Chan KW et al. (2000), Severed molecules functionally define the bound...

XB-ART-10580

J Gen Physiol 2000 Aug 01;1162:163-80.

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Severed molecules functionally define the boundaries of the cystic fibrosis transmembrane conductance regulator's NH(2)-terminal nucleotide binding domain.

Chan KW , Csanády L , Seto-Young D , Nairn AC , Gadsby DC .

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The cystic fibrosis transmembrane conductance regulator is a Cl(-) channel that belongs to the family of ATP-binding cassette proteins. The CFTR polypeptide comprises two transmembrane domains, two nucleotide binding domains (NBD1 and NBD2), and a regulatory (R) domain. Gating of the channel is controlled by kinase-mediated phosphorylation of the R domain and by ATP binding, and, likely, hydrolysis at the NBDs. Exon 13 of the CFTR gene encodes amino acids (aa's) 590-830, which were originally ascribed to the R domain. In this study, CFTR channels were severed near likely NH(2)- or COOH-terminal boundaries of NBD1. CFTR channel activity, assayed using two-microelectrode voltage clamp and excised patch recordings, provided a sensitive measure of successful assembly of each pair of channel segments as the sever point was systematically shifted along the primary sequence. Substantial channel activity was taken as an indication that NBD1 was functionally intact. This approach revealed that the COOH terminus of NBD1 extends beyond aa 590 and lies between aa's 622 and 634, while the NH(2) terminus of NBD1 lies between aa's 432 and 449. To facilitate biochemical studies of the expressed proteins, a Flag epitope was added to the NH(2) termini of full length CFTR, and of CFTR segments truncated before the normal COOH terminus (aa 1480). The functionally identified NBD1 boundaries are supported by Western blotting, coimmunoprecipitation, and deglycosylation studies, which showed that an NH(2)-terminal segment representing aa's 3-622 (Flag3-622) or 3-633 (Flag3-633) could physically associate with a COOH-terminal fragment representing aa's 634-1480 (634-1480); however, the latter fragment was glycosylated to the mature form only in the presence of Flag3-633. Similarly, 433-1480 could physically associate with Flag3-432 and was glycosylated to the mature form; however, 449-1480 protein seemed unstable and could hardly be detected even when expressed with Flag3-432. In excised-patch recordings, all functional severed CFTR channels displayed the hallmark characteristics of CFTR, including the requirement of phosphorylation and exposure to MgATP for gating, ability to be locked open by pyrophosphate or AMP-PNP, small single channel conductances, and high apparent affinity of channel opening by MgATP. Our definitions of the boundaries of the NBD1 domain in CFTR are supported by comparison with the solved NBD structures of HisP and RbsA.

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Species referenced: Xenopus laevis
Genes referenced: abcb6 cftr pnp tbx2

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	Scheme S1.
	Figure 1. (AâC) Representative current recordings from oocytes held at their initial resting membrane potential in Ca2+-free oocyte Ringer's solution. Oocytes were injected with (A) water, (B) 6 ng of WT CFTR RNA, or (C) 2.5 ng of Flag3-633 RNA plus 2.5 ng 634-1480 RNA. Horizontal bars mark periods of exposure to 50 Î¼M forskolin plus 1 mM IBMX. Basal and activated conductances were monitored by applying Â±60-mV voltage steps â¤1-s long. Magnitudes of basal and activated conductances and rates of activation were similar in oocytes injected with 2.5 or 6 ng of WT CFTR RNA. (D) I-V relationships obtained at times a-f in AâC; empty symbols, resting (basal); filled symbols, activated. Steady current levels averaged over â¼200 ms near the end of 1-s voltage steps from â100 to +80 mV, in 20-mV increments, are plotted against voltage.
	Figure 2. Defining the COOH-terminal boundary of NBD1 by coexpressing severed CFTR segments. Bars show mean (Â±SEM) basal and activated conductances (by 50 Î¼M forskolin plus 1 mM IBMX) of oocytes injected with cRNAs (2.5 ng per construct) of various CFTR segments cartooned at left: NH2-terminal Flag epitope (black zigzag); transmembrane domains (blue, cyan); NBD1 (red); R domain (green); NBD2 (yellow). Conductances were calculated from linear fits to steady state I-V data between â60 and â20 mV; average values are from five or more oocytes. The COOH terminus lies between residues 622 and 634.
	Figure 3. Defining the NH2-terminal boundary of NBD1 by coexpressing severed CFTR channel segments. Methods and symbols are as in Fig. 2. The NH2 terminus was found to lie between residues 432 and 449.
	Figure 8. Representative excised-patch current recordings showing WT, 1-432+433-1480, 1-633+634-1480, Flag-WT, Flag3-432+433-1480, and Flag3-633+634-1480 CFTR channels. All constructs displayed hallmark characteristics of WT CFTR including requirement for phosphorylation by PKA (here 300 nM) and exposure to MgATP (here 1 mM for WT, 2 mM for others) for channel activity, slow burst kinetics, and locking in the open state by a mixture of ATP (0.1 mM) and PPi (2 mM).
	Figure 4. Negligible basal and activated conductances of oocytes injected with 2.5 ng cRNA encoding the indicated single truncated CFTR channel segments. Methods and symbols are as in Fig. 2.
	Figure 5. Protein expression of CFTR constructs severed near the COOH terminus of NBD1. (A and B) Immunoblots of membrane proteins from oocytes expressing individual CFTR segments or pairs of segments, as indicated, resolved by SDS-PAGE, transferred, and blotted with antiâR-domain antibody (A) or with antiâFlag M2 antibody (B). (C) Coexpressed constructs Flag3-633 plus 634-1480 were digested with N-glycosidase-F (left) or endoglycosidase-H (right), and the products were identified with antiâR-domain antibody. Arrows in A and C mark fully glycosylated (â¼150 kD, fat arrow), core glycosylated (â¼95 kD, thin arrow), and deglycosylated (â¼90 kD, thin arrow) forms of the 634-1480 protein.
	Figure 6. Protein expression of CFTR constructs severed near the NH2 terminus of NBD1. (A) Immunoblot of membrane proteins from oocytes expressing individual CFTR segments or pairs of segments, as indicated, blotted with antiâR-domain antibody. (B) Coexpressed constructs Flag3-432+433-1480 were digested with N-glycosidase-F (left) and endoglycosidase-H (right), and the products were identified with antiâR-domain antibody. Arrows in A and B mark fully glycosylated (â¼160 kD, fat arrow), core glycosylated (â¼125 kD, thin arrow), and deglycosylated (â¼120 kD, thin arrow) forms of 433-1480 protein. *Partially degraded form (â¼70 kD).
	Figure 7. Coimmunoprecipitation of severed CFTR constructs. Digitonin-solubilized membrane proteins were immunoprecipitated with antiâFlag M2 affinity beads, eluted, resolved with SDS-PAGE on 7.5â10% gradient gels, and blotted with antiâR-domain antibody (top) or antiâNH2-terminus antibody (bottom). (A) Constructs severed near the COOH terminus of NBD1: uninjected (lane 1), Flag3-622+634-1480 (lane 2), Flag3-633+634-1480 (lane 3); arrows are as in Fig. 5 A. (B) Constructs severed near the NH2-terminus of NBD1: Flag3-432+449-1480 (lane 1), Flag3-432+433-1480 (lane 2); arrows are as in Fig. 6 A.
	Figure 9. Kinetic analysis of WT, 1-432+433-1480, 1-633+634-1480, Flag-WT, Flag3-432+433-1480, and Flag3-633+634-1480 channels. Kinetic parameters were extracted from records (filtered at 100 Hz, sampled at 1 kHz) of patches containing one to seven channels, exposed to 2 mM MgATP and 300 nM PKA. Mean burst durations of constructs severed between NBD1 and the R domain (1-633+634-1480 and Flag3-633+634-1480) were significantly shorter than those of WT or Flag-WT. All constructs containing the Flag epitope (Flag-WT, Flag3-432+ 433-1480, and Flag3-633+634-1480) showed significantly prolonged interburst durations and, hence, significantly reduced Po compared with WT. Significance levels: P < 0.1, P < 0.05, and **P < 0.01.
	Figure 11. Apparent affinities for activation of channel Po by MgATP. Representative macropatch currents are shown for WT and Flag-WT (A), 1-633+634-1480 and Flag3-633+634-1480 (B), 1-432+433-1480 and Flag3-432+433-1480 (C), and Flag3-414+433-1480 (D) channels. After prephosphorylation by PKA, and removal of both PKA and ATP, once all channels had closed, 50 Î¼M ATP was applied for 10â30 s, bracketed between exposures to 2 mM ATP. (E) The ratios of the mean steady currents, I50Î¼M/I2mM, were 0.50 Â± 0.02 (WT), 0.56 Â± 0.03 (Flag-WT), 0.51 Â± 0.02 (1-633+634-1480), 0.42 Â± 0.01 (Flag3-633+634-1480), 0.49 Â± 0.02 (1-432+433-1480), 0.44 Â± 0.01 (Flag3-432+433-1480), and 0.45 Â± 0.01 (Flag3-414+433-1480). None of the constructs differed significantly from WT at P < 0.05.
	Figure 12. Alignment of CFTR's NBD1 with HisP. PSI-Blast aligned CFTR residues 422â637 with corresponding residues in HisP. Secondary structure elements shown below (Î²-strands, magenta arrows; Î±-helices, orange boxes) correspond to those of the solved crystal structure of HisP (Hung et al. 1998). CFTR residue numbers are centered above the corresponding letters. Teal boxes identify the three consensus sequences conserved in most ABC proteins. Red boxes in the CFTR sequence identify nontolerated, green boxes tolerated, cut sites. (Cuts occurred between the two highlighted residues in each case.) Gray boxes identify sections flanking the NBD1 sequence that could be omitted without destroying channel function. *Residue F508.
	Figure 10. Single-channel conductances of WT, 1-432+433-1480, 1-633+634-1480, FlagWT, Flag3-432+433-1480, and Flag3-633+634-1480 channels in symmetrical 140 mM Clâ. (A and B) Segments of representative CFTR channel current records, illustrated for WT and 1-432+433-1480, at holding potentials of â80, â40, 0, +40, and +80 mV, were used to create amplitude histograms, fitted by sums of Gaussians (right). The distances between adjacent peaks, plotted against potential gave linear I-V plots. (C) Fitted slopes gave conductances of 6.8 Â± 0.3 (WT), 7.1 Â± 0.2 (1-432+433-1480), 6.3 Â± 0.2 (1-633+634-1480), 6.6 Â± 0.3 (Flag-WT), 7.0 Â± 0.2 (Flag3-432+433-1480), and 7.0 Â± 0.1 (Flag3-633+634-1480), none of which differed significantly from WT at P < 0.1.

References [+] :

Al-Nakkash, Activation of wild-type and deltaF508-CFTR by phosphodiesterase inhibitors through cAMP-dependent and -independent mechanisms. 1999, Pubmed

Al-Nakkash, Activation of wild-type and deltaF508-CFTR by phosphodiesterase inhibitors through cAMP-dependent and -independent mechanisms. 1999, Pubmed
Annereau, A novel model for the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator. 1997, Pubmed
Bear, Cl- channel activity in Xenopus oocytes expressing the cystic fibrosis gene. 1991, Pubmed , Xenbase
Berkower, Mutational analysis of the yeast a-factor transporter STE6, a member of the ATP binding cassette (ABC) protein superfamily. 1991, Pubmed
Berkower, Functional and physical interactions between partial molecules of STE6, a yeast ATP-binding cassette protein. 1996, Pubmed
Betton, In vivo assembly of active maltose binding protein from independently exported protein fragments. 1994, Pubmed
Bibi, In vivo expression of the lacY gene in two segments leads to functional lac permease. 1990, Pubmed
Clancy, Cystic fibrosis transmembrane conductance regulator (CFTR) nucleotide-binding domain 1 (NBD-1) and CFTR truncated within NBD-1 target to the epithelial plasma membrane and increase anion permeability. 1998, Pubmed
Csanády, Rapid kinetic analysis of multichannel records by a simultaneous fit to all dwell-time histograms. 2000, Pubmed
Csanády, CFTR channel gating: incremental progress in irreversible steps. 1999, Pubmed
Devidas, The second half of the cystic fibrosis transmembrane conductance regulator forms a functional chloride channel. 1998, Pubmed , Xenbase
Dulhanty, Phosphorylation by cAMP-dependent protein kinase causes a conformational change in the R domain of the cystic fibrosis transmembrane conductance regulator. 1994, Pubmed
Gadsby, Control of CFTR channel gating by phosphorylation and nucleotide hydrolysis. 1999, Pubmed
Gao, Reconstitution of ATP-dependent leukotriene C4 transport by Co-expression of both half-molecules of human multidrug resistance protein in insect cells. 1996, Pubmed
Groves, Functional reassembly of the anion transport domain of human red cell band 3 (AE1) from multiple and non-complementary fragments. 1998, Pubmed , Xenbase
Hartman, Recombinant synthesis, purification, and nucleotide binding characteristics of the first nucleotide binding domain of the cystic fibrosis gene product. 1992, Pubmed
Higgins, ABC transporters: from microorganisms to man. 1992, Pubmed
Hung, Crystal structure of the ATP-binding subunit of an ABC transporter. 1998, Pubmed
Ishihara, Block by MOPS reveals a conformation change in the CFTR pore produced by ATP hydrolysis. 1997, Pubmed
Jones, Subunit interactions in ABC transporters: towards a functional architecture. 1999, Pubmed
Kaczmarek, Microinjection of catalytic subunit of cyclic AMP-dependent protein kinase enhances calcium action potentials of bag cell neurons in cell culture. 1980, Pubmed
Ko, The first nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator can function as an active ATPase. 1995, Pubmed
Ko, Cystic fibrosis transmembrane conductance regulator: the first nucleotide binding fold targets the membrane with retention of its ATP binding function. 1997, Pubmed
Liman, Subunit stoichiometry of a mammalian K+ channel determined by construction of multimeric cDNAs. 1992, Pubmed , Xenbase
Loo, Reconstitution of drug-stimulated ATPase activity following co-expression of each half of human P-glycoprotein as separate polypeptides. 1994, Pubmed
Mourez, Subunit interactions in ABC transporters: a conserved sequence in hydrophobic membrane proteins of periplasmic permeases defines an important site of interaction with the ATPase subunits. 1997, Pubmed
Moyer, Membrane trafficking of the cystic fibrosis gene product, cystic fibrosis transmembrane conductance regulator, tagged with green fluorescent protein in madin-darby canine kidney cells. 1998, Pubmed
Naren, CFTR chloride channel regulation by an interdomain interaction. 1999, Pubmed , Xenbase
Ostedgaard, Association of domains within the cystic fibrosis transmembrane conductance regulator. 1997, Pubmed
Picciotto, Phosphorylation of the cystic fibrosis transmembrane conductance regulator. 1992, Pubmed
Ramjeesingh, Walker mutations reveal loose relationship between catalytic and channel-gating activities of purified CFTR (cystic fibrosis transmembrane conductance regulator). 1999, Pubmed
Randak, A recombinant polypeptide model of the second predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator is a GTP-binding protein. 1996, Pubmed
Randak, A recombinant polypeptide model of the second nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator functions as an active ATPase, GTPase and adenylate kinase. 1997, Pubmed
Riordan, Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. 1989, Pubmed
Sheppard, Structure and function of the CFTR chloride channel. 1999, Pubmed
Sheppard, The amino-terminal portion of CFTR forms a regulated Cl- channel. 1994, Pubmed
Shiba, Functional assembly of a randomly cleaved protein. 1992, Pubmed
Shyamala, Structure-function analysis of the histidine permease and comparison with cystic fibrosis mutations. 1991, Pubmed
Smit, Functional roles of the nucleotide-binding folds in the activation of the cystic fibrosis transmembrane conductance regulator. 1993, Pubmed , Xenbase
Stühmer, Structural parts involved in activation and inactivation of the sodium channel. 1989, Pubmed , Xenbase
Walker, Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. 1982, Pubmed
Wilkinson, CFTR: the nucleotide binding folds regulate the accessibility and stability of the activated state. 1996, Pubmed , Xenbase
Yike, A recombinant peptide model of the first nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator: comparison of wild-type and delta F508 mutant forms. 1996, Pubmed
Zeltwanger, Gating of cystic fibrosis transmembrane conductance regulator chloride channels by adenosine triphosphate hydrolysis. Quantitative analysis of a cyclic gating scheme. 1999, Pubmed