Function of the second nucleotide‐binding fold in the CFTR chloride channel

Zerhusen, Bryan; Ma, Jianjie

doi:10.1016/s0014-5793(99)01230-2

Cited by 9 publications

(9 citation statements)

References 34 publications

(74 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It has been found, however, that the number of amino acids deleted in NBD2 and their locations can greatly influence maturation and activity of the protein. For example, a ⌬NBD2 CFTR mutant lacking amino acids 1185 to 1349 did not mature in HEK 293 cells (Zerhusen and Ma, 1999). Channel measurements on microsomal membranes, however, showed that the mutant was active.…”

Section: Discussionmentioning

confidence: 99%

Modulating the Folding of P-Glycoprotein and Cystic Fibrosis Transmembrane Conductance Regulator Truncation Mutants with Pharmacological Chaperones

Wang¹,

Loo²,

Bartlett³

et al. 2006

Mol Pharmacol

View full text Add to dashboard Cite

Cystic fibrosis transmembrane conductance regulator (CFTR) and P-glycoprotein (P-gp) are ATP-binding cassette (ABC) transporters that have two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). Defective folding of CFTR lacking phenylalanine 508 (⌬Phe508) in NBD1 is the most common cause of cystic fibrosis. The Phe508 position seems to be universally important in ABC transporters because deletion of the equivalent residue (Tyr490) in P-gp also inhibits maturation of the protein. The pharmacological chaperone VRT-325 can repair the ⌬Phe508-type folding defects in P-gp or CFTR. VRT-325 may repair the folding defects by promoting dimerization of the two NBDs or by promoting folding of the TMDs. To distinguish between these two mechanisms, we tested the ability of VRT-325 to promote folding of truncation mutants lacking one or both NBDs. Sensitivity to glycosidases was used as an indirect indicator of folding. It was found that VRT-325 could promote maturation of truncation mutants lacking NBD2. Truncation mutants of CFTR or P-gp lacking both NBDs showed deficiencies in core-glycosylation that could be partially reversed by carrying out expression in the presence of VRT-325. The results show that dimerization of the two NBDs to form a "nucleotide-sandwich" structure or NBD interactions with the TMDs are not essential for VRT-325 enhancement of folding. Instead, VRT-325 can promote folding of the TMDs alone. The ability of VRT-325 to promote core-glycosylation of the NBD-less truncation mutants suggests that one mechanism whereby the compound enhances folding is by promoting proper insertion of TM segments attached to the glycosylated loops so that they adopt an orientation favorable for glycosylation.

show abstract

Section: Discussionmentioning

confidence: 99%

Modulating the Folding of P-Glycoprotein and Cystic Fibrosis Transmembrane Conductance Regulator Truncation Mutants with Pharmacological Chaperones

Wang¹,

Loo²,

Bartlett³

et al. 2006

Mol Pharmacol

View full text Add to dashboard Cite

show abstract

“…The TMDs contribute to the formation of the Cl À selective pore [18,[45][46][47][48][49][50], the NBDs bind and hydrolyze ATP to regulate channel gating [19,21,[51][52][53] and phosphorylation of the R domain controls channel activity [54][55][56][57][58]. Zerhusen and Ma [59] suggested that NBD2 could have inhibitory and stimulatory roles in CFTR activity by interaction with NBD1 directly or indirectly via the R domain.…”

Section: Walker Motifsmentioning

confidence: 99%

Insight in eukaryotic ABC transporter function by mutation analysis

Frelet

Klein

2006

FEBS Letters

View full text Add to dashboard Cite

With regard to structure-function relations of ATPbinding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.

show abstract

“…The predicted location of all 18 endogenous cysteines is depicted [1,[21][22][23][24]. filtered at 100 Hz, digitized at 1 kHz, and analyzed as above.…”

Section: Fig 2 Cftr Topologymentioning

confidence: 99%

Intracellular Cysteines of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Modulate Channel Gating

Ketchum¹,

Yue²,

Alessi³

et al. 2002

Cell Physiol Biochem

View full text Add to dashboard Cite

The cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ATP-binding cassette superfamily, is a cAMP-activated chloride channel. CFTR contains two transmembrane domains (TMDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. We found that whole-cell CFTR-dependent Cl- currents in Xenopus laevis oocytes were sensitive to HgCl₂, suggesting that modification of endogenous cysteines alters channel activity. To understand better this phenomenon, site-directed mutagenesis was employed to generate both individual cysteine replacements and a version of the molecule with no cysteines in the hydrophobic sector. Each mutant displayed a forskolin/IBMX-activated Cl^- conductance similar to wild type, indicating that none of the cysteines located within the TMDs is essential. Subsequent single-channel analysis of inside-out patches excised from HEK293 cells expressing either cysteine-less or wild-type CFTR showed that intracellular application of a membrane impermeant sulphydryl reagent, p-chloromercuribenzosulfonate (PCMBS), significantly reduced open probability without affecting ion selectivity or conductance. The cysteine-less molecule also acquired a voltage-dependent sensitivity to extracellular PCMBS not observed in the wild type, perhaps due to a more flexible conformation that allowed PCMBS access to the intracellular surface. Together, these experiments suggest that endogenous intracellular cysteines, located primarily within the NBDs and/or R domain, influence channel gating.

show abstract

Function of the second nucleotide‐binding fold in the CFTR chloride channel

Abstract: To test the role of nucleotide-binding fold (NBF) 2 and its interaction with the regulatory

Cited by 9 publications

References 34 publications

Modulating the Folding of P-Glycoprotein and Cystic Fibrosis Transmembrane Conductance Regulator Truncation Mutants with Pharmacological Chaperones

Modulating the Folding of P-Glycoprotein and Cystic Fibrosis Transmembrane Conductance Regulator Truncation Mutants with Pharmacological Chaperones

Insight in eukaryotic ABC transporter function by mutation analysis

Intracellular Cysteines of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Modulate Channel Gating

Contact Info

Product

Resources

About