ABCMdb

ABCC7 p.Gln1291Phe

ClinVar:	c.3872A>G , p.Gln1291Arg ? , not provided c.3871C>T , p.Gln1291* ? , not provided c.3873G>C , p.Gln1291His D , Pathogenic
CF databases:	c.3872A>G , p.Gln1291Arg (CFTR1) D , Q1291R, an A->G substitution at nucleotide position 4004 in exon 20 has a haplotype of 2-2-1 (KM19-D9-J44) with seven GATT repeats. The mutation creates a new BsaJI site.
Predicted by SNAP2:	A: D (95%), C: D (95%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: N (82%), I: D (95%), K: D (95%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), R: N (78%), S: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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Publications
[hide] Mutating the Conserved Q-loop Glutamine 1291 Selec... J Biol Chem. 2015 May 29;290(22):14140-53. doi: 10.1074/jbc.M114.611616. Epub 2015 Apr 17. 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., [PMID:25887396]

Abstract [show]
The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP left arrow over right arrow 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia.

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No. Sentence Comment
8 8-Azidoadenosine 5d15;-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. Login to comment
10 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 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 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 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 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
159 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 This result indicates that the Q1291F mutation does not substantially alter the potency of ATP at stimulating current. Login to comment
165 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 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 *, 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
199 FIGURE 4. Effect of ATP concentration on current of wild-type (Q1291; black) and Q1291F CFTR (red). Login to comment
201 Data are from two wild-type and 15 Q1291F CFTR patches with n c56; 8 for each ATP concentration. Login to comment
203 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 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 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 Experimental testing showed that we indeed detected a second population of very long Q1291F CFTR bursts after adding AMPPNP. Login to comment
233 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 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 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 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 In contrast, AMP had no effect on Q1291F CFTR gating. Login to comment
240 These results are consistent with the disruption of nucleotide interactions at the AMP-binding site in Q1291F CFTR. Login to comment
243 FIGURE 6. Effect of AMPPNP on wild-type (Q1291; A-C) and Q1291F (D-F) CFTR burst duration. Login to comment
244 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 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 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 We expressed both wild-type and Q1291F CFTR in HeLa cells and collected cell membranes. Login to comment
269 We also detected a labeling signal with Q1291F CFTR. Login to comment
271 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 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 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 Current measurements in the absence of AMP are the same as shown in Fig. 4 for Q1291F CFTR. Login to comment
291 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 In contrast, Q1291F CFTR showed very little labeling. Login to comment
301 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 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 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 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 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 The Q1291F mutation did not reduce processing of CFTR to the apical membrane (Fig. 13, A-C). Login to comment
313 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 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 Photolabeling of wild-type (Q1291) and Q1291F CFTR with 8-N3-[33 P]AMP. Login to comment
323 Letters label highly glycosylated(C)andcore-glycosylated(B)CFTR.Themajorityofwild-typeand Q1291F CFTR migrated as band C. Login to comment
330 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 These results indicate defective channel function of Q1291F CFTR. Login to comment
345 Adenylate kinase activity of wild-type (Q1291) and Q1291F CFTR. Login to comment
346 A, left, autoradiograph of immunoprecipitated wild-type and Q1291F CFTR fractionated on a 6% SDS-polyacrylamide gel. Login to comment
347 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 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 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 Data are means afe; S.E. (error bars) of four wild-type and five Q1291F CFTR patches. Login to comment
372 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 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 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 Two of these epithelia were infected with recombinant adenovirus to express wild-type or Q1291F CFTR. Login to comment
393 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 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 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