ABCMdb

PMID: 25887396

Dong Q, Ernst SE, Ostedgaard LS, Shah VS, Ver Heul AR, Welsh MJ, Randak CO
Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia.
J Biol Chem. 2015 May 29;290(22):14140-53. doi: 10.1074/jbc.M114.611616. Epub 2015 Apr 17., [PubMed]

Sentences

No. Mutations Sentence Comment

8 ABCC7 p.Gln1291Phe 8-Azidoadenosine 5d15;-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. Login to comment 10 ABCC7 p.Gln1291Phe However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Login to comment 11 ABCC7 p.Gln1291Phe Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cld1a; channel function in well differentiated primary human airway epithelia. Login to comment 82 ABCC7 p.Gln1291Phe Ussing Chamber Studies-Well differentiated primary human bronchial airway epithelia derived from cystic fibrosis (CF) donors homozygous for the F508del mutation and cultured at the air-liquid interface (42) were infected with recombinant adenovirus serotype 5 at a multiplicity of infection of 50-100 for 1 h. The vectors encoded wild-type or Q1291F CFTR cDNA driven by a cytomegalovirus promotor. Login to comment 96 ABCC7 p.Gln1291Phe To quantify wild-type and Q1291F CFTR expression relative to endogenous F508del CFTR expression, the 2afa;èc;èc;CT method (45) was applied. Login to comment 141 ABCC7 p.Gln1291His ABCC7 p.Gln1291Phe ABCC7 p.Gln1291Tyr ABCC7 p.Gln1291Trp We found that Ap5A did not inhibit Clafa; current when Gln-1291 was replaced by amino acids with bulky side chains like phenylalanine (Q1291F), tryptophan (Q1291W), tyrosine (Q1291Y), or histidine (Q1291H). Login to comment 142 ABCC7 p.Gln1291Ala ABCC7 p.Gln1291Gly Gln mutations that resulted in shortening (Q1291A) or deletion (Q1291G) of the side chain reduced Ap5A inhibition (Fig. 3, A FIGURE 1. Login to comment 154 ABCC7 p.Gln1291Gly To further evaluate the effect of these mutations, we tested inhibition of Q1291G CFTR current with a range of ApA concentrations. Login to comment 159 ABCC7 p.Gln1291Phe To test the effect of such a mutation on the interaction of CFTR with ATP and ATPase-dependent gating, we compared the relationship between current and ATP concentration of wild-type CFTR with that of Q1291F CFTR and found that they were similar (Fig. 4). Login to comment 160 ABCC7 p.Gln1291Phe This result indicates that the Q1291F mutation does not substantially alter the potency of ATP at stimulating current. Login to comment 165 ABCC7 p.Ser1248Phe ABCC7 p.Ser1248Phe ABCC7 p.Gln1291Phe As a positive control, we mutated serine 1248 to phenylalanine (S1248F) in Q1291F CFTR.TheS1248FmutationpreventstheinteractionofATPwith ATP-bindingsite2(48).Asanticipated,theopenprobabilityofthe double mutant was markedly reduced, mainly due to interburst closed times that were significantly longer than those of wild-type andQ1291FCFTR(comparebar6withbars1and5inthetopand bottom panels of Fig. 5B). Login to comment 181 ABCC7 p.Gln1291His ABCC7 p.Gln1291Ala ABCC7 p.Gln1291Phe ABCC7 p.Gln1291Tyr ABCC7 p.Gln1291Trp ABCC7 p.Gln1291Gly No significant differences were detected between bars 1, 3, 4, 5, 6, 7, and 8 (one-way ANOVA followed by Holm-Sidak`s method of all pairwise multiple comparisons; wild-type CFTR, n afd; 13; F508del CFTR, n afd; 10; Q1291A CFTR, n afd; 4; Q1291G CFTR, n afd; 4; Q1291F CFTR, n afd; 6; Q1291H CFTR, n afd; 6; Q1291W CFTR, n afd; 6; Q1291Y CFTR, n afd; 6). Login to comment 189 ABCC7 p.Gln1291Phe ABCC7 p.Gln1291Phe ABCC7 p.Gln1291Gly *, p b0d; 0.01 when compared with bar 1, 3, 4, 5, 6, or 7 (one-way ANOVA followed by Holm-Sidak`s method of multiple comparisons versus control group; wild-type CFTR, n afd; 6; F508del CFTR, n afd; 4; Q1291G CFTR, n afd; 4; Q1291F CFTR, n afd; 6; Q1291HCFTR,nafd;4;Q1291WCFTR,nafd;4;Q1291YCFTR,nafd;4).Nosignificantdifferencesweredetectedbetweenbars1,3,4,5,6,and7(one-wayANOVA).Error bars, S.E. Selective Disruption of Adenylate Kinase-coupled CFTR Gating 14144 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290ߦNUMBER 22ߦMAY 29, 2015 To further probe ATPase-dependent gating of Q1291F CFTR, we studied the effect of adenosine 5b18;-(beta,ॹ-imido)triphosphate (AMPPNP). Login to comment 196 ABCC7 p.Gln1291Gly *, p b0d; 0.05 compared with wild type; double daggers, p b0d; 0.001 compared with Q1291G (one-way ANOVA followed by Holm-Sidak`s method of multiple comparisons versus control group). Login to comment 199 ABCC7 p.Gln1291Phe FIGURE 4. Effect of ATP concentration on current of wild-type (Q1291; black) and Q1291F CFTR (red). Login to comment 201 ABCC7 p.Gln1291Phe Data are from two wild-type and 15 Q1291F CFTR patches with n c56; 8 for each ATP concentration. Login to comment 203 ABCC7 p.Gln1291Phe ABCC7 p.Gln1291Phe Lines are fits to a Michaelis-Menten equation using apparent Km values of 98 afe; 7 òe;M (wild type; black line) and 89 afe; 6 òe;M (Q1291F mutant; red line) and maximum normalized current values at high ATP concentrations of 1.13 afe; 0.02 (wild type; black line) and 1.08 afe; 0.02 (Q1291F mutant; red line). Login to comment 207 ABCC7 p.Gln1291His ABCC7 p.Ser1248Phe ABCC7 p.Gln1291Phe ABCC7 p.Gln1291Phe ABCC7 p.Gln1291Tyr ABCC7 p.Gln1291Trp Holding voltage was afa;80 mV for wild-type, Q1291W, and Q1291Y CFTR and afa;60 mV for Q1291H, Q1291F, and Q1291F/S1248F CFTR. Login to comment 221 ABCC7 p.Gln1291Phe Therefore, we predicted that AMPPNP would induce prolonged open bursts of Q1291F CFTR if channel closing was coupled to ATP hydrolysis. Login to comment 222 ABCC7 p.Gln1291Phe Experimental testing showed that we indeed detected a second population of very long Q1291F CFTR bursts after adding AMPPNP. Login to comment 224 ABCC7 p.Gln1291Ala Consistent with our findings, a previous study showed that the Q1291A mutation did not change burst duration, interburst interval, or the ATP dose-response curve (52). Login to comment 233 ABCC7 p.Gln1291Phe In contrast, no change in current was observed when Gln-1291 was replaced by phenylalanine or glycine (Figs. 7 and 9). Login to comment 234 ABCC7 p.Gln1291Phe Because the effect of AMP on wild-type CFTR current depended on the ATP concentration (15), we tested its effect on Q1291F CFTR current at a range of different ATP concentrations. Login to comment 235 ABCC7 p.Gln1291Phe AMP did not affect Q1291F CFTR current at any ATP concentration and thus did not alter its ATP dose-response curve (Fig. 7C). Login to comment 237 ABCC7 p.Gln1291Phe Consistent with their ATP dose-response curves (Fig. 4), wild-type and Q1291F CFTR exhibited similar open state probabilities (Po), which were lower than those obtained at 1 mM ATP (compare Po values in Figs. 5B and 8B). Login to comment 239 ABCC7 p.Gln1291Phe In contrast, AMP had no effect on Q1291F CFTR gating. Login to comment 240 ABCC7 p.Gln1291Phe These results are consistent with the disruption of nucleotide interactions at the AMP-binding site in Q1291F CFTR. Login to comment 241 ABCC7 p.Gln1291Gly If the Gln-1291 side chain interacted with AMP as predicted based on the SMC-NBD structure (Fig. 1) (19), then removal of this side chain (Q1291G mutation) should affect the interaction of AMP with CFTR. Login to comment 243 ABCC7 p.Gln1291Phe FIGURE 6. Effect of AMPPNP on wild-type (Q1291; A-C) and Q1291F (D-F) CFTR burst duration. Login to comment 244 ABCC7 p.Gln1291Phe A and D, examples of two 140-s current traces from the same excised membrane patch containing at least three wild-type (A) and two Q1291F (D) CFTR channels before and after adding AMPPNP (2 mM) to the cytosolic surface. ATP (0.3 mM) and PKA catalytic subunit were present throughout the experiments. Login to comment 247 ABCC7 p.Gln1291Phe For illustration purposes, traces were digitally low pass-filtered at 50 Hz. B, C, E, and F, burst duration histograms of wild-type (B and C) and Q1291F (E and F) CFTR channel activity before (B and E) and after (C and F) adding AMPPNP derived from the experiments shown in A (histograms B and C) and D (histograms E and F). Login to comment 260 ABCC7 p.Gln1291Phe ABCC7 p.Gln1291Phe Selective Disruption of Adenylate Kinase-coupled CFTR Gating 14146 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290ߦNUMBER 22ߦMAY 29, 2015 The Q1291F Mutation Disrupts Photolabeling of the AMP-binding Site with 8-N3-AMP-We hypothesized that if the Q1291F mutation abolished nucleotide interactions at the AMP-binding site, photolabeling of this site with 8-N3-AMP should be disrupted. Login to comment 265 ABCC7 p.Gln1291Phe We expressed both wild-type and Q1291F CFTR in HeLa cells and collected cell membranes. Login to comment 269 ABCC7 p.Gln1291Phe We also detected a labeling signal with Q1291F CFTR. Login to comment 271 ABCC7 p.Gln1291Phe ABCC7 p.Gln1291Phe The Q1291F Mutation Disrupts CFTR Adenylate Kinase Activity-The data suggest that the Q1291F mutation would interfere with adenylate kinase activity. Login to comment 275 ABCC7 p.Gln1291Phe ABCC7 p.Gln1291Phe A and B, time courses showing the effect of AMP added at 75 òe;M ATP on wild-type (Q1291) (A) versus Q1291F (B) CFTR Clafa; current. Recordings (100 ms averages) are from excised inside-out membrane patches containing multiple wild-type (A) or Q1291F (B) CFTR channels. ATP and AMP were present during times and at concentrations indicated by bars. ATP was added together with PKA catalytic subunit as described under "Experimental Procedures." Login to comment 276 ABCC7 p.Gln1291Phe Holding voltage was afa;40 mV. C, quantitative data showing lack of effect of AMP (1 mM) on ATP-dependent Q1291F CFTR current. Login to comment 279 ABCC7 p.Gln1291Phe Current measurements in the absence of AMP are the same as shown in Fig. 4 for Q1291F CFTR. Login to comment 284 ABCC7 p.Gln1291Gly D, removal of the Gln-1291 side chain (Q1291G mutation) reduces the potency of AMP to increase CFTR Clafa; current. Login to comment 291 ABCC7 p.Gln1291Phe ABCC7 p.Gln1291Phe A, examples of two 1-min current recordings from the same excised membrane patch, each containing at least three wild-type or Q1291F CFTR channels, before and after adding AMP (1 mM) to the cytosolic surface. ATP (75 òe;M) andPKAcatalyticsubunitwerepresentthroughouttheexperiments.Holding voltage was afa;80 mV. c, channel closed state; o1, o2, and o3, open states. For illustration purposes, traces were digitally low pass-filtered at 50 Hz. B, average single channel properties. Data are means afe; S.E. (error bars) of six wild-type and three Q1291F CFTR patches. Login to comment 299 ABCC7 p.Gln1291Phe In contrast, Q1291F CFTR showed very little labeling. Login to comment 301 ABCC7 p.Gln1291Phe ABCC7 p.Gln1291Phe The Q1291F Mutation Interferes with Channel Opening in the Presence of Physiologic Concentrations of ATP, ADP, and AMP-We examined the functional consequences of the Q1291F mutation on channel gating under physiologic conditions (i.e. in the presence of ATP, ADP, and AMP). Login to comment 304 ABCC7 p.Gln1291Phe Therefore, we hypothesized that when all three nucleotides are present, the adenylate kinase-deficient Q1291F mutant displays a lower open state probability than wild-type CFTR. Login to comment 306 ABCC7 p.Gln1291Phe In the presence of 1 mM ATP, 250 òe;M ADP, and 50 òe;M AMP, Q1291F CFTR indeed displayed a significantly lower open state probability (Po) and longer closed interburst intervals (i.e. a reduced rate of opening into a burst) than wild-type CFTR (Fig. 12). Login to comment 308 ABCC7 p.Gln1291Phe ABCC7 p.Gln1291Phe The Q1291F Mutation Causes Defective CFTR Clafa; Channel Function in Primary Human Airway Epithelia-The data suggest that the Q1291F mutation might cause defective Clafa; channel function in living cells. Login to comment 309 ABCC7 p.Gln1291Phe To test this hypothesis, we expressed either wild-type CFTR or the adenylate kinase-deficient Q1291F mutant in well differentiated primary human airway epithelia from cystic fibrosis patients (that lack endogenous CFTR activity) because they are closest to an in vivo airway epithelium. Login to comment 310 ABCC7 p.Gln1291Phe The Q1291F mutation did not reduce processing of CFTR to the apical membrane (Fig. 13, A-C). Login to comment 313 ABCC7 p.Gln1291Phe ABCC7 p.Gln1291Phe A and B, time courses showing the effect of AMP-NH2 added at 75 òe;M ATP on wild-type (Q1291) (A) versus Q1291F (B) CFTR Clafa; current. Recordings(100msaverages)arefromexcisedinside-outmembranepatches containing multiple wild-type (A) or Q1291F (B) CFTR channels. ATP and AMP-NH2 were present during the times and at the concentrations indicated by bars. ATP was added together with PKA catalytic subunit as described under "Experimental Procedures." Login to comment 316 ABCC7 p.Gln1291Phe ABCC7 p.Gln1291Phe ABCC7 p.Gln1291Gly ABCC7 p.Gln1291Gly Columns show the means afe; S.E. (error bars) of 15 (wild type), 11 (Q1291F mutant), and 5 (Q1291G mutant) individual experiments obtained from two (wild type) and three (Q1291F and Q1291G mutants) membrane patches. Login to comment 319 ABCC7 p.Gln1291Phe Photolabeling of wild-type (Q1291) and Q1291F CFTR with 8-N3-[33 P]AMP. Login to comment 323 ABCC7 p.Gln1291Phe Letters label highly glycosylated(C)andcore-glycosylated(B)CFTR.Themajorityofwild-typeand Q1291F CFTR migrated as band C. Login to comment 330 ABCC7 p.Gln1291Phe We found that the forskolin/IBMX-induced and the GlyH-101-sensitive changes in transepithelial conductance (Fig. 13, D and E), which are directly related to CFTR channel activity, as well as the corresponding changes in short circuit current (Fig. 13, F and G) were significantly smaller in epithelia expressing Q1291F CFTR than in epithelia expressing wild-type CFTR. Login to comment 332 ABCC7 p.Gln1291Phe These results indicate defective channel function of Q1291F CFTR. Login to comment 345 ABCC7 p.Gln1291Phe Adenylate kinase activity of wild-type (Q1291) and Q1291F CFTR. Login to comment 346 ABCC7 p.Gln1291Phe A, left, autoradiograph of immunoprecipitated wild-type and Q1291F CFTR fractionated on a 6% SDS-polyacrylamide gel. Login to comment 347 ABCC7 p.Gln1291Phe Membranes containing 50 òe;g of protein from either wild-type or Q1291F CFTR-expressing HeLa cells were used in each reaction. Login to comment 353 ABCC7 p.Gln1291Phe Error bars, S.E. FIGURE 12. Effect of physiologic concentrations of ATP, ADP, and AMP on gating of wild-type (Q1291) and Q1291F CFTR. Login to comment 354 ABCC7 p.Gln1291Phe A, examples of 1-min current recordings from excised membrane patches, each containing at least four wild-type or Q1291F CFTR channels in the presence of 1 mM ATP, 250 òe;M ADP, and 50 òe;M AMP at the cytosolic surface. Login to comment 357 ABCC7 p.Gln1291Phe Data are means afe; S.E. (error bars) of four wild-type and five Q1291F CFTR patches. Login to comment 372 ABCC7 p.Gln1291Phe A, immunostaining of differentiated CF human airway epithelia from the same donor infected with recombinant adenovirus expressing wild-type (Q1291) or Q1291F CFTR and of uninfected control. Login to comment 377 ABCC7 p.Gln1291Phe Two of these epithelia were infected with recombinant adenovirus to express wild-type or Q1291F CFTR 4 days prior to the experiment as described under "Experimental Procedures." Login to comment 383 ABCC7 p.Gln1291Phe Experiments were performed 4 days after infection with recombinant adenovirus to express either wild-type or Q1291F CFTR as described under "Experimental Procedures." Login to comment 387 ABCC7 p.Gln1291Phe Two of these epithelia were infected with recombinant adenovirus to express wild-type or Q1291F CFTR. Login to comment 393 ABCC7 p.Gln1291Phe H, relative expression levels of wild-type and Q1291F CFTR in primary CF human airway epithelia used in the Ussing chamber studies (D-G) determined by quantitative PCR as described under "Experimental Procedures." Login to comment 394 ABCC7 p.Gln1291Phe Values for wild-type and Q1291F CFTR from epithelia of the same donor are connected by lines and are depicted in the same color. Login to comment 409 ABCC7 p.Gln1291Phe Although this mutation did not notably affect channel function in the presence of ATP alone (i.e. under experimental conditions testing ATPase-dependent gating), Q1291F CFTR displayed markedly reduced channel activity in the presence of physiologic concentrations of ATP, ADP, and AMP. Login to comment 411 ABCC7 p.Gln1291Arg Interestingly, a mutation at this position, Q1291R, has been described in a pancreatic insufficient CF patient carrying the F508del mutation on the other chromosome (84). Login to comment