Expression of the cystic fibrosis gene in non-epithelial invertebrate cells produces a regulated anion conductance

Kartner, Norbert; Hanrahan, John W.; Jensen, Timothy J.; Naismith, Angela; Sun, Shi‐Zhang; Ackerley, Cameron; Reyes, E; Tsui, Lap Chee; Rommens, Johanna M.; Bear, Christine E.; Riordan, John R.

doi:10.1016/0092-8674(91)90498-n

Cited by 494 publications

(231 citation statements)

References 34 publications

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“…It was shown earlier that coincident with expression of human CFTR in Sf9 insect cells there is appearance of a new anion permeability with features appropriate to a CFTR-mediated anion channel [13]. And by use of a vanadate (Vi)-trapping assay, CFTR-specific ATP binding and hydrolysis has also been observed in the crude membranes of such cells [14,15].…”

Section: Overexpression Of Cftr In Sf9 Insect Cellsmentioning

confidence: 99%

“…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 permeability with the electrophysiological signature of CFTR [13] [14,15]. In this study, we focused on purification of human CFTR from this heterologous expression system as a way to provide amounts of CFTR that might support extended biochemical and biophysical work with purified material.…”

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confidence: 99%

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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: Overexpression Of Cftr In Sf9 Insect Cellsmentioning

confidence: 99%

mentioning

confidence: 99%

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

2004

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“…Recent studies demonstrated that CF is caused by mmations in the gone coding for a transmembrahe protein denominated Cystic Fibrosis Transmembrane conductance Regulator, CFTR [2][3][4]. Transfeetion experiments [5][6][7] and the functional reconstitution into liposomes [8] demonstrated that CFTR is indeed a cAMP-dependent chloride transporter.…”

Section: Introductionmentioning

confidence: 99%

“…The human colon carcinoma cell line T84 has been shown to express a high level of CFTR [3] and a cAMP- dependent chloride conductance with the same biophysical characteristics of the channel generated after transfection of the CFTR gone in cells with undetectable endogenous CFTR mRNA [5,6]. We have therefore initiated studies on the role of PKC on the chloride conductance ofT84 cells.…”

Section: Introductionmentioning

confidence: 99%

Effect of modulation of protein kinase C on the cAMP‐dependent chloride conductance in T84 cells

et al. 1992

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The regulation of chloride conductance was investi~ted in the T84 human colon carcinoma cell line by the quenching of the fluore.~.cent probe 6-methoxy-N-(3-sulfopropyl)quinollnium. The permeable cAMP analog 8.Br-eAMP (100/JM) and the calcium ionophom ionomyein (l/aM) activate a chloride conductancE. A prolonged (4 h) preineubation of cells with phorbol 12-myristate 13-acetate (100 nM) or with the diaeylgly~rol analog l-oleoyl-2-acctyl-l~lycerol (100 ,tiM): (i) down-modulates to almost zero the protein kinase C activity in the membranes; (ii) inhibits the activation of the chloride conductance mediated by 8-Br.cAMP but not by calcium; (iii) reduces the mRNA without chan#ng the ¢xpr~sion of the protein prod uct of the cystic fibrosis gone. The data suggest that PKC is essential for the activation of the cAMP-dependent chloride conductan~ in TM cells.

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“…The Shaker channels [6] and the cystic fibrosis gone product [7], which arc known to directly conduct ions, have been produced in Sf9 cells and examined electrophysiologically. These studies have indicated that the insect cells are highly suited for patch clamping.…”

Section: Introductionmentioning

confidence: 99%

Muscarinic acetylcholine receptor produced in recombinant baculovirus infected Sf9 insect cells couples with endogenous G‐proteins to activate ion channels

et al. 1992

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Following the inl'ection of insect ovarian cells ($)9) with recombinant baculoviras bearing the eDNA coding for the rat muscarini¢ acetyleholine (ACh) receptor subtype m3, ionic flax across the membrane in response to the application of ACh was ¢xamin~ elcctrophysiologically. We show that ACh activates potassium currents. The respons~ is abolish~ when cells arc treated with pertussis toxin. No ACh-induced currents are observed from uninfected cells or cells infected with virus which do not contain the eDNA coding for ACh r~ceptors in its genom¢. The characteristics of single channel currents show time-dependent changes following the application of ACh. Initially, ACh activates brief channel currents with a conductance of about 5 p$. The conductance level of channels gradually increases in st¢ps to I 0 p$ and then to 20 pS and 40 p$. At the same time, channel open probability also increases. Thereafter, additional channels appear, opening and closing independently of, or at times in synehrony with, the original channel.

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Expression of the cystic fibrosis gene in non-epithelial invertebrate cells produces a regulated anion conductance

Cited by 494 publications

References 34 publications

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

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

Effect of modulation of protein kinase C on the cAMP‐dependent chloride conductance in T84 cells

Muscarinic acetylcholine receptor produced in recombinant baculovirus infected Sf9 insect cells couples with endogenous G‐proteins to activate ion channels

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