in vitro models. A panel of CFTR 3' untranslated region (UTR)-specific locked nucleic acid antisense oligonucleotide TSBs was assessed for their ability to increase CFTR expression. Their effects on CFTR activity alone or in combination with CFTR modulators were measured in CF BEC models. TSB encapsulation in poly-lactic-co-glycolic acid (PLGA) nanoparticles was assessed as a proof of principle of delivery into CF BECs. TSBs targeting the CFTR 3' UTR 298-305:miR-145-5p or 166-173:miR-223-3p sites increased CFTR expression and anion channel activity and enhanced the effects of ivacaftor/lumacaftor or ivacaftor/tezacaftor in CF BECs. Biocompatible PLGA-TSB nanoparticles promoted CFTR expression in primary BECs and retained desirable biophysical characteristics following nebulization. Alone or in combination with CFTR modulators, aerosolized CFTR-targeting TSBs encapsulated in PLGA nanoparticles could represent a promising drug-device combination therapy for the treatment for CFTR dysfunction in the lung.

Pharmacological and Genomic Approaches in Management of Cystic Fibrosis.

Cystic fibrosis (CF) is an inherited recessive autosomal disorder that affects the lungs, the digestive system, and secretory glands. It is a lethal condition caused by a mutation in the gene cystic-fibrosis-transmembrane-conductance- regulator (CFTR), which leads to defects in ion channels and results in obstruction of mucus in airway channels. Unbalanced ion exchange causes impaired water transport and accumulation of viscous mucus in the air way leads to bacterial colonization, for example, with Staphylococcus aureus. The most common mutation is the deletion of nucleotides in epithelial membrane; hence, it is a multiple-organ-defective disease that mostly effects the lungs. Researchers are working on gene therapy that aims to introduce a normal CFTR gene copy into the epithelial cells of lungs. Several approaches have been designed to improve transepithelial ion transport in CF patients. Normal CFTR gene delivery has been performed using viral and nonviral vectors, but these approaches are not more efficient against the cell barriers. Enzymes may be used that inhibit the sphingolipid to provide proper microenvironment for the CFTR gene product. Thymosin alpha-1 has also been reported as a potential corrector in treatment of CF.

Salivary Thiocyanate as a Biomarker of Cystic Fibrosis Transmembrane Regulator Function.

Improved methods are needed to reliably assess Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) function in vivo in light of recent therapeutic developments targeting the CFTR protein. Oral fluid from patients with cystic fibrosis (CF) and healthy controls (HCs) were studied using colorimetry and nonresonant Raman spectroscopy. Colorimetry experiments showed only a 36% decrease in thiocyanate (SCN-) concentration, but a sharp Raman peak at 2068 cm-1, attributable to (SCN-) vibrations, normalized to C-H peak, was on average 18 times higher for HC samples. Samples from patients undergoing treatment with CFTR modulators including ivacaftor, lumacaftor, and tezacaftor showed a high normalized peak in response to therapy. The peak intensity was consistent in longitudinal samples from single donors and in stored samples. The Raman peak ratio is a more sensitive, convenient, noninvasive biomarker for assessments of the therapeutic efficacy of drugs targeting CFTR and provides a value that is in much better agreement with theoretical expectations of saliva SCN- concentrations compared to colorimetry. This insight may greatly facilitate assessments of CFTR modulator efficacy in individual patients.

Cryo-electron microscopy of membrane proteins.

Membrane proteins represent a large proportion of the proteome, but have characteristics that are problematic for many methods in modern molecular biology (that have often been developed with soluble proteins in mind). For structural studies, low levels of expression and the presence of detergent have been thorns in the flesh of the membrane protein experimentalist. Here we discuss the use of cryo-electron microscopy in breakthrough studies of the structures of membrane proteins. This method can cope with relatively small quantities of sample and with the presence of detergent. Until recently, cryo-electron microscopy could not deliver high-resolution structures of membrane proteins, but recent developments in transmission electron microscope technology and in the image processing of single particles imaged in the microscope have revolutionized the field, allowing high resolution structures to be obtained. Here we focus on the specific issues surrounding the application of cryo-electron microscopy to the study of membrane proteins, especially in the choice of a system to keep the protein soluble.

ANALYSIS OF LOCAL IMMUNITY INDICATORS OF THE ORAL CAVITY AND DEGREE OF GINGIVITIS DEPENDING ON MUTATION OF CFTR GENE IN CHILDREN WITH CYSTIC FIBROSIS.

The aim of the work was to establish the relationship between the genotype of the cystic fibrosis transmembrane conductance regulator (CFTR), the level of local immune reactivity and the degree of chronic gingivitis in children with cystic fibrosis. The study has shown significant differences in the local immunity indices of the oral mucosa and the condition of periodontal tissues in children with cystic fibrosis in comparison with the control group. The features of the course of dental pathology among sick children, depending on the type of CFTR gene mutation are determined. Disturbance of mucosal immunity of the oral cavity in children with cystic fibrosis is manifested by a decrease in lysozyme activity in mixed saliva by 1.5 times and level of secretory immunoglobulins IgA by 1.4 times. A consequence of this is an increase of the degree of dysbiosis of the oral cavity by 3.7 times. At the same time, a lesser imbalance in the microflora and lysozyme activity observed in the homozygote group of the F508del mutation, and heterozygotes of the F508del mutation have the most severe manifestations of chronic gingivitis.

Bicarbonate Transport During Enamel Maturation.

Amelogenesis (tooth enamel formation) is a biomineralization process consisting primarily of two stages (secretory stage and maturation stage) with unique features. During the secretory stage, the inner epithelium of the enamel organ (i.e., the ameloblast cells) synthesizes and secretes enamel matrix proteins (EMPs) into the enamel space. The protein-rich enamel matrix forms a highly organized architecture in a pH-neutral microenvironment. As amelogenesis transitions to maturation stage, EMPs are degraded and internalized by ameloblasts through endosomal-lysosomal pathways. Enamel crystallite formation is initiated early in the secretory stage, however, during maturation stage the more rapid deposition of calcium and phosphate into the enamel space results in a rapid expansion of crystallite length and mineral volume. During maturation-stage amelogenesis, the pH value of enamel varies considerably from slightly above neutral to acidic. Extracellular acid-base balance during enamel maturation is tightly controlled by ameloblast-mediated regulatory networks, which include significant synthesis and movement of bicarbonate ions from both the enamel papillary layer cells and ameloblasts. In this review we summarize the carbonic anhydrases and the carbonate transporters/exchangers involved in pH regulation in maturation-stage amelogenesis. Proteins that have been shown to be instrumental in this process include CA2, CA6, CFTR, AE2, NBCe1, SLC26A1/SAT1, SLC26A3/DRA, SLC26A4/PDS, SLC26A6/PAT1, and SLC26A7/SUT2. In addition, we discuss the association of miRNA regulation with bicarbonate transport in tooth enamel formation.

Wheat bran components modulate intestinal bacteria and gene expression of barrier function relevant proteins in a piglet model.

The objective of this study was to determine the impact of wheat bran and its main polysaccharides on intestinal bacteria and gene expression of intestinal barrier function relevant proteins. Thirty freshly weaned male piglets were assigned randomly to five dietary treatment groups with six piglets per group. Accordingly, five synthetic diets including a basal control diet without fiber components (CON), wheat bran diet (10% wheat bran, WB), arabinoxylan diet (AX), cellulose diet (CEL) and combined diet of arabinoxylan and cellulose (CB) were studied. The piglets were fed ad libitum for 30 d. Lower Escherichia coli (E. coli) populations in WB group and higher probiotic (Lactobacillus and Bifidobacterium) populations in groups fed diets containing arabinoxylan (WB, AX and CB) were observed and compared with CON group. Compared with CON group, the gene expressions of cystic fibrosis transmembrane conductance regulator (CFTR), calcium-activated chloride channel regulator 1 (CLCA1) and voltage-gated chloride channel 2 (CIC2) were suppressed in the WB group. And wheat bran down-regulated gene expression of pro-inflammation (TNF-α, IL-1ß, IL-6) and TLRs/MyD88/NF-κB pathway compared with CON group. In conclusion, wheat bran and its main polysaccharides could change intestinal microflora and down-regulate the gene expression of intestinal barrier function relevant proteins in the distal small intestinal mucosa.

Functional Architecture of the Cytoplasmic Entrance to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore.

As an ion channel, the cystic fibrosis transmembrane conductance regulator must form a continuous pathway for the movement of Cl(-) and other anions between the cytoplasm and the extracellular solution. Both the structure and the function of the membrane-spanning part of this pathway are well defined. In contrast, the structure of the pathway that connects the cytoplasm to the membrane-spanning regions is unknown, and functional roles for different parts of the protein forming this pathway have not been described. We used patch clamp recording and substituted cysteine accessibility mutagenesis to identify positively charged amino acid side chains that attract cytoplasmic Cl(-) ions to the inner mouth of the pore. Our results indicate that the side chains of Lys-190, Arg-248, Arg-303, Lys-370, Lys-1041, and Arg-1048, located in different intracellular loops of the protein, play important roles in the electrostatic attraction of Cl(-) ions. Mutation and covalent modification of these residues have charge-dependent effects on the rate of Cl(-) permeation, demonstrating their functional role in maximization of Cl(-) flux. Other nearby positively charged side chains were not involved in electrostatic interactions with Cl(-). The location of these Cl(-)-attractive residues suggests that cytoplasmic Cl(-) ions enter the pore via a lateral portal located between the cytoplasmic extensions to the fourth and sixth transmembrane helices; a secondary, functionally less relevant portal might exist between the extensions to the 10th and 12th transmembrane helices. These results define the cytoplasmic mouth of the pore and show how it attracts Cl(-) ions from the cytoplasm.

Topical cystic fibrosis transmembrane conductance regulator gene replacement for cystic fibrosis-related lung disease.

BACKGROUND: Cystic fibrosis is caused by a defective gene encoding a protein called the cystic fibrosis transmembrane conductance regulator (CFTR), and is characterised by chronic lung infection resulting in inflammation and progressive lung damage that results in a reduced life expectancy. OBJECTIVES: To determine whether topical CFTR gene replacement therapy to the lungs in people with cystic fibrosis is associated with improvements in clinical outcomes, and to assess any adverse effects. SEARCH METHODS: We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Trials Register comprising references identified from comprehensive electronic database searches, handsearching relevant journals and abstract books of conference proceedings.Date of most recent search: 05 May 2016.An additional search of the National Institutes for Health (NIH) Genetic Modification Clinical Research Information System (GeMCRIS) was also performed for the years 1992 to 2015.Date of most recent search: 20 April 2016. SELECTION CRITERIA: Randomised controlled studies comparing topical CFTR gene delivery to the lung, using either viral or non-viral delivery systems, with placebo or an alternative delivery system in people with confirmed cystic fibrosis. DATA COLLECTION AND ANALYSIS: The authors independently extracted data and assessed study quality. Authors of included studies were contacted and asked for any available additional data. Meta-analysis was limited due to differing study designs. MAIN RESULTS: Four randomised controlled studies met the inclusion criteria for this review, involving a total of 302 participants lasting from 29 days to 13 months; 14 studies were excluded. The included studies differed in terms of CFTR gene replacement agent and study design, which limited the meta-analysis. One study only enrolled adult males, the remaining studies included both males and females aged 12 years and over.Risk of bias in the studies was moderate. Random sequence generation and allocation concealment was only described in the more recent study; the remaining three studies were judged to have an unclear risk of bias. All four studies documented double-blinding to the intervention, but there is some uncertainty with regards to participant blinding in one study. Some outcome data were missing from all four studies.There were no differences in either the number of respiratory exacerbations or the number of participants with an exacerbation between replacement therapy or placebo groups at any time point. Meta-analysis of most respiratory function tests showed no difference between treatment and placebo groups, but the smallest study (n = 16) reported forced vital capacity (litres) increased more in the placebo group at up to 24 hours. A further study reported a significant improvement in forced expiratory volume at one second (litres) at 30 days after participants had received their first dose of favouring the gene therapy agent, but this finding was not confirmed when combined with at second study in the meta-analysis. The more recent study (n = 140) demonstrated a small improvement in forced vital capacity (per cent predicted) at two and three months and again at 11 and 12 months for participants receiving CFTR gene replacement therapy compared to those receiving placebo. The same study reported a significant difference in the relative change in forced expiratory volume at one second (per cent predicted) at two months, three months and 12 months.One small study reported significant concerns with "influenza-like" symptoms in participants treated with CFTR gene replacement therapy; this was not reported on repeated use of the same agent in a larger recent study.There was no other evidence of positive impact on outcomes, in particular improved quality of life or reduced treatment burden.Two studies measured ion transport in the lower airways; one (n = 16) demonstrated significant changes toward normal values in the participants who received gene transfer agents (P < 0.0001), mean difference 6.86 (95% confidence interval 3.77 to 9.95). The second study (n = 140) also reported significant changes toward normal values (P = 0.032); however, aggregate data were not available for analysis. In the most recent study, there was also evidence of increased salt transport in cells obtained by brushing the lower airway. These outcomes, whilst important, are not of direct clinical relevance. AUTHORS' CONCLUSIONS: One study of liposome-based CFTR gene transfer therapy demonstrated some improvements in respiratory function in people with CF, but this limited evidence of efficacy does not support this treatment as a routine therapy at present. There was no evidence of efficacy for viral-mediated gene delivery.Future studies need to investigate clinically important outcome measures.

Efficient intratracheal delivery of airway epithelial cells in mice and pigs.

Cellular therapy via direct intratracheal delivery has gained interest as a novel therapeutic strategy for treating various pulmonary diseases including cystic fibrosis lung disease. However, concerns such as insufficient cell engraftment in lungs and lack of large animal model data remain to be resolved. This study aimed to establish a simple method for evaluating cell retention in lungs and to develop reproducible approaches for efficient cell delivery into mouse and pig lungs. Human lung epithelial cells including normal human bronchial/tracheal epithelial (NHBE) cells and human lung epithelial cell line A549 were infected with pSicoR-green fluorescent protein (GFP) lentivirus. GFP-labeled NHBE cells were delivered via a modified intratracheal cell instillation method into the lungs of C57BL/6J mice. Two days following cell delivery, GFP ELISA-based assay revealed a substantial cell-retention efficiency (10.48 ± 2.86%, n = 7) in mouse lungs preinjured with 2% polidocanol. When GFP-labeled A549 cells were transplanted into Yorkshire pig lungs with a tracheal intubation fiberscope, a robust initial cell attachment (22.32% efficiency) was observed at 24 h. In addition, a lentiviral vector was developed to induce the overexpression and apical localization of cystic fibrosis transmembrane conductance regulator (CFTR)-GFP fusion proteins in NHBE cells as a means of ex vivo CFTR gene transfer in nonprogenitor (relatively differentiated) lung epithelial cells. These results have demonstrated the convenience and efficiency of direct delivery of exogenous epithelial cells to lungs in mouse and pig models and provided important background for future preclinical evaluation of intratracheal cell transplantation to treat lung diseases.

Thermal stability of purified and reconstituted CFTR in a locked open channel conformation.

CFTR is unique among ABC transporters as the only one functioning as an ion channel and from a human health perspective because mutations in its gene cause cystic fibrosis. Although considerable advances have been made towards understanding CFTR's mechanism of action and the impact of mutations, the lack of a high-resolution 3D structure has hindered progress. The large multi-domain membrane glycoprotein is normally present at low copy number and when over expressed at high levels it aggregates strongly, limiting the production of stable mono-disperse preparations. While the reasons for the strong self-association are not fully understood, its relatively low thermal stability seems likely to be one. The major CF causing mutation, ΔF508, renders the protein very thermally unstable and therefore a great deal of attention has been paid to this property of CFTR. Multiple second site mutations of CFTR in NBD1 where F508 normally resides and small molecule binders of the domain increase the thermal stability of the mutant. These manipulations also stabilize the wild-type protein. Here we have applied ΔF508-stabilizing changes and other modifications to generate wild-type constructs that express at much higher levels in scaled-up suspension cultures of mammalian cells. After purification and reconstitution into liposomes these proteins are active in a locked-open conformation at temperatures as high as 50 °C and remain monodisperse at 4 °C in detergent or lipid for at least a week. The availability of adequate amounts of these and related stable active preparations of homogeneous CFTR will enable stalled structural and ligand binding studies to proceed.

Composition of mineralizing incisor enamel in cystic fibrosis transmembrane conductance regulator-deficient mice.

Formation of crystals in the enamel space releases protons that need to be buffered to sustain mineral accretion. We hypothesized that apical cystic fibrosis transmembrane conductance regulator (CFTR) in maturation ameloblasts transduces chloride into forming enamel as a critical step to secrete bicarbonates. We tested this by determining the calcium, chloride, and fluoride levels in developing enamel of Cftr-null mice by quantitative electron probe microanalysis. Maturation-stage enamel from Cftr-null mice contained less chloride and calcium than did wild-type enamel, was more acidic when stained with pH dyes ex vivo, and formed no fluorescent modulation bands after in vivo injection of the mice with calcein. To acidify the enamel further we exposed Cftr-null mice to fluoride in drinking water to stimulate proton release during formation of hypermineralized lines. In Cftr-deficient mice, fluoride further lowered enamel calcium without further reducing chloride levels. The data support the view that apical CFTR in maturation ameloblasts tranduces chloride into developing enamel as part of the machinery to buffer protons released during mineral accretion.

Transepithelial ion transport across duct cells of the salivary gland.

Fluid and electrolyte secretions are vital for all epithelia and when aberrant lead to numerous pathophysiological conditions. Electrolyte transport across epithelia generates the osmotic force for fluid movement and is mediated by several membrane proteins expressed on both apical and basolateral poles of epithelial cells. Sodium and chloride are crucial for regulation of fluid secretion, thus regulating salivary volume. Bicarbonate (HCO3-), on the other hand, is the major pH buffer; hence, aberrant HCO3- secretion is a major factor in diseases such as cystic fibrosis (CF) causing altered mucin hydration and solubilization. Here, the structure-function mechanisms of the major membrane transporters involved in salivary duct electrolyte transport are reviewed focusing on transepithelial movement of Cl(-) and HCO3-.

Detergent-free purification of ABC (ATP-binding-cassette) transporters.

ABC (ATP-binding-cassette) transporters carry out many vital functions and are involved in numerous diseases, but study of the structure and function of these proteins is often hampered by their large size and membrane location. Membrane protein purification usually utilizes detergents to solubilize the protein from the membrane, effectively removing it from its native lipid environment. Subsequently, lipids have to be added back and detergent removed to reconstitute the protein into a lipid bilayer. In the present study, we present the application of a new methodology for the extraction and purification of ABC transporters without the use of detergent, instead, using a copolymer, SMA (polystyrene-co-maleic acid). SMA inserts into a bilayer and assembles into discrete particles, essentially solubilizing the membrane into small discs of bilayer encircled by a polymer, termed SMALPs (SMA lipid particles). We show that this polymer can extract several eukaryotic ABC transporters, P-glycoprotein (ABCB1), MRP1 (multidrug-resistance protein 1; ABCC1), MRP4 (ABCC4), ABCG2 and CFTR (cystic fibrosis transmembrane conductance regulator; ABCC7), from a range of different expression systems. The SMALP-encapsulated ABC transporters can be purified by affinity chromatography, and are able to bind ligands comparably with those in native membranes or detergent micelles. A greater degree of purity and enhanced stability is seen compared with detergent solubilization. The present study demonstrates that eukaryotic ABC transporters can be extracted and purified without ever being removed from their lipid bilayer environment, opening up a wide range of possibilities for the future study of their structure and function.

NBCe1 (SLC4A4) a potential pH regulator in enamel organ cells during enamel development in the mouse.

During the formation of dental enamel, maturation-stage ameloblasts express ion-transporting transmembrane proteins. The SLC4 family of ion-transporters regulates intra- and extracellular pH in eukaryotic cells by cotransporting HCO3 (-) with Na(+). Mutation in SLC4A4 (coding for the sodium-bicarbonate cotransporter NBCe1) induces developmental defects in human and murine enamel. We have hypothesized that NBCe1 in dental epithelium is engaged in neutralizing protons released during crystal formation in the enamel space. We immunolocalized NBCe1 protein in wild-type dental epithelium and examined the effect of the NBCe1-null mutation on enamel formation in mice. Ameloblasts expressed gene transcripts for NBCe1 isoforms B/D/C/E. In wild-type mice, weak to moderate immunostaining for NBCe1 with antibodies that recognized isoforms A/B/D/E and isoform C was seen in ameloblasts at the secretory stage, with no or low staining in the early maturation stage but moderate to high staining in the late maturation stage. The papillary layer showed the opposite pattern being immunostained prominently at the early maturation stage but with gradually less staining at the mid- and late maturation stages. In NBCe1 (-/-) mice, the ameloblasts were disorganized, the enamel being thin and severely hypomineralized. Enamel organs of CFTR (-/-) and AE2a,b (-/-) mice (CFTR and AE2 are believed to be pH regulators in ameloblasts) contained higher levels of NBCe1 protein than wild-type mice. Thus, the expression of NBCe1 in ameloblasts and the papillary layer cell depends on the developmental stage and possibly responds to pH changes.

Direct interaction of a CFTR potentiator and a CFTR corrector with phospholipid bilayers.

Cystic fibrosis transmembrane conductance regulator (CFTR) potentiators and correctors are new drugs that target the basic CFTR protein defect and are expected to benefit cystic fibrosis patients. To optimize the substances so far proposed for human use, and to minimise unwanted side effects, it is essential to investigate possible interactions between the drugs and cell components. We used small-angle X-ray scattering with synchrotron radiation to analyse the effects of two representative drugs, the potentiator VX-770 (Ivacaftor), approved for human use, and the corrector VX-809 (Lumacaftor), on a model phospholipid membrane. By reconstruction of the electron density profile of unilamellar vesicles treated with VX-770 or VX-809 we found that these drugs penetrate the phospholipid bilayer. VX-809 becomes homogeneously distributed throughout the bilayer whereas VX-770 accumulates predominantly in the internal leaflet, behaviour probably favoured by the asymmetry of the bilayer, because of vesicle curvature. Penetration of the bilayer by these drugs, probably as part of the mechanisms of permeation, causes destabilization of the membrane; this must be taken into account during future drug development.

Backbone cationized highly branched poly(ß-amino ester)s as enhanced delivery vectors in non-viral gene therapy.

Gene therapy holds great potential for treating Lung Cystic Fibrosis (CF) which is a fatal hereditary condition arising from mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in dysfunctional CFTR protein. However, the advancement and clinical application of CF gene therapy systems have been hindered due to the absence of a highly efficient delivery vector. In this work, we introduce a new generation of highly branched poly(ß-amino ester) (HPAE) gene delivery vectors for CF treatment. Building upon the classical chemical composition of HPAE, a novel backbone cationization strategy was developed to incorporate additional functional amine groups into HPAE without altering their branching degree. By carefully adjusting the type, proportion, and backbone distribution of the added cationic groups, a series of highly effective HPAE gene delivery vectors were successfully constructed for CF disease gene therapy. In vitro assessment results showed that the backbone cationized HPAEs with randomly distributed 10% proportion of 1-(3-aminopropyl)-4-methylpiperazine (E7) amine groups exhibited superior transfection performance than their counterparts. Furthermore, the top-performed backbone cationized HPAEs, when loaded with therapeutic plasmids, successfully reinstated CFTR protein expression in the CFBE41o- disease model, achieving levels 20-23 times higher than that of normal human bronchial epithelial (HBE) cells. Their therapeutic effectiveness significantly surpassed that of the currently advanced commercial vectors, Xfect and Lipofectamine 3000.