CFTR in Calu-3 human airway cells: channel properties and role in cAMP-activated Cl- conductance

Haws, C. M.; Finkbeiner, Walter E.; Widdicombe, Jonathan; Wine, Jeffrey J.

doi:10.1152/ajplung.1994.266.5.l502

Cited by 112 publications

(125 citation statements)

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“…In cell-attached patches we observe the same range of long and short burst durations as in excised patches, and others have observed similarly long open bursts in cell-attached patches (18,39). Moreover, we observed a decrease in long open bursts in cell-attached patches when cells were treated with ␤-ME, raising the possibility that channels in the plasma membrane may not be in a uniformly reduced state.…”

Section: Discussionsupporting

confidence: 83%

Redox Reagents and Divalent Cations Alter the Kinetics of Cystic Fibrosis Transmembrane Conductance Regulator Channel Gating

Harrington¹,

Gunderson²,

Kopito³

1999

Journal of Biological Chemistry

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Gating of the cystic fibrosis Cl؊ channel requires hydrolysis of ATP by its nucleotide binding folds, but how this process controls the kinetics of channel gating is poorly understood. In the present work we show that the kinetics of channel gating and presumably the rate of ATP hydrolysis depends on the species of divalent cation present and the oxidation state of the protein.With Ca 2؉ as the dominant divalent cation instead of Mg 2؉ , the open burst duration of the channel is increased approximately 20-fold, and this change is reversible upon washout of Ca 2؉ . In contrast, "soft" divalent cations such as Cd 2؉ interact covalently with cystic fibrosis transmembrane conductance regulator (CFTR). These metals decrease both opening and closing rates of the channel, and the effects are not reversed by washout. Oxidation of CFTR channels with a variety of oxidants resulted in a similar slowing of channel gating. In contrast, reducing agents had the opposite effect, increasing both opening and closing rates of the channel. In cell-attached patches, CFTR channels exhibit both oxidized and reduced types of gating, raising the possibility that regulation of the redox state of the channel may be a physiological mode of control of CFTR channel activity.The cystic fibrosis transmembrane conductance regulator (CFTR) 1 belongs to the superfamily of ATP-binding cassette transporters, members of which are characterized structurally by two ATP-binding motifs and functionally by their ability to couple ATP hydrolysis to transport of a substrate molecule across a membrane (1, 2). Although a role for CFTR in solute transport has been proposed (3-6), no substrate has yet been identified. CFTR remains unique among ATP-binding cassette transporters in that it couples ATP hydrolysis to the opening of a Cl Ϫ channel (6, 7), reviewed in (8). The role of ATP hydrolysis in the CFTR gating process was inferred initially from studies of CFTR gating in electrophysiological experiments using poorly hydrolyzable ATP analogs and transition state phosphate analogs (9 -12). Direct measurement of ATPase activity in purified reconstituted CFTR has confirmed that it functions as an ATPase, and the measured turnover rate of the phosphorylated form (0.5-1 s Ϫ1 ) is consistent with a model in which opening and closing of the CFTR channel gate is coupled directly to ATP hydrolysis (13).The role of ATP hydrolysis by the two nucleotide-binding folds (NBF1 and NBF2) in mediating channel gating has been elucidated by studies examining CFTR channels individually mutated in each of the NBFs (14 -16). These papers presented models in which two ATP hydrolytic events, one occurring at NBF1 and one at NBF2, are involved first in opening and then closing the CFTR channel. In one model a single cycle of ATP hydrolysis occurs at NBF1 to directly open the channel (8,14,16,17). Alternatively, a second model proposes that ATP hydrolysis at NBF1 converts the channel into a gatable state from which nucleotide binding at NBF2 can then open the channel (15). In both mod...

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Section: Discussionsupporting

confidence: 83%

Redox Reagents and Divalent Cations Alter the Kinetics of Cystic Fibrosis Transmembrane Conductance Regulator Channel Gating

Harrington¹,

Gunderson²,

Kopito³

1999

Journal of Biological Chemistry

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“…The regulatory role of vitamin C on Cl ion channel activity was studied in Calu-3 airway cells, which express large amounts of native CFTR as their major Cl conductance (32). Using the outside-out patch clamp mode, we found that L-ascorbic acid induced openings of CFTR Cl channels when applied to the extracellular surface of the patched membrane.…”

Section: Resultsmentioning

confidence: 99%

Vitamin C controls the cystic fibrosis transmembrane conductance regulator chloride channel

Fischer¹,

Schwarzer²,

Illek³

2004

Proc. Natl. Acad. Sci. U.S.A.

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“…2). These cells are a model for proximal airway submucosal glands, where CFTR is expressed at relatively high levels (20). The physiological significance of the CFTR-E3KARP interaction is supported by several findings.…”

Section: Co-expression Of E3karp and Ezrin With Cftr Potentiates Cftr CLmentioning

confidence: 85%

E3KARP Mediates the Association of Ezrin and Protein Kinase A with the Cystic Fibrosis Transmembrane Conductance Regulator in Airway Cells

Sun

Hug

Lewarchik

et al. 2000

Journal of Biological Chemistry

196

186

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Although it is generally recognized that cystic fibrosis transmembrane conductance regulator (CFTR) contains a PSD-95/Disc-large/ZO-1 (PDZ)-binding motif at its COOH terminus, the identity of the PDZ domain protein(s) that interact with CFTR is uncertain, and the functional impact of this interaction is not fully understood. By using human airway epithelial cells, we show that CFTR associates with Na ؉ /H ؉ exchanger (NHE) type 3 kinase A regulatory protein (E3KARP), an EBP50/ NHE regulatory factor (NHERF)-related PDZ domain protein. The PDZ binding motif located at the COOH terminus of CFTR interacts preferentially with the second PDZ domain of E3KARP, with nanomolar affinity. In contrast to EBP50/NHERF, E3KARP is predominantly localized (>95%) in the membrane fractions of Calu-3 and T84 cells, where CFTR is located. Moreover, confocal immunofluorescence microscopy of polarized Calu-3 monolayers shows that E3KARP and CFTR are co-localized at the apical membrane domain. We also found that ezrin associates with E3KARP in vivo. Co-expression of CFTR with E3KARP and ezrin in Xenopus oocytes potentiated cAMP-stimulated CFTR Cl ؊ currents. These results support the concept that E3KARP functions as a scaffold protein that links CFTR to ezrin. Since ezrin has been shown previously to function as a protein kinase A anchoring protein, we suggest that one function served by the interaction of E3KARP with both ezrin and CFTR is to localize protein kinase A in the vicinity of the R-domain of CFTR. Since ezrin is also an actinbinding protein, the formation of a CFTR⅐E3KARP⅐ezrin complex may be important also in stabilizing CFTR at the apical membrane domain of airway cells.

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CFTR in Calu-3 human airway cells: channel properties and role in cAMP-activated Cl- conductance

Cited by 112 publications

References 0 publications

Redox Reagents and Divalent Cations Alter the Kinetics of Cystic Fibrosis Transmembrane Conductance Regulator Channel Gating

Redox Reagents and Divalent Cations Alter the Kinetics of Cystic Fibrosis Transmembrane Conductance Regulator Channel Gating

Vitamin C controls the cystic fibrosis transmembrane conductance regulator chloride channel

E3KARP Mediates the Association of Ezrin and Protein Kinase A with the Cystic Fibrosis Transmembrane Conductance Regulator in Airway Cells

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