Lumacaftor/ivacaftor combination for cystic fibrosis patients homozygous for Phe508del-CFTR.

Cystic fibrosis (CF) is a life-shortening inherited disease caused by the loss or dysfunction of the CF transmembrane conductance regulator (CFTR) channel activity resulting from mutations in the CFTR gene. Phe508del is the most prevalent mutation, with approximately 90% of all CF patients carrying it on at least one allele. Over the past two or three decades, significant progress has been made in understanding the pathogenesis of CF, and in the development of effective CF therapies. The approval of Orkambi® (lumacaftor/ivacaftor) marks another milestone in CF therapeutics development, which, with the advent of personalized medicine, could potentially revolutionize CF care and management. This article reviews the rationale, progress and future direction in the development of lumacaftor/ivacaftor combination to treat CF patients homozygous for the Phe508del-CFTR mutation.

Tyrosine phosphorylation of occludin attenuates its interactions with ZO-1, ZO-2, and ZO-3.

Occludin, the transmembrane integral protein of the tight junction, plays a crucial role in the molecular organization and function of tight junction. While the homotypic interaction of extracellular loops of occludin appears to determine the barrier function of tight junction, the intracellular C-terminal tail, C-occludin, interacts with other tight junction proteins such as ZO-1, ZO-2, and ZO-3 and with the actin filaments of cytoskeleton. In the present study we phosphorylated GST-fused C-occludin on tyrosine residues, in TKX1 Epicurian coli or by active c-Src in vitro. c-Src binds to occludin and phosphorylates it on tyrosine residues. The effect of tyrosine phosphorylation of C-occludin on its ability to bind ZO-1, ZO-2, ZO-3, and F-actin was evaluated. Results show that the amounts of ZO-1, ZO-2, and ZO-3 bound to tyrosine phosphorylated C-occludin were several fold less than the amounts bound to non-phosphorylated C-occludin. However, the amount of tyrosine phosphorylated C-occludin bound to F-actin was not significantly different from the amount of non-phosphorylated C-occludin bound to F-actin. These results demonstrate that tyrosine phosphorylation of occludin reduces its ability to bind ZO-1, ZO-2, and ZO-3, but not F-actin. Results also suggest that c-Src-mediated disruption of tight junction may involve tyrosine phosphorylation of occludin.

A cluster of negative charges at the amino terminal tail of CFTR regulates ATP-dependent channel gating.

1. The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is activated by protein kinase A (PKA) phosphorylation of its R domain and by ATP binding at its nucleotide-binding domains (NBDs). Here we investigated the functional role of a cluster of acidic residues in the amino terminal tail (N-tail) that also modulate CFTR channel gating by an unknown mechanism. 2. A disease-associated mutant that lacks one of these acidic residues (D58N CFTR) exhibited lower macroscopic currents in Xenopus oocytes and faster deactivation following washout of a cAMP -activating cocktail than wild-type CFTR. 3. In excised membrane patches D58N CFTR exhibited a two-fold reduction in single channel open probability due primarily to shortened open channel bursts. 4. Replacing this and two nearby acidic residues with alanines (D47A, E54A, D58A) also reduced channel activity, but had negligible effects on bulk PKA phosphorylation or on the ATP dependence of channel activation. 5. Conversely, the N-tail triple mutant exhibited a markedly inhibited response to AMP-PNP, a poorly hydrolysable ATP analogue that can nearly lock open the wild-type channel. The N-tail mutant had both a slower response to AMP-PNP (activation half-time of 140 +/- 20 s vs. 21 +/- 4 s for wild type) and a lower steady-state open probability following AMP-PNP addition (0.68 +/- 0.08 vs. 0.92 +/- 0.03 for wild type). 6. Introducing the N-tail mutations into K1250A CFTR, an NBD2 hydrolysis mutant that normally exhibits very long open channel bursts, destabilized the activity of this mutant as evidenced by decreased macroscopic currents and shortened open channel bursts. 7. We propose that this cluster of acidic residues modulates the stability of CFTR channel openings at a step that is downstream of ATP binding and upstream of ATP hydrolysis, probably at NBD2.

Syntaxin 1A is expressed in airway epithelial cells, where it modulates CFTR Cl(-) currents.

The CFTR Cl(-) channel controls salt and water transport across epithelial tissues. Previously, we showed that CFTR-mediated Cl(-) currents in the Xenopus oocyte expression system are inhibited by syntaxin 1A, a component of the membrane trafficking machinery. This negative modulation of CFTR function can be reversed by soluble syntaxin 1A peptides and by the syntaxin 1A binding protein, Munc-18. In the present study, we determined whether syntaxin 1A is expressed in native epithelial tissues that normally express CFTR and whether it modulates CFTR currents in these tissues. Using immunoblotting and immunofluorescence, we observed syntaxin 1A in native gut and airway epithelial tissues and showed that epithelial cells from these tissues express syntaxin 1A at >10-fold molar excess over CFTR. Syntaxin 1A is seen near the apical cell surfaces of human bronchial airway epithelium. Reagents that disrupt the CFTR-syntaxin 1A interaction, including soluble syntaxin 1A cytosolic domain and recombinant Munc-18, augmented cAMP-dependent CFTR Cl(-) currents by more than 2- to 4-fold in mouse tracheal epithelial cells and cells derived from human nasal polyps, but these reagents did not affect CaMK II-activated Cl(-) currents in these cells.

CFTR chloride channel regulation by an interdomain interaction.

The cystic fibrosis gene encodes a chloride channel, CFTR (cystic fibrosis transmembrane conductance regulator), that regulates salt and water transport across epithelial tissues. Phosphorylation of the cytoplasmic regulatory (R) domain by protein kinase A activates CFTR by an unknown mechanism. The amino-terminal cytoplasmic tail of CFTR was found to control protein kinase A-dependent channel gating through a physical interaction with the R domain. This regulatory activity mapped to a cluster of acidic residues in the NH(2)-terminal tail; mutating these residues proportionately inhibited R domain binding and CFTR channel function. CFTR activity appears to be governed by an interdomain interaction involving the amino-terminal tail, which is a potential target for physiologic and pharmacologic modulators of this ion channel.

Characterization of CFTR expression and chloride channel activity in human endothelia.

The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a low-conductance, cAMP-regulated chloride (Cl-) channel in a variety of cell types, such as exocrine epithelial cells. Our results demonstrate that human primary endothelial cells isolated from umbilical vein (HUVEC) and lung microvasculature (HLMVEC) also express CFTR as determined via RT-PCR and immunohistochemical and immunoprecipitation analyses. Moreover, Cl- efflux and whole cell patch-clamp analyses reveal that HUVEC (n = 6 samples, P < 0.05) and HLMVEC (n = 5 samples, P < 0.05) display cyclic nucleotide-stimulated Cl- transport that is inhibited by the CFTR selective Cl- channel blocker glibenclamide but not by the blocker DIDS, indicative of CFTR Cl- channel activity. Taken together, these findings demonstrate that human endothelial cells derived from multiple organ systems express CFTR and that CFTR functions as a cyclic nucleotide-regulated Cl- channel in human endothelia.

Syntaxin 1A inhibits CFTR chloride channels by means of domain-specific protein-protein interactions.

Previously we showed that the functional activity of the epithelial chloride channel that is encoded by the cystic fibrosis gene (CFTR) is reciprocally modulated by two components of the vesicle fusion machinery, syntaxin 1A and Munc-18. Here we report that syntaxin 1A inhibits CFTR chloride channels by means of direct and domain-specific protein-protein interactions. Syntaxin 1A stoichiometrically binds to the N-terminal cytoplasmic tail of CFTR, and this binding is blocked by Munc-18. The modulation of CFTR currents by syntaxin 1A is eliminated either by deletion of this tail or by injecting this tail as a blocking peptide into coexpressing Xenopus oocytes. The CFTR binding site on syntaxin 1A maps to the third predicted helical domain (H3) of this membrane protein. Moreover, CFTR Cl- currents are effectively inhibited by a minimal syntaxin 1A construct (i.e., the membrane-anchored H3 domain) that cannot fully substitute for wild-type syntaxin 1A in membrane fusion reactions. We also show that syntaxin 1A binds to and inhibits the activities of disease-associated mutants of CFTR, and that the chloride current activity of recombinant DeltaF508 CFTR (i.e., the most common cystic fibrosis mutant) can be potentiated by disrupting its interaction with syntaxin 1A in cultured epithelial cells. Our results provide evidence for a direct physical interaction between CFTR and syntaxin 1A that limits the functional activities of normal and disease-associated forms of this chloride channel.

Regulation of CFTR chloride channels by syntaxin and Munc18 isoforms.

The cystic fibrosis gene encodes a cyclic AMP-gated chloride channel (CFTR) that mediates electrolyte transport across the luminal surfaces of a variety of epithelial cells. The molecular mechanisms that modulate CFTR activity in epithelial tissues are poorly understood. Here we show that CFTR is regulated by an epithelially expressed syntaxin (syntaxin 1A), a membrane protein that also modulates neurosecretion and calcium-channel gating in brain. Syntaxin 1A physically interacts with CFTR chloride channels and regulates CFTR-mediated currents both in Xenopus oocytes and in epithelial cells that normally express these proteins. The physical and functional interactions between syntaxin 1A and CFTR are blocked by a syntaxin-binding protein of the Munc18 protein family (also called n-Secl). Our results indicate that CFTR function in epithelial cells is regulated by an interplay between syntaxin and Munc18 isoforms.

Cation effects on the conformations of muscle and non-muscle alpha-actinins.

We examined the effects of changing KCl concentration on the secondary structures of alpha-actinins using circular dichroism (CD), 1,1'-bis(4-anilino) naphthalene-5,5'-disulfonic acid (bisANS) fluorescence and proteolysis experiments. Under near-physiological conditions, divalent cations also were added and changes in conformation were investigated. In 25 mM KH2PO4, pH 7.5, increasing KCl from 0 to 120 mM led to decreases in alpha-helix conformation for brain, platelet and heart alpha-actinins (40.5-30.2%, 65.5-37.8% and 37.5-27.8%, respectively). In buffered 120 mM KCl, 0.65 mM calcium produced small changes in the CD spectra of both brain and platelet alpha-actinin, but had no effect on heart alpha-actinin. bisANS fluorescence of all three alpha-actinins also showed significant changes in conformation with increasing KCl. However, in buffered 120 mM KCl increasing concentrations of Ca2+ or Mg2+ did not have significant effects on the bisANS fluorescence of any alpha-actinin. Digestion of brain, platelet and heart alpha-actinins with alpha-chymotrypsin showed an increase of proteolytic susceptibility in 120 mM KCl. These experiments also showed that increasing the concentration of Ca2+ or Mg2+ led to greater changes in digestion fragment patterns in the absence of KCl than in the presence of 120 mM KCl. The results suggest that alpha-actinins exist in different conformations depending on the ionic strength of the medium, which could explain the differences in calcium and F-actin binding results obtained from different alpha-actinins.

Cation binding to chicken gizzard alpha-actinin.

Gizzard alpha-actinin binds 45Ca2+ as shown by the calcium overlay method. Flow dialysis measurements in 20 mM Hepes (pH 7.5) reveal 3.5 +/- 1.8 (S.D.) high affinity calcium binding sites per dimer, with Kd1 = 6.36 +/- 0.34 x 10(-6) M, and 87.3 +/- 7.2 sites with Kd2 = 1.66 +/- 0.44 x 10(-4) M. Chymotrypsin and thermolysin digestion yielded peptides of gizzard alpha-actinin which, if they included the putative EF-hands, bound 45Ca2+ in 10 mM imidazole-HCl (pH 7.4) or 60 mM KCl, 10 mM imidazole-HCl (pH 7.4). In addition, peptides which include a region of the molecule more than 27 kDa from the N-terminal also bind calcium. In contrast, when KCl in the binding buffer was increased to 120 mM, calcium binding was eliminated. Flow dialysis data revealed no high-affinity binding and 82.5 +/- 3.3 calcium binding sites with calculated affinities in the millimolar range. These are divalent cation binding sites, not Ca(2+)-specific sites, because they are eliminated by the addition of up to 5 mM Mg2+. Structural changes produced upon cation binding to alpha-actinin measured by circular dichroism, proteolysis and bisANS fluorescence are substantial when binding K+ with only small changes upon binding of Ca2+ or Mg2+ in the presence of 120 mM KCl. These results suggest that monovalent and divalent cations have different effects on different parts of the molecule with a complete elimination of 45Ca2+ binding to the EF-hands being produced by 120 mM KCl.

Crystallization and preliminary X-ray diffraction analysis of crystals of Thermoascus aurantiacus xylanase.

Crystals suitable for high resolution X-ray diffraction analysis have been grown of the 29,774-Da protein, xylanase (1,-4-beta-xylan xylanohydrolase EC 3.2.1.8) from the thermophilic fungus Thermoascus aurantiacus. This protein, an endoxylanase demonstrates the hydrolysis of beta-(1-4)-D-xylose linkage in xylans and crystallizes as monoclinic pinacoids in the presence of ammonium sulphate buffered at pH 6.5, and also with neutral polyethylene glycol 6000. The crystals belong to space group P2(1) and have cell dimensions, a = 41.2 A, b = 67.76 A, c = 51.8 A; beta = 113.2 degrees.