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Mutant D836X that encodes the amino-terminal region of the cystic fibrosis transmembrane conductance regulator (CFTR) with a membrane-spanning domain and a nucleotide binding domain NBD. The R Domain constitutes a controlled chloride channel with characteristics that are similar to that of the wild-type channel. Activity increased with phosphorylation but conductance did not require phosphorylation.

Presents a study on the pharmacological agents that interact directly with the cystic fibrosis transmembrane conductance regulator (CFTR) chlorine channel. Agents which stimulate CFTR by interacting with the nucleotide-binding domains that control channel gating; CFTR inhibitors characterized by binding within the channel pore and preventing chlorine permeation; Use of molecular pharmacology of CFTR in the treatment of diseases caused by CFTR dysfunction.

Deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is the most common cause of cystic fibrosis. The F508 residue is located on nucleotide-binding domain 1 (NBD1) in contact with the cytosolic extensions of the transmembrane helices, in particular intracellular loop 4 (ICL4). To investigate how absence of F508 at this interface impacts the CFTR protein, we carried out a mutagenesis scan of ICL4 by introducing secondsite mutations at 11 positions in cis with F508del. Using an image-based fluorescence assay, we measured how each mutation affected membrane proximity and ion-channel function. The scan strongly validated the effectiveness of R1070W at rescuing F508del defects. Molecular dynamics simulations highlighted two features characterizing the ICL4/NBD1 interface of F508del/R1070W-CFTR: flexibility, with frequent transient formation of interdomain hydrogen bonds, and loosely stacked aromatic sidechains (F1068, R1070W, and F1074, mimicking F1068, F508, and F1074 in WT CFTR). F508del-CFTR displayed a distorted aromatic stack, with F1068 displaced toward the space vacated by F508, while in F508del/R1070F-CFTR, which largely retained F508del defects, R1070F could not form hydrogen bonds and the interface was less flexible. Other ICL4 second-site mutations which partially rescued F508del-CFTR included F1068M and F1074M. Methionine side chains allow hydrophobic interactions without the steric rigidity of aromatic rings, possibly conferring flexibility to accommodate the absence of F508 and retain a dynamic interface. These studies highlight how both hydrophobic interactions and conformational flexibility might be important at the ICL4/NBD1 interface, suggesting possible structural underpinnings of F508del-induced dysfunction. [ABSTRACT FROM AUTHOR]

The use of gene-focussed libraries for screening against protein targets can improve timelines for drug discovery projects. This is especially true when the library is based on a novel core scaffold, avoiding the potential need to scaffold hop from early hits. Identification of an appropriate novel scaffold is therefore integral to the success of such a library. In this article we outline a new method to aid scaffold design that combines structure-based virtual screening (VS) with a second phase in which fragmentation of the output is made before the final scaffold design step. Through consideration of a refined set of bound fragments, in the context of the compounds from which they originated, appropriate vectors for appended R-groups can be assigned before validation of the final library. [Copyright &y& Elsevier]

One therapeutic strategy for cystic fibrosis (CF) seeks to restore anion transport to affected epithelia by targeting other apical membrane Cl − channels to bypass dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl − channel. The properties and regulation of the Ca 2+ -activated Cl − channel TMEM16A argue that long-acting small molecules which target directly TMEM16A are required to overcome CFTR loss. Through genetic studies of lung diseases, SLC26A9, a member of the solute carrier 26 family of anion transporters, has emerged as a promising target to bypass CFTR dysfunction. An alternative strategy to circumvent CFTR dysfunction is to deliver to CF epithelia artificial anion transporters that shuttle Cl − across the apical membrane. Recently, powerful, non-toxic, biologically-active artificial anion transporters have emerged. [ABSTRACT FROM AUTHOR]

Fragment screening is one approach to hit identification for early stage drug discovery projects. Like any screening library, diversity is needed in fragment libraries. This includes coverage of shape and electrostatic space, as well as chemotype diversity. A new, easily interpretable shape-based fingerprint is described and its utility in probing fragment library content is demonstrated using a Rule of Three library from Maybridge. This method explicitly considers size as a component of shape. It allows interrogation of shape space on both a per conformer and a per molecule level, and includes a measure of flexibility. This allows for the identification of highly flexible compounds and their exclusion from the analysis. A comparison with two literature methods, the triangle plot approach of Sauer and Schwarz and the plane of best fit method of Firth et al., is also included. [ABSTRACT FROM AUTHOR]

In cystic fibrosis (CF), dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel disrupts epithelial ion transport and perturbs the regulation of intracellular pH (pHi). CFTR modulates pHi through its role as an ion channel and by regulating transport proteins. However, it is unknown how CFTR senses pHi. Here, we investigate the direct effects of pHi on recombinant CFTR using excised membrane patches. By altering channel gating, acidic pHi increased the open probability (Po) of wild-type CFTR, whereas alkaline pH, decreased Po and inhibited Cl- flow through the channel. Acidic pHi potentiated the MgATP dependence of wild-type CFTR by increasing MgATP affinity and enhancing channel activity, whereas alkaline pHi inhibited the MgATP dependence of wildtype CFTR by decreasing channel activity. Because these data suggest that pHi modulates the interaction of MgATP with the nucleotide-binding domains (NBDs) of CFTR, we examined the pHi dependence of site-directed mutations in the two ATP-binding sites of CFTR that are located at the NBD1:NBD2 dimer interface (site 1: K464A-, D572N-, and G1349D-CFTR; site 2: G551D-, K1250M-, and D1370N-CFTR), Site 2 mutants, but not site 1 mutants, perturbed both potentiation by acidic pH, and inhibition by alkaline pHi, suggesting that site 2 is a critical determinant of the pHi sensitivity of CFTR. The effects of pHi also suggest that site 2 might employ substrate-assisted catalysis to ensure that ATP hydrolysis follows NBD dimerization. We conclude that the CFTR Cl- channel senses directly pHi. The direct regulation of CFTR by pH, has important implications for the regulation of epithelial ion transport. [ABSTRACT FROM AUTHOR]

The relationship between cell proliferation, Cl− secretion, and renal cyst growth: A study using CFTR inhibitors.Background.In autosomal-dominant polycystic kidney disease (ADPKD), cAMP-stimulated cell proliferation and Cl− secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel drive the enlargement of fluid-filled epithelial cysts. To investigate how CFTR blockers inhibit cyst growth, we studied cAMP-dependent Cl− secretion, cell proliferation, and cyst growth using type I Madin Darby canine kidney (MDCK) cells as a model of renal cyst development and growth.Methods.We grew MDCK cysts in collagen gels in the presence of the cAMP agonist forskolin, measured Cl− secretion with the Ussing chamber technique, and assayed cell proliferation using nonpolarized and polarized MDCK cells. To inhibit CFTR, we used glibenclamide, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), genistein, and the specific CFTR inhibitor CFTRinh-172. As controls, we tested the effects of blockers of other types of apical membrane Cl− channels and inhibitors of basolateral membrane ion channels and transporters.Results.In the absence of inhibitors of transepithelial ion transport, forskolin stimulated dramatic cyst growth. CFTR blockers and inhibitors of basolateral membrane ion channels and transporters retarded cyst growth. In contrast, blockers of other types of apical membrane Cl− channels, which were without effect on CFTR, failed to inhibit cyst growth. Inhibition of cyst growth by CFTR blockers was correlated with inhibition of cAMP-stimulated Cl− current (correlation coefficient= 0.81;P<0.05), but not cell proliferation (correlation coefficient= 0.50;P>0.05).Conclusion.Our data suggest that CFTR blockers might retard cyst growth predominantly by inhibiting fluid accumulation within the cyst lumen. [ABSTRACT FROM AUTHOR]

Individuals with chronic temporal lobe epilepsy (TLE) experience episodic memory deficits that may be progressive in nature. These memory decrements have been shown to increase with the extent of hippocampal damage, a hallmark feature of TLE. Pattern separation, a neural computational mechanism thought to play a role in episodic memory formation, has been shown to be negatively affected by aging and in individuals with known hippocampal dysfunction. Despite the link between poor pattern separation performance and episodic memory deficits, behavioral pattern separation has not been examined in patients with TLE. We examined pattern separation performance in a group of 22 patients with medically-refractory TLE and 20 healthy adults, using a task hypothesized to measure spatial pattern separation with graded levels of spatial interference. We found that individuals with TLE showed less efficient spatial pattern separation performance relative to healthy adults. Poorer spatial pattern separation performance in TLE was associated with poorer visuospatial memory, but only under high interference conditions. In addition, left hippocampal atrophy was associated with poor performance in the high interference condition in TLE. These data suggest that episodic memory impairments in patients with chronic, refractory TLE may be partially due to less efficient pattern separation, which likely reflects their underlying hippocampal dysfunction. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) is caused by mutations that disrupt the plasma membrane expression, stability, and function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Two small molecules, the CFTR corrector lumacaftor and the potentiator ivacaftor, are now used clinically to treat CF, although some studies suggest that they have counteracting effects on CFTR stability. Here, we investigated the impact of these compounds on the instability of F508del-CFTR, the most common CF mutation. To study individual CFTR Cl- channels, we performed single-channel recording, whereas to assess entire CFTR populations, we used purified CFTR proteins and macroscopicCFTRCl-currents. At 37 °C, low temperature-rescued F508del-CFTR more rapidly lost function in cell-free membrane patches and showed altered channel gating and current flow through open channels. Compared with purified wildtype CFTR, the full-length F508del-CFTR was about 10 °C less thermostable. Lumacaftor partially stabilized purified fulllength F508del-CFTR and slightly delayed deactivation of individual F508del-CFTR Cl- channels. By contrast, ivacaftor further destabilized full-length F508del-CFTR and accelerated channel deactivation. Chronic (prolonged) co-incubation of F508del-CFTR-expressing cells with lumacaftor and ivacaftor deactivated macroscopic F508del-CFTR Cl- currents. However, at the single-channel level, chronic co-incubation greatly increased F508del-CFTR channel activity and temporal stability in most, but not all, cell-free membrane patches. We conclude that chronic lumacaftor and ivacaftor co-treatment restores stability in a small subpopulation of F508del-CFTR Cl- channels but that the majority remain destabilized. A fuller understanding of these effects and the characterization of the small F508del-CFTR subpopulation might be crucial for CF therapy development. [ABSTRACT FROM AUTHOR]

Abstract: Defective epithelial ion transport is the hallmark of the life-limiting genetic disease cystic fibrosis (CF). This abnormality is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), the ATP-binding cassette transporter that functions as a ligand-gated anion channel. Since the identification of the CFTR gene, almost 2000 disease-causing mutations associated with a spectrum of clinical phenotypes have been reported, but the majority remain poorly characterised. Studies of a small number of mutations including the most common, F508del-CFTR, have identified six general mechanisms of CFTR dysfunction. Here, we review selectively progress to understand how CF mutations disrupt CFTR processing, stability and function. We explore CFTR structure and function to explain the molecular mechanisms of CFTR dysfunction and highlight new knowledge of disease pathophysiology emerging from large animal models of CF. Understanding CFTR dysfunction is crucial to the development of transformational therapies for CF patients. This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances. [Copyright &y& Elsevier]

Impairment of the cystic fibrosis transmembrane conductance regulator (CFTR) CI- channel causes cystic fibrosis, a fatal genetic disease. Here, to gain insight into CFTR structure and function, we exploited interspecies differences between CFTR homologues using human (h)-murine (m) CFTR chimeras containing murine nucleotide-binding domains (NBDs) or regulatory domain on an hCFTR backbone. Among 15 hmCFTR chimeras analyzed, all but two were correctly processed, one containing part of mNBD1 and another containing part of mNBD2. Based on physicochemical distance analysis of divergent residues between human and murine CFTR in the two misprocessed hmCFTR chimeras, we generated point mutations for analysis of respective CFTR processing and functional properties. We identified one amino acid substitution (K584E-CFTR) that disrupts CFTR processing in NBD1. No single mutation was identified in NBD2 that disrupts protein processing. However, a number of NBD2 mutants altered channel function. Analysis of structural models of CFTR identified that although Lys584 interacts with residue Leu581 in human CFTR Glu584 interacts with Phe581 in mouse CFTR. Introduction of the murine residue (Phe581) in cis with K584E in human CFTR rescued the processing and trafficking defects of K584E-CFTR. Our data demonstrate that human-murine CFTR chimeras may be used to validate structural models of full-length CFTR. We also conclude that hmCFTR chimeras are a valuable tool to elucidate interactions between different domains of CFTR. [ABSTRACT FROM AUTHOR]

Deletion of phenylalanine 508 (ΔF508) from the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common mutation in cystic fibrosis. The F508 region lies within a surface-exposed loop that has not been assigned any interaction with associated proteins. Here we demonstrate that the pleiotropic protein kinase CK2 that controls protein trafficking, cell proliferation, and development binds wild-type CFTR near F508 and phosphorylates NBD1 at Ser-511 in vivo and that mutation of Ser-511 disrupts CFTR channel gating. Importantly, the interaction of CK2 with NBD1 is selectively abrogated by the ΔF508 mutation without disrupting four established CFTR-associated kinases and two phosphatases. Loss of CK2 association is functionally corroborated by the insensitivity of ΔF508-CFTR to CK2 inhibition, the absence of CK2 activity in SF508 CFTR-expressing cell membranes, and inhibition of CFTR channel activity by a peptide that mimics the F508 region of CFTR (but not the equivalent ΔF508 peptide). Disruption of this CK2-CFTR association is the first described ΔF508- dependent protein-protein interaction that provides a new molecular paradigm in the most frequent form of cystic fibrosis. [ABSTRACT FROM AUTHOR]