Binding site of activators of the cystic fibrosis transmembrane conductance regulator in the nucleotide binding domains

Morán, Óscar; Galietta, Luis J. V.; Zegarra‐Moran, Olga

doi:10.1007/s00018-004-4422-3

Cited by 110 publications

(122 citation statements)

References 121 publications

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“…Studies using nucleotide binding domain mutants of CFTR Cl -channels, such as G551D and G1349D, demonstrate that most CFTR activators have decreased affinity for activating mutant CFTR Cl -channels. 41 Therefore, putative binding sites on the nucleotide-binding domains of CFTR Cl -channels are proposed to be target(s) for many CFTR activators. 42,43 However, our data indicate that the primary mechanism of single CFTR Cl -channel activation by N20 may not involve the nucleotide-binding domains or regulatory domain of CFTR, because these sites have been previously stimulated by ATP and protein kinase A.…”

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confidence: 99%

Polystyrene nanoparticles activate ion transport in human airway epithelial cells

McCarthy¹,

Gong²,

Nahirney³

et al. 2011

IJN

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Background Over the last decade, nanotechnology has provided researchers with new nanometer materials, such as nanoparticles, which have the potential to provide new therapies for many lung diseases. In this study, we investigated the acute effects of polystyrene nanoparticles on epithelial ion channel function. Methods Human submucosal Calu-3 cells that express cystic fibrosis transmembrane conductance regulator (CFTR) and baby hamster kidney cells engineered to express the wild-type CFTR gene were used to investigate the actions of negatively charged 20 nm polystyrene nanoparticles on short-circuit current in Calu-3 cells by Ussing chamber and single CFTR Clchannels alone and in the presence of known CFTR channel activators by using baby hamster kidney cell patches. Results Polystyrene nanoparticles caused sustained, repeatable, and concentration-dependent increases in short-circuit current. In turn, these short-circuit current responses were found to be biphasic in nature, ie, an initial peak followed by a plateau. EC 50 values for peak and plateau short-circuit current responses were 1457 and 315.5 ng/mL, respectively. Short-circuit current was inhibited by diphenylamine-2-carboxylate, a CFTR Cl − channel blocker. Polystyrene nanoparticles activated basolateral K + channels and affected Cl − and HCO 3 − secretion. The mechanism of short-circuit current activation by polystyrene nanoparticles was found to be largely dependent on calcium-dependent and cyclic nucleotide-dependent phosphorylation of CFTR Cl − channels. Recordings from isolated inside-out patches using baby hamster kidney cells confirmed the direct activation of CFTR Cl − channels by the nanoparticles. Conclusion This is the first study to identify the activation of ion channels in airway cells after exposure to polystyrene-based nanomaterials. Thus, polystyrene nanoparticles cannot be considered as a simple neutral vehicle for drug delivery for the treatment of lung diseases, due to the fact that they may have the ability to affect epithelial cell function and physiological processes on their own.

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confidence: 99%

Polystyrene nanoparticles activate ion transport in human airway epithelial cells

McCarthy¹,

Gong²,

Nahirney³

et al. 2011

IJN

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“…G551D-CFTR channels exhibit much lower open probability than wild-type (WT) channels (7)(8)(9), and consequently, the patients carrying this mutation present a severe phenotype (10,11). G551D has been used as a model in pharmacological characterizations of CFTR (9,(12)(13)(14)(15), but the fundamental mechanism for the functional defect has been unclear until recently. We found that the G551D mutation completely eliminates the ability of ATP to increase the opening rate of the channel (7), consistent with the idea that ATP binding to ABP2 is critical for the ATP-dependent opening of CFTR channels (6).…”

mentioning

confidence: 99%

Mechanism of G551D-CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) Potentiation by a High Affinity ATP Analog

Bompadre

Hwang

2008

Journal of Biological Chemistry

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Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel gated by ATP binding and hydrolysis at its nucleotide binding domains (NBD). The NBDs dimerize in a head-to-tail configuration, forming two ATP binding pockets (ABP) with the ATP molecules buried at the dimer interface. Previous studies have indicated that ABP2, formed by the Walker A and B motifs of NBD2 and the signature sequence of NBD1, is the site critical for the ATP-dependent opening of CFTR. The G551D mutation in ABP2, the third most common cystic fibrosis-associated mutation, abolishes ATP-dependent gating, resulting in an open probability that is ϳ100-fold lower than that of wild-type channels. Interestingly, we found that the ATP analog N -(2-phenylethyl)-ATP (P-ATP) increases G551D currents mainly by increasing the open time of the channel. This effect is reduced when P-ATP is applied together with ATP, suggesting a competition between ATP and P-ATP for a common binding site.Introducing mutations that lower the nucleotide binding affinity at ABP2 did not alter significantly the effects of P-ATP on G551D-CFTR, whereas an equivalent mutation at ABP1 (consisting of the Walker A and B motifs of NBD1 and the signature sequence of NBD2) dramatically decreased the potency of P-ATP, indicating that ABP1 is the site where P-ATP binds to increase the activity of G551D-CFTR. These results substantiate the idea that nucleotide binding at ABP1 stabilizes the open channel conformation. Our observation that P-ATP enhances the G551D activity by binding at ABP1 implicates that ABP1 can potentially be a target for drugs to bind and increase the channel activity.

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“…CFTR is a unique member of the ATP-binding cassette transporter superfamily that forms an epithelial anion channel with complex regulation (2,3). It is composed of 1,480 amino acid residues organized into five domains: two membranespanning domains that form an anion-selective pore, two nucleotide-binding domains (NBDs) that likely form a head-to-tail dimer with two ATP-binding sites located at the dimer interface (4,5), and a regulatory domain with multiple consensus phosphorylation sites (2).…”

mentioning

confidence: 99%

Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms

Roxo-Rosa

Schmidt

et al. 2006

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

104

131

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The revertant mutations G550E and 4RK [the simultaneous mutation of four arginine-framed tripeptides (AFTs): R29K, R516K, R555K, and R766K] rescue the cell surface expression and function of F508del-cystic fibrosis (CF) transmembrane conductance regulator (-CFTR), the most common CF mutation. Here, we investigate their mechanism of action by using biochemical and functional assays to examine their effects on F508del and three CF mutations (R560T, A561E, and V562I) located within a conserved region of the first nucleotide-binding domain (NBD1) of CFTR. Like F508del, R560T and A561E disrupt CFTR trafficking. G550E rescued the trafficking defect of A561E but not that of R560T. Of note, the processing and function of V562I were equivalent to that of wild-type (wt)-CFTR, suggesting that V562I is not a disease-causing mutation. Biochemical studies revealed that 4RK generates higher steady-state levels of mature CFTR (band C) for wtand V562I-CFTR than does G550E. Moreover, functional studies showed that the revertants rescue the gating defect of F508del-CFTR with different efficacies. 4RK modestly increased F508del-CFTR activity by prolonging channel openings, whereas G550E restored F508del-CFTR activity to wt levels by altering the duration of channel openings and closings. Thus, our data suggest that the revertants G550E and 4RK might rescue F508del-CFTR by distinct mechanisms. G550E likely alters the conformation of NBD1, whereas 4RK allows F508del-CFTR to escape endoplasmic reticulum retention͞retrieval mediated by AFTs. We propose that AFTs might constitute a checkpoint for endoplasmic reticulum quality control. endoplasmic reticulum quality control ͉ folding ͉ membrane traffic ͉ arginine-framed motifs ͉ trafficking signals

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Binding site of activators of the cystic fibrosis transmembrane conductance regulator in the nucleotide binding domains

Cited by 110 publications

References 121 publications

Polystyrene nanoparticles activate ion transport in human airway epithelial cells

Polystyrene nanoparticles activate ion transport in human airway epithelial cells

Mechanism of G551D-CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) Potentiation by a High Affinity ATP Analog

Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms

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