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The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is an ATP-binding cassette transporter that contains conserved nucleotide-binding domains (NBDs). In CFTR, the NBDs bind and hydrolyze ATP to open and close the channel. Crystal structures of related NBDs suggest a structural model with an important signaling role for a gamma-phosphate linker peptide that couples bound nucleotide to movement of an alpha-helical subdomain. We mutated two residues in CFTR that the structural model predicts will uncouple effects of nucleotide binding from movement of the alpha-helical subdomain. These residues are Gln-493 and Gln-1291, which may directly connect the ATP gamma-phosphate to the gamma-phosphate linker, and residues Asn-505 and Asn-1303, which may form hydrogen bonds that stabilize the linker. In NBD1, Q493A reduced the frequency of channel opening, suggesting a role for this residue in coupling ATP binding to channel opening. In contrast, N505C increased the frequency of channel opening, consistent with a role for Asn-505 in stabilizing the inactive state of the NBD. In NBD2, Q1291A decreased the effects of pyrophosphate without altering other functions. Mutations of Asn-1303 decreased the rate of channel opening and closing, suggesting an important role for NBD2 in controlling channel burst duration. These findings are consistent with both the bacterial NBD structural model and gating models for CFTR. Our results extend models of nucleotide-induced structural changes from bacterial NBDs to a functional mammalian ATP-binding cassette transporter.

Phosphorylation of the R domain regulates cystic fibrosis transmembrane conductance regulator Cl−channel activity. Earlier studies suggested that the R domain controls activity via more than one mechanism; a phosphorylated R domain may stimulate activity, and an unphosphorylated R domain may prevent constitutive activity, i.e.opening with ATP alone. However, the mechanisms responsible for these two regulatory properties are not understood. In this study we asked whether the two effects are dependent on its position in the protein and whether smaller regions from the R domain mediate the effects. We found that several portions of the R domain conferred phosphorylation-stimulated activity. This was true whether the R domain sequences were present in their normal location or were translocated to the C terminus. We also found that some parts of the R domain could be deleted without inducing constitutive activity. However, when residues 760–783 were deleted, channels opened without phosphorylation. Translocation of the R domain to the C terminus did not prevent constitutive activity. These results suggest that different parts of the phosphorylated R domain can stimulate activity and that their location within the protein is not critical. In contrast, prevention of constitutive activity required a short specific sequence that could not be moved to the C terminus. These results are consistent with a recent model of an R domain composed primarily of random coil in which more than one phosphorylation site is capable of stimulating channel activity, and net activity reflects interactions between multiple sites in the R domain and the rest of the channel.

BNC1 is a mammalian neuronal cation channel in the novel DEG/ENaC ion channel family. BNC1 channels are transiently activated by extracellular protons and are constitutively activated by insertion of large residues, such as valine, in place of Gly-430; residue 430 is a site where analogous mutations in someCaenorhabditis elegansfamily members cause a swelling neurodegeneration. Mutation of Gly-430 to a small amino acid, cysteine, neither generated constitutive currents nor allowed modification of this residue by sulfhydryl-reactive methanethiosulfonate (MTS) compounds. However, when protons activated the channel, Cys-430 became accessible to extracellular MTS reagents, which modified Cys-430 to generate constitutive currents. Fluorescent MTS reagents also labeled Cys-430 in activated channels. These data indicate that protons induce a reversible conformational change that activates BNC1 thereby exposing residue 430 to the extracellular solution. Once Cys-430 is modified with a large chemical group, the channel is prevented from relaxing back to the inactive state. These results link ligand-dependent activation and activation by mutations that cause neurodegeneration.

Three subunits, α, β, and γ, contribute to the formation of the epithelial Na+channel. To investigate the oligomeric assembly of the channel complex, we used sucrose gradient sedimentation analysis to determine the sedimentation properties of individual subunits and heteromultimers comprised of multiple subunits. When the α subunit was expressed alone, it first formed an oligomeric complex with a sedimentation coefficient of 11 S, and then generated a higher order multimer of 25 S. In contrast, individual β and γ subunits predominately assembled into 11 S complexes. We obtained similar results with expression in cells andin vitro. When we co-expressed β with α or with α plus γ, the β subunit assembled into a 25 S complex. Glycosylation of the α subunit was not required for assembly into a 25 S complex. We found that the α subunit formed intra-chain disulfide bonds. Although such bonds were not required to generate an oligomeric complex, under nonreducing conditions the α subunit formed a complex that migrated more homogeneously at 25 S. This suggests that intra-chain disulfide bonds may stabilize the complex. These data suggest that the epithelial Na+channel subunits form high order oligomeric complexes and that the α subunit contains the information that facilitates such formation. Interestingly, the ability of the α, but not the β or γ, subunit to assemble into a 25 S homomeric complex correlates with the ability of these subunits to generate functional channels when expressed alone.

A unique feature of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl−channel is regulation by ATP through the two cytoplasmic nucleotide-binding domains (NBDs). To better understand this process, we asked how channel activity is affected by inorganic pyrophosphate (PPi), a compound that binds to NBDs in other proteins. PPiand three nonhydrolyzable PPianalogs reversibly stimulated the activity of phosphorylated channels. Kinetic modeling of single channel data demonstrated that PPiaffected two distinct steps in channel regulation. First, PPiincreased the rate at which channels opened. Second, once channels were open, PPidelayed their closure. PPicould only stimulate channels when it was applied in the presence of ATP. PPialso increased the photolabeling of CFTR by an ATP analog. These two findings suggest that PPimodifies the activity of ATP-dependent CFTR channel gating. Based on these and previous data, we speculate that the effects of PPiare mediated by binding of PPito NBD2 where it regulates channel opening by NBD1, and then, because it is not hydrolyzed, it slows the rate of NBD2-mediated channel closing. Because PPistimulated wild-type channels, we tested its effect on CFTR containing the cystic fibrosis mutations: ΔF508, R117H, and G551S. PPistimulated all three. PPialso stimulated endogenous CFTR in the apical membrane of permeabilized T-84 epithelia. These results suggest that PPior an analog might be of value in the development of new approaches to the treatment of cystic fibrosis.

The free nucleoplasmic Ca2+concentration ([Ca2+]n) may regulate many nuclear events, such as gene transcription. Since the nucleus may possess the enzymes necessary to generate the second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), and because the nuclear envelope may enclose an Ins(1,4,5)P3-releasable Ca2+store, we tested the hypothesis that nuclear and/or cytosolic levels of Ins(1,4,5)P3can control [Ca2+]n. To assay [Ca2+]n, we measured the fluorescence of the Ca2+indicator fluo 3 in the nucleus of Xenopusoocytes by confocal microscopy. When we injected Ins(1,4,5)P3into the cytosol, [Ca2+]nincreased. This increase in [Ca2+]nstill occurred when heparin was present in the nucleus, but was abolished when heparin was present in the cytosol, indicating that cytosolic Ins(1,4,5)P3levels could control [Ca2+]n. When we injected Ins(1,4,5)P3directly into the nucleus, [Ca2+]nincreased, even when heparin was present in the cytosol, indicating that Ins(1,4,5)P3could control [Ca2+]nfrom within the nucleus. These results provide functional evidence for Ins(1,4,5)P3receptors facing the nucleoplasm and raise the possibility that a phosphoinositide cycle situated at the nuclear membranes can control Ca2+-dependent nuclear functions.

Improving the efficiency of gene transfer remains an important goal in developing new treatments for cystic fibrosis and other diseases. Adenovirus vectors and nonviral vectors each have specific advantages, but they also have limitations. Adenovirus vectors efficiently escape from the endosome and enter the nucleus, but the virus shows limited binding to airway epithelia. Nonviral cationic vectors bind efficiently to the negatively charged cell surface, but they do not catalyze subsequent steps in gene transfer. To take advantage of the unique features of the two different vector systems, we noncovalently complexed cationic molecules with recombinant adenovirus encoding a transgene. Complexes of cationic polymers and cationic lipids with adenovirus increased adenovirus uptake and transgene expression in cells that were inefficiently infected by adenovirus alone. Infection by both complexes was independent of adenovirus fiber and its receptor and occurred via a different cellular pathway than adenovirus alone. Complexes of cationic molecules and adenovirus also enhanced gene transfer to differentiated human airway epithelia in vitroand to the nasal epithelium of cystic fibrosis mice in vivoThese data show that complexes of adenovirus and cationic molecules increase the efficiency of gene transfer, which may enhance the development of gene therapy.

To explore the relationship between structure and function in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl-channel, we studied XenopusCFTR. We found that the anion permeability sequence of cAMP-activated Cl-currents in the apical membrane of XenopusA6 epithelia differed from that of cAMP-activated Cl-currents in human epithelia expressing CFTR. To understand the molecular basis for this difference and to learn whether CFTR from another species would have properties similar to human CFTR, we assembled a full-length XenopusCFTR cDNA from A6 cells. Expression of XenopusCFTR in HeLa cells generated cAMP-activated whole-cell currents and cAMP-dependent protein kinase-activated single channels that resembled those of human CFTR with the exception that the anion permeability sequence was different (Br-= I-> Cl-in XenopusCFTR and Br-= Cl-> I-in human). In addition, the single-channel conductance of XenopusCFTR was increased. To investigate protein regions that account for these differences, we constructed chimeric proteins by replacing either the first or second membrane-spanning domain of human CFTR with the equivalent region of XenopusCFTR (hX1-6 and hX7-12, respectively) and examined their function in HeLa cells. We found that the anion permeability sequence (Br-= I-> Cl-) and single-channel conductance of hX1-6 resembled that of XenopusCFTR expressed in HeLa cells, whereas hX7-12 had properties like those of human CFTR. However, the gating of hX1-6 showed a flickery behavior. The altered gating of hX1-6 was attributed to residues in the first extracellular loop of XenopusCFTR because mutation of residues in that region to the corresponding residues of human CFTR produced gating behavior similar to that of human CFTR. These data suggest that sequence differences in the first membrane-spanning domains are responsible for the differences in the permeation properties of human and XenopusCFTR and that the first extracellular loop influences channel gating.