ABCC7 p.Leu1254Ala
| ClinVar: |
c.3761T>G
,
p.Leu1254*
?
, not provided
|
| CF databases: |
c.3761T>G
,
p.Leu1254*
D
, CF-causing
|
| Predicted by SNAP2: | A: N (66%), C: N (72%), D: D (75%), E: D (66%), F: N (57%), G: D (63%), H: D (66%), I: N (93%), K: D (71%), M: N (78%), N: D (66%), P: D (71%), Q: D (63%), R: D (71%), S: N (53%), T: N (61%), V: N (87%), W: D (66%), Y: D (59%), |
| Predicted by PROVEAN: | A: N, C: N, D: D, E: N, F: N, G: D, H: D, I: N, K: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N, |
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None has been submitted yet.
[hide] A mutation in CFTR modifies the effects of the ade... Biophys J. 2008 Dec;95(11):5178-85. Epub 2008 Sep 19. Dong Q, Randak CO, Welsh MJ
A mutation in CFTR modifies the effects of the adenylate kinase inhibitor Ap5A on channel gating.
Biophys J. 2008 Dec;95(11):5178-85. Epub 2008 Sep 19., [PMID:18805924]
Abstract [show]
Mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis. The CFTR anion channel is controlled by ATP binding and enzymatic activity at the two nucleotide-binding domains. CFTR exhibits two types of enzymatic activity: 1), ATPase activity in the presence of ATP and 2), adenylate kinase activity in the presence of ATP plus physiologic concentrations of AMP or ADP. Previous work showed that P(1),P(5)-di(adenosine-5')pentaphosphate (Ap(5)A), a specific adenylate kinases inhibitor, inhibited wild-type CFTR. In this study, we report that Ap(5)A increased activity of CFTR with an L1254A mutation. This mutation increased the EC50 for ATP by >10-fold and reduced channel activity by prolonging the closed state. Ap(5)A did not elicit current on its own nor did it alter ATP EC50 or maximal current. However, it changed the relationship between ATP concentration and current. At submaximal ATP concentrations, Ap(5)A stimulated current by stabilizing the channel open state. Whereas previous work indicated that adenylate kinase activity regulated channel opening, our data suggest that Ap(5)A binding may also influence channel closing. These results also suggest that a better understanding of the adenylate kinase activity of CFTR may be of value in developing new therapeutic strategies for cystic fibrosis.
Comments [show]
None has been submitted yet.
No. Sentence Comment
4 In this study, we report that Ap5A increased activity of CFTR with an L1254A mutation.
X
ABCC7 p.Leu1254Ala 18805924:4:70
status: NEW75 RESULTS Ap5A inhibited wild-type CFTR but stimulated CFTR-L1254A We generated 29 variants in which alanine or another amino acid replaced a residue in NBD2 or NBD1.
X
ABCC7 p.Leu1254Ala 18805924:75:58
status: NEW84 Surprisingly, the L1254A mutation reversed the effect of Ap5A, so that Ap5A stimulated CFTR current.
X
ABCC7 p.Leu1254Ala 18805924:84:18
status: NEW85 When added alone to phosphorylated wild-type CFTR (18) or CFTR-L1254A (Fig. 4), Ap5A did not generate activity.
X
ABCC7 p.Leu1254Ala 18805924:85:63
status: NEW86 Ap5A changed the ATP-dependence of CFTR-L1254A To better understand ATP-dependent regulation of the L1254A variant, we examined the relationship between ATP concentration and current (Fig. 5, A and B).
X
ABCC7 p.Leu1254Ala 18805924:86:40
status: NEWX
ABCC7 p.Leu1254Ala 18805924:86:100
status: NEW87 The L1254A mutation did not significantly change the shape of the curve; the Hill coefficient was 1.06 6 0.08 for wild-type CFTR and 0.92 6 0.06 for CFTR-L1254A, although the two lowest ATP concentrations were not well fit by the regression line.
X
ABCC7 p.Leu1254Ala 18805924:87:4
status: NEWX
ABCC7 p.Leu1254Ala 18805924:87:154
status: NEW88 However, introduction of the L1254A mutation shifted the relationship between ATP concentration and current to the right, increasing the ATP EC50 from 34 6 3 mM in wild-type CFTR to 392 6 38 mM in CFTR-L1254A (p , 0.05).
X
ABCC7 p.Leu1254Ala 18805924:88:29
status: NEWX
ABCC7 p.Leu1254Ala 18805924:88:202
status: NEW89 In addition, the activity of CFTR-L1254A was not completely saturated at 10 mM ATP.
X
ABCC7 p.Leu1254Ala 18805924:89:34
status: NEW90 These results suggest that the L1254A mutation may have impaired the interaction with ATP.
X
ABCC7 p.Leu1254Ala 18805924:90:31
status: NEW98 (A) Representative time course of ClÀ current (I) in response to application of the indicated agents on wild-type CFTR and CFTR-L1254A. Data are from excised, inside-out patches containing multiple channels.
X
ABCC7 p.Leu1254Ala 18805924:98:128
status: NEW105 With CFTR-L1254A, we found that 1 mM Ap5A had no significant effect on the EC50 (369 6 100 mM) or the predicted current at maximal ATP concentrations (Fig. 5, A and B).
X
ABCC7 p.Leu1254Ala 18805924:105:10
status: NEW107 Thus, Ap5A reduced the Hill coefficient to ,1 in wild-type and L1254A CFTR suggesting conformational changes associated with negative cooperativity.
X
ABCC7 p.Leu1254Ala 18805924:107:63
status: NEW108 CFTR-L1254A interacts with AMP and other nucleotides We tested several other agents to learn how the L1254A mutation affected function.
X
ABCC7 p.Leu1254Ala 18805924:108:5
status: NEWX
ABCC7 p.Leu1254Ala 18805924:108:101
status: NEW111 We found that, as with wild-type channels, AMP stimulated and ADP inhibited current from the L1254A mutant (Fig. 6, A and B).
X
ABCC7 p.Leu1254Ala 18805924:111:93
status: NEW112 These results suggest that the L1254A mutation did not completely disrupt the AMP-binding site in CFTR.
X
ABCC7 p.Leu1254Ala 18805924:112:31
status: NEW113 To further examine the functional consequences of the CFTR-L1254A mutation in the region around ATP-binding Site 2, we examined the effect of AMP-PNP and pyrophosphate (PPi).
X
ABCC7 p.Leu1254Ala 18805924:113:59
status: NEW117 We found that the L1254A mutation did not prevent the stimulatory effects of AMP-PNP or PPi (Fig. 6, C and D) and that stimulation was greater in CFTR-L1254A than in wild-type CFTR, perhaps because the basal activity before adding these agents was lower in CFTR-L1254A.
X
ABCC7 p.Leu1254Ala 18805924:117:18
status: NEWX
ABCC7 p.Leu1254Ala 18805924:117:151
status: NEWX
ABCC7 p.Leu1254Ala 18805924:117:262
status: NEW118 These results suggest that the L1254A mutation did not completely disrupt the area around ATP-binding Site 2.
X
ABCC7 p.Leu1254Ala 18805924:118:31
status: NEW119 Ap5A stabilized the open state of CFTR-L1254A To determine how Ap5A increased CFTR-L1254A current at low ATP concentrations, we removed ATP and measured the rate at which current decreased, that is, the relaxation rate.
X
ABCC7 p.Leu1254Ala 18805924:119:39
status: NEWX
ABCC7 p.Leu1254Ala 18805924:119:83
status: NEW123 In contrast, in CFTR-L1254A, Ap5A nearly doubled the time constant for current decay (1657 6 565 ms without Ap5A, 3161 6 948 ms with Ap5A; p , 0.01).
X
ABCC7 p.Leu1254Ala 18805924:123:21
status: NEW124 This result suggested that Ap5A may have FIGURE 4 Effect of Ap5A on current of CFTR-L1254A. Data are a tracing of a membrane patch perfused on the cytosolic surface with 1 mM purified Ap5A alone, with 75 mM ATP alone, or with 75 mM ATP plus 1 mM purified Ap5A.
X
ABCC7 p.Leu1254Ala 18805924:124:84
status: NEW126 Adding Ap5A alone yielded a failure to activate CFTR-L1254A in five other experiments.
X
ABCC7 p.Leu1254Ala 18805924:126:53
status: NEW127 FIGURE 5 Effect of ATP concentration on current of wild-type CFTR (WT, red) and CFTR-L1254A in the absence (black) and presence (blue) of 1 mM Ap5A.
X
ABCC7 p.Leu1254Ala 18805924:127:85
status: NEW131 For CFTR-L1254A, the Hill coefficient was 0.92 6 0.06, the Km ¼ 392 6 38 mM, and the Imax ¼ 1.33 6 0.04.
X
ABCC7 p.Leu1254Ala 18805924:131:9
status: NEW132 For CFTR-L1254A studied with 1 mM Ap5A data, the Hill coefficient was 0.58 6 0.06, the Km¼ 369 6 100 mM, and the Imax¼1.45 6 0.09.
X
ABCC7 p.Leu1254Ala 18805924:132:9
status: NEW134 stabilized the open state and predicted that Ap5A would increase the burst duration of CFTR-L1254A.
X
ABCC7 p.Leu1254Ala 18805924:134:92
status: NEW135 In excised, inside-out patches of membrane, CFTR-L1254A showed reduced channel activity compared to wild-type CFTR.
X
ABCC7 p.Leu1254Ala 18805924:135:49
status: NEW137 A longer interburst interval (32.5 6 11.6 s compared to 2.4 6 0.27 s for wild-type CFTR (18)) reduced the Po of CFTR-L1254A.
X
ABCC7 p.Leu1254Ala 18805924:137:117
status: NEW139 Adding 1 mM Ap5A to CFTR-L1254A increased the Po by increasing the burst duration ;40% (from 870 6 204 ms to 1214 6 281 ms; p , 0.05) without a significant effect on the interburst interval (from 32.5 6 11.6 s to 32.9 6 13.9 s; p .
X
ABCC7 p.Leu1254Ala 18805924:139:25
status: NEW143 DISCUSSION In all of the CFTR variants that we examined, Ap5A altered current. However, we were surprised to observe that Ap5A increased current from one of the variants, L1254A; this finding is the opposite of the effect of Ap5A on wild-type CFTR (18).
X
ABCC7 p.Leu1254Ala 18805924:143:171
status: NEW144 Several observations suggest that the AMP-binding site remained available in CFTR-L1254A.
X
ABCC7 p.Leu1254Ala 18805924:144:82
status: NEW145 First, Ap5A altered CFTR-L1254A current.
X
ABCC7 p.Leu1254Ala 18805924:145:25
status: NEW146 Our previous work on wild-type CFTR (18) and studies on other adenylate kinases (24,36) indicate that the ability of Ap5A to alter function depends on FIGURE 6 Effect of AMP (A), ADP (B), AMP-PNP (C), and PPi (D) on wild-type CFTR and CFTR-L1254A. Data are from excised, inside-out patches containing multiple CFTR channels.
X
ABCC7 p.Leu1254Ala 18805924:146:240
status: NEW149 In A, N ¼ 9 for wild-type CFTR and N ¼ 15 for CFTR-L1254A; in B, N ¼ 4 for wild-type and N ¼ 8 for L1254A; in C, N ¼ 3 for wild-type and L1254; and in D, N ¼ 5 for wild-type and N ¼ 3 for L1254A.
X
ABCC7 p.Leu1254Ala 18805924:149:59
status: NEWX
ABCC7 p.Leu1254Ala 18805924:149:61
status: NEWX
ABCC7 p.Leu1254Ala 18805924:149:115
status: NEWX
ABCC7 p.Leu1254Ala 18805924:149:119
status: NEW153 Second, AMP stimulated CFTR-L1254A current in the presence of ATP.
X
ABCC7 p.Leu1254Ala 18805924:153:28
status: NEW155 Third, ADP inhibited CFTR-L1254A current.
X
ABCC7 p.Leu1254Ala 18805924:155:26
status: NEW157 The data also indicate that the L1254A variant altered the interaction with ATP.
X
ABCC7 p.Leu1254Ala 18805924:157:32
status: NEW160 Studies of NBD crystal structures from related ABC proteins are also consistent with the conclusion that the L1254A mutation altered the interaction with ATP.
X
ABCC7 p.Leu1254Ala 18805924:160:109
status: NEW164 Our finding that CFTR-L1254A had a prolonged burst duration is consistent with a reduced enzymatic activity associated with mutating those residues.
X
ABCC7 p.Leu1254Ala 18805924:164:22
status: NEW165 How did Ap5A stimulate CFTR-L1254A current?
X
ABCC7 p.Leu1254Ala 18805924:165:28
status: NEW166 We speculate the following: 1), ATP binds ATP-binding Site 1 as it does in wild-type CFTR. Our data showing that Ap5A only affects gating in the presence of ATP, combined with our earlier studies (18), suggest that the L1254A mutation does not disrupt ATP binding at Site 1.
X
ABCC7 p.Leu1254Ala 18805924:166:219
status: NEW168 2), The L1254A mutation partially disrupts ATP binding at Site 2.
X
ABCC7 p.Leu1254Ala 18805924:168:8
status: NEW172 It seems unlikely that the L1254A mutation completely eliminates ATP binding at Site 2 because the mutation increased burst duration, whereas mutations that eliminate ATP binding at Site 2 do not increase burst duration (11,14,28).
X
ABCC7 p.Leu1254Ala 18805924:172:27
status: NEW175 Why would the L1254A mutation reduce the affinity for ATP but allow Ap5A to bind?
X
ABCC7 p.Leu1254Ala 18805924:175:14
status: NEW177 Moreover, Ap5A binding in adenylate kinases induces conformational changes that may enhance its binding affinity (23,44,45), and perhaps this occurs in CFTR-L1254A.
X
ABCC7 p.Leu1254Ala 18805924:177:157
status: NEW178 4), We speculate that Ap5A binding to CFTR-L1254A generates a more stable open state because Ap5A is not a substrate for ATPase or adenylate kinase activity.
X
ABCC7 p.Leu1254Ala 18805924:178:43
status: NEW183 N ¼ 5 for both wild-type CFTR and CFTR-L1254A.
X
ABCC7 p.Leu1254Ala 18805924:183:43
status: NEW185 In those experiments, Ap5A prolonged the relaxation rate for CFTR-L1254A (153 6 57%; N ¼ 5; p , 0.05) but not for wild-type CFTR (30 6 42%; N ¼ 5).
X
ABCC7 p.Leu1254Ala 18805924:185:66
status: NEW188 Although L1254A is not a mutation that causes disease, it does reduce CFTR function.
X
ABCC7 p.Leu1254Ala 18805924:188:9
status: NEW[hide] ATP and AMP mutually influence their interaction w... J Biol Chem. 2013 Sep 20;288(38):27692-701. doi: 10.1074/jbc.M113.479675. Epub 2013 Aug 6. Randak CO, Dong Q, Ver Heul AR, Elcock AH, Welsh MJ
ATP and AMP mutually influence their interaction with the ATP-binding cassette (ABC) adenylate kinase cystic fibrosis transmembrane conductance regulator (CFTR) at separate binding sites.
J Biol Chem. 2013 Sep 20;288(38):27692-701. doi: 10.1074/jbc.M113.479675. Epub 2013 Aug 6., [PMID:23921386]
Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP &lrarr2; 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, also have adenylate kinase activity. All three ABC adenylate kinases bind and hydrolyze ATP in the absence of other nucleotides. However, little is known about how an ABC adenylate kinase interacts with ATP and AMP when both are present. Based on data from non-ABC adenylate kinases, we hypothesized that ATP and AMP mutually influence their interaction with CFTR at separate binding sites. We further hypothesized that only one of the two CFTR ATP-binding sites is involved in the adenylate kinase reaction. We found that 8-azidoadenosine 5'-triphosphate (8-N3-ATP) and 8-azidoadenosine 5'-monophosphate (8-N3-AMP) photolabeled separate sites in CFTR. Labeling of the AMP-binding site with 8-N3-AMP required the presence of ATP. Conversely, AMP enhanced photolabeling with 8-N3-ATP at ATP-binding site 2. The adenylate kinase active center probe P(1),P(5)-di(adenosine-5') pentaphosphate interacted simultaneously with an AMP-binding site and ATP-binding site 2. These results show that ATP and AMP interact with separate binding sites but mutually influence their interaction with the ABC adenylate kinase CFTR. They further indicate that the active center of the adenylate kinase comprises ATP-binding site 2.
Comments [show]
None has been submitted yet.
No. Sentence Comment
329 An example is L1254A CFTR, which showed an increase in current after adding Ap5A due to a reduced channel closing rate.
X
ABCC7 p.Leu1254Ala 23921386:329:14
status: NEW