Effect of Deleting the R Domain on CFTR-Generated Chloride Channels

Rich, Devra P.; Gregory, Richard J.; Anderson, Matthew P.; Manavalan, Parthasarathy; Smith, Alan E.; Welsh, Michael J.

doi:10.1126/science.1712985

Cited by 274 publications

(176 citation statements)

References 27 publications

(19 reference statements)

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“…Because deletion of the R domain activates the CFTR Clchannel [4,50], it has been suggested that a dephosphorylated R domain may effectively occlude the conducting pore of CFTR, analogous to "ball and chain" inactivation of the Shaker K + channel in Drosophila [51]. R domain phosphorylation may therefore cause a conformational change within the molecule, alleviating a steric blockade and thereby facilitating nucleotide-dependent channel regulation.…”

Section: Role Of R Domain Phosphorylationmentioning

confidence: 99%

Characterization of the Adenosinetriphosphatase and Transport Activities of Purified Cystic Fibrosis Transmembrane Conductance Regulator

2004

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The cystic fibrosis transmembrane conductance regulator (CFTR) functions in vivo as a cAMPactivated chloride channel. A member of the ATP-binding cassette superfamily of membrane transporters, CFTR contains two transmembrane domains (TMDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. It is presumed that CFTR couples ATP hydrolysis to channel gating, and as a first step in addressing this issue directly, we have established conditions for purification of biochemical quantities of human CFTR expressed in Sf9 insect cells. Use of an 8-azido-[α 32 P]-ATP binding and vanadate-trapping assay allowed us to devise conditions to preserve CFTR function during purification of a C-terminal His 10 -tagged variant after solubilization with lyso-phosphatidylglycerol (1%) and diheptanolylphosphatidylcholine (0.3%) in the presence of excess phospholipid. Study of purified and reconstituted CFTR showed that it binds nucleotide with an efficiency comparable to that of P-glycoprotein, and that it hydrolyzes ATP at rates sufficient to account for presumed in vivo activity (V Max of 58 ± 5 nmol/min/mg protein, K M (MgATP) of 0.15 mM). In further work, we found that neither nucleotide binding nor ATPase activity were altered by phosphorylation (using Protein Kinase A) or dephosphorylation (with Protein Phosphatase 2B); we also observed inhibition (∼40%) of ATP hydrolysis by reduced glutathione, but not by DTT. To evaluate CFTR function as an anion channel, we introduced an in vitro macroscopic assay based on the equilibrium exchange of proteoliposome-entrapped radioactive tracers. This revealed a CFTRdependent transport of 125 I that could be inhibited by known chloride channel blockers; no significant CFTR-dependent transport of [α 32 P]-ATP was observed. We conclude that heterologous expression of CFTR in Sf9 cells can support manufacture and purification of fully functional CFTR. This should aid in further biochemical characterization of this important molecule.The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is the cAMP-activated chloride channel encoded by the gene defective in patients with the disease [1,2]. As a member of the ATP-Binding Cassette (ABC) superfamily of membrane transporters, CFTR shares a conserved architecture consisting of two homologous halves, each containing a transmembrane domain (TMD) and a nucleotide-binding domain (NBD). In CFTR, unlike other ABC transporters, a third domain, termed the regulatory (R) domain, is located between the two half molecules. Current evidence suggests that the TMDs define the CFTR chloride channel, while the NBDs and the R domain mediate channel gating [3][4][5][6][7][8][9][10][11] Although CFTR is glycosylated, there is currently no evidence indicating that the presence of carbohydrate affects CFTR structure or function [12]. Consistent with this presumption, expression of human CFTR in Sf9 insect cells results in appearance of the 140 kD core polypeptide -containing little or no glycosylation -that mediates a newly acquired anion ...

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Section: Role Of R Domain Phosphorylationmentioning

confidence: 99%

Characterization of the Adenosinetriphosphatase and Transport Activities of Purified Cystic Fibrosis Transmembrane Conductance Regulator

2004

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“…Mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) lead to dysfunction or absence of a cAMP regulated chloride channel in the apical membrane of epithelial cells. 1,2 Although various organs of the body are affected, respiratory failure is the cause of death in more than 95% of CF patients. Chronic inflammation, mucus accumulation and persistent bacterial infections cause a progressive decline in lung function.…”

Section: Introductionmentioning

confidence: 99%

Comparison between intratracheal and intravenous administration of liposome–DNA complexes for cystic fibrosis lung gene therapy

Griesenbach

Chonn

Cassady³

et al. 1998

Gene Ther

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Intratracheal (i.t.) and intravenous (i.v.) delivery of DNA-Expression using either mode of administration was maxivector formulations are two strategies to obtain gene transmal 24 h after injection and declined to around 10% of day fer to the lung. It is still uncertain, however, which of these 1 levels 2 weeks after injection. For i.v. delivery of DODAC: two modes of delivery will be more effective in the treat-DOPE-DNA complexes multilamellar vesicles were more ment of cystic fibrosis and other lung diseases. In this effective than large unilamellar vesicles in all organs invesstudy, we attempted to optimize formulations of the cationic tigated. Recombinant DNA could be detected in the distal liposome DODAC:DOPE (dioleoyldimethylammoniumlung region following either route of administration. Howchloride:dioleoylphosphatidylethanolamine) complexed to ever, i.t. administration predominantly led to DNA depoplasmids encoding chloramphenicol acetyltransferase for sition in epithelial cells lining the bronchioles, eg in clara i.t. and i.v. injection into CD-1 mice and compared the two cells, whereas i.v. administration resulted in DNA depomethods. Our results showed that both methods conferred sition in the alveolar region of the lung including type II reporter gene expression in the lung that was significantly alveolar epithelial cells. higher relative to injection of plasmid DNA alone.

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“…The consensus view is that the unphosphorylated R domain inhibits channel opening, an effect that is relieved by its phosphorylation. Rich et al (1991) discovered that channels that lacked large portions of the R domain were more active in the absence of PKA stimulation. Csanády et al (2000) subsequently showed that split CFTR molecules that lack the R domain and distal NBD1 (missing residues 634 -836) formed channels with opening rates in the absence of PKA that approached maximal opening rates for fully phosphorylated wild-type channels.…”

Section: Cftr Regulation By Pka Phosphorylationmentioning

confidence: 99%