Channel Gating Regulation by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) First Cytosolic Loop (original) (raw)
Related papers
Cellular Physiology …, 2010
The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a cAMP-activated chloride channel. The recent model of CFTR gating predicts that the ATP binding to both nucleotide-binding domains (NBD1 and NBD2) of CFTR is required for the opening of the channel, while the ATP hydrolysis at NBD2 induces subsequent channel closing. In most ABC proteins, efficient hydrolysis of ATP requires the presence of the invariant histidine residue within the H-loop located in the C-terminal part of the NBD. However, the contribution of the corresponding region (H-loop) of NBD2 to the CFTR channel gating has not been examined so far. Here we report that the alanine substitution of the conserved dipeptide HR motif (HR→AA) in the H-loop of NBD2 leads to prolonged open states of CFTR channel, indicating that the H-loop is required for efficient channel closing. On the other hand, the HR→AA substitution lead to the substantial decrease of CFTR-mediated current density (pA/pF) in transfected HEK 293 cells, as recorded in the whole-cell patch-clamp analysis. These results suggest that the H-loop of NBD2, apart from being required for CFTR channel closing, may be involved in regulating CFTR trafficking to the cell surface.
Journal of Biological Chemistry, 2002
The gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP binding cassette (ABC) transporter that functions as a phosphorylationand nucleotide-regulated chloride channel, is mutated in cystic fibrosis (CF) patients. Deletion of a phenylalanine at amino acid position 508 (⌬F508) in the first nucleotide binding domain (NBD1) is the most prevalent CF-causing mutation and results in defective protein processing and reduced CFTR function, leading to chloride impermeability in CF epithelia and heterologous systems. Using a STE6/CFTR⌬F508 chimera system in yeast, we isolated two novel ⌬F508 revertant mutations, I539T and G550E, proximal to and within the conserved ABC signature motif of NBD1, respectively. Western blot and functional analysis in mammalian cells indicate that mutations I539T and G550E each partially rescue the CFTR⌬F508 defect. Furthermore, a combination of both revertant mutations resulted in a 38-fold increase in CFTR⌬F508-mediated chloride current, representing 29% of wild type channel activity. The G550E mutation increased the sensitivity of CFTR⌬F508 and wild type CFTR to activation by cAMP agonists and blocked the enhancement of CFTR⌬F508 channel activity by 2 mM 3-isobutyl-1-methylxanthine. The data show that the ⌬F508 defect can be significantly rescued by second-site mutations in the nucleotide binding domain 1 region, that includes the LSGGQ consensus motif.
Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator
The EMBO Journal, 2004
Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a chloride channel. Nucleotide-binding domain 1 (NBD1), one of two ABC domains in CFTR, also contains sites for the predominant CF-causing mutation and, potentially, for regulatory phosphorylation. We have determined crystal structures for mouse NBD1 in unliganded, ADP-and ATP-bound states, with and without phosphorylation. This NBD1 differs from typical ABC domains in having added regulatory segments, a foreshortened subdomain interconnection, and an unusual nucleotide conformation. Moreover, isolated NBD1 has undetectable ATPase activity and its structure is essentially the same independent of ligand state. Phe508, which is commonly deleted in CF, is exposed at a putative NBD1transmembrane interface. Our results are consistent with a CFTR mechanism, whereby channel gating occurs through ATP binding in an NBD1-NBD2 nucleotide sandwich that forms upon displacement of NBD1 regulatory segments.
Journal of Biological Chemistry, 2002
The gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP binding cassette (ABC) transporter that functions as a phosphorylationand nucleotide-regulated chloride channel, is mutated in cystic fibrosis (CF) patients. Deletion of a phenylalanine at amino acid position 508 (⌬F508) in the first nucleotide binding domain (NBD1) is the most prevalent CF-causing mutation and results in defective protein processing and reduced CFTR function, leading to chloride impermeability in CF epithelia and heterologous systems. Using a STE6/CFTR⌬F508 chimera system in yeast, we isolated two novel ⌬F508 revertant mutations, I539T and G550E, proximal to and within the conserved ABC signature motif of NBD1, respectively. Western blot and functional analysis in mammalian cells indicate that mutations I539T and G550E each partially rescue the CFTR⌬F508 defect. Furthermore, a combination of both revertant mutations resulted in a 38-fold increase in CFTR⌬F508-mediated chloride current, representing 29% of wild type channel activity. The G550E mutation increased the sensitivity of CFTR⌬F508 and wild type CFTR to activation by cAMP agonists and blocked the enhancement of CFTR⌬F508 channel activity by 2 mM 3-isobutyl-1-methylxanthine. The data show that the ⌬F508 defect can be significantly rescued by second-site mutations in the nucleotide binding domain 1 region, that includes the LSGGQ consensus motif.
Structure and function of the cystic fibrosis transmembrane conductance regulator
Brazilian Journal of Medical and Biological Research, 1999
Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR). Mutations in the CFTR gene may result in a defective processing of its protein and alter the function and regulation of this channel. Mutations are associated with different symptoms, including pancreatic insufficiency, bile duct obstruction, infertility in males, high sweat Cl -, intestinal obstruction, nasal polyp formation, chronic sinusitis, mucus dehydration, and chronic Pseudomonas aeruginosa and Staphylococcus aureus lung infection, responsible for 90% of the mortality of CF patients. The gene responsible for the cellular defect in CF was cloned in 1989 and its protein product CFTR is activated by an increase of intracellular cAMP. The CFTR contains two membrane domains, each with six transmembrane domain segments, two nucleotide-binding domains (NBDs), and a cytoplasmic domain. In this review we discuss the studies that have correlated the role of each CFTR domain in the protein function as a chloride channel and as a regulator of the outwardly rectifying Clchannels (ORCCs).
The Journal of Physiology, 2009
We have employed rate-equilibrium free energy relationship (REFER) analysis to characterize the dynamic events involved in the allosteric regulation of cystic fibrosis transmembrane conductance regulator (CFTR) function. A wide range of different hydrolysable and poorly hydrolysable nucleoside triphosphates were used to elucidate the role of ATP hydrolysis in CFTR function. The linearity of the REFER plots and values near unity for all ligands tested implies that CFTR channel gating is a reversible thermally driven process with all structural reorganization in the binding site(s) completed prior to channel opening. This is consistent with the requirement for nucleotide binding for channel opening. However, the channel structural transition from the open to the closed state occurs independently of any events in the binding sites. Similar results were obtained on substitution of amino acids at coupling joints between both nucleotide binding domains (NBD) and cytoplasmic loops (CL) in opposite halves of the protein, indicating that any structural reorganization there also had occurred in the channel closed state. The fact that fractional values were not observed in either of these distant sites suggests that there may not be a deterministic 'lever-arm' mechanism acting between nucleotide binding sites and the channel gate. These findings favour a stochastic coupling between binding and gating in which all structural transitions are thermally driven processes. We speculate that increase of channel open state probability is due to reduction of the number of the closed state configurations available after physical interaction between ligand bound NBDs and the channel.
Journal of Biological Chemistry, 2010
The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl ؊ channel physiologically important in fluidtransporting epithelia and pathologically relevant in several human diseases. Here, we show that mutations in the C terminus of the first nucleotide binding domain comprising the latest  strands ( c 5 and  c 6) influence the trafficking, channel activity, and pharmacology of CFTR. We mutated CFTR amino acids located in the  c 5- c 6 hairpin, within the  c 5 strand (H620Q), within the -turn linking the two  strands (E621G, G622D), as well as within (S623A, S624A) and at the extremity (G628R) of the  c 6 strand. Functional analysis reveals that the current density was largely reduced for G622D and G628R channels compared with wt CFTR, similar for E621G and S624A, but increased for H620Q and S623A. For G622D and G628R, the abnormal activity is likely due to a defective maturation process, as assessed by the augmented activity and mature C-band observed in the presence of the trafficking corrector miglustat. In addition, in presence of the CFTR activator benzo[c]quinolizinium, the CFTR current density compared with that of wt CFTR was abolished for G622D and G628R channels, but similar for H620Q, S623A, and S624A or slightly increased for E621G. Finally, G622D and G628R were activated by the CFTR agonists genistein, RP-107, and isobutylmethylxanthine. Our results identify the C terminus of the CFTR first nucleotide binding domain as an important molecular site for the trafficking of CFTR protein, for the control of CFTR channel gating, and for the pharmacological effect of a dual activity agent.
Proceedings of the …, 1998
CFTR is a cyclic AMP (cAMP)-activated chloride (Cl ؊) channel and a regulator of outwardly rectifying Cl ؊ channels (ORCCs) in airway epithelia. CFTR regulates ORCCs by facilitating the release of ATP out of cells. Once released from cells, ATP stimulates ORCCs by means of a purinergic receptor. To define the domains of CFTR important for Cl ؊ channel function and͞or ORCC regulator function, mutant CFTRs with N-and C-terminal truncations and selected individual amino acid substitutions were created and studied by transfection into a line of human airway epithelial cells from a cystic fibrosis patient (IB3-1) or by injection of in vitro transcribed complementary RNAs (cRNAs) into Xenopus oocytes. Two-electrode voltage clamp recordings, 36 Cl ؊ eff lux assays, and whole cell patch-clamp recordings were used to assay for the Cl ؊ channel function of CFTR and for its ability to regulate ORCCs. The data showed that the first transmembrane domain (TMD-1) of CFTR, especially predicted ␣-helices 5 and 6, forms an essential part of the Cl ؊ channel pore, whereas the first nucleotide-binding and regulatory domains (NBD1͞R domain) are essential for its ability to regulate ORCCs. Finally, the data show that the ability of CFTR to function as a Cl ؊ channel and a conductance regulator are not mutually exclusive; one function could be eliminated while the other was preserved. CFTR is a transmembrane protein involved in the regulation of several processes, including the activation of outwardly rectifying Cl Ϫ channels (1, 2) and the inhibition of Na ϩ channels by cAMP-dependent protein kinase A (PKA) (3-5). Mutations in CFTR cause cystic fibrosis (CF). Both channels lose this pattern of PKA sensitivity when CFTR is absent or its function is severely compromised in mutant forms. Other members of the ATP-binding cassette (ABC) transporter superfamily also regulate other processes. For example, the multidrug transporter, MDR, may regulate volume-activated chloride channels (6-9). The sulfonylurea receptor (SUR) binds sulfonylurea compounds such as glybenclamide and confers sulfonylurea inhibition upon a separate ATP-gated K ϩ channel protein in pancreatic  cells (10-15). More recent results suggest that CFTR can act as a SUR for ATP-gated K ϩ channels in kidney (16). We have shown previously that CFTR regulates outwardly rectifying Cl Ϫ channels (ORCCs) by an autocrine mechanism involving ATP release that is CFTR dependent. The ATP released binds to purinergic receptors to stimulate ORCCs (17, 18). The mechanism of how ATP is released, either through CFTR itself or by a separate mechanism, remains highly controversial (19-21). Two possibilities are that CFTR either transports ATP directly or activates an alternate ATP-release pathway. A key question in CF research is: How does CFTR allow protein kinase A to activate separate populations of ORCCs and inhibit a distinct family of Na ϩ-conductive channels? In this study, we tested the hypothesis that the complex, multidomain structure of CFTR supports its multifunctional behavior and that separate domains within the CFTR protein perform Cl Ϫ channel function independent of its regulatory functions. We show that the ability of CFTR to regulate ORCCs is not dependent upon CFTR's Cl Ϫ channel function and that conductance regulation is separate from CFTR's ability to conduct Cl Ϫ. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.
The Journal of biological chemistry, 2017
Characterization of the second nucleotide binding domain (NBD2) of the cystic fibrosis transmembrane conductance regulator (CFTR) has lagged behind research into the NBD1 domain, in part because NBD1 contains the F508del mutation which is the dominant cause of cystic fibrosis. Research on NBD2 has also been hampered by the overall instability of the domain and the difficulty of producing reagents. Nonetheless multiple disease-causing mutations reside in NBD2 and the domain is critical to CFTR function, since channel gating involves NBD1:NBD2 dimerization and NBD2 contains the catalytically active ATPase site in CFTR. Recognizing the paucity of structural and biophysical data on NBD2, here, we have defined a bioinformatics-based method for manually identifying stabilizing substitutions in NBD2, and used an iterative process of screening single substitutions against thermal melting points to both produce minimally mutated stable constructs and individually characterize mutations. We p...