ABCMdb

ABCC7 p.Leu1254Ala

None has been submitted yet.

Publications

PMID: 18805924 [PubMed] Dong Q et al: "A mutation in CFTR modifies the effects of the adenylate kinase inhibitor Ap5A on channel gating."

No. Sentence Comment

4 In this study, we report that Ap5A increased activity of CFTR with an L1254A mutation. Login to comment
75 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. Login to comment
84 Surprisingly, the L1254A mutation reversed the effect of Ap5A, so that Ap5A stimulated CFTR current. Login to comment
85 When added alone to phosphorylated wild-type CFTR (18) or CFTR-L1254A (Fig. 4), Ap5A did not generate activity. Login to comment
86 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). Login to comment
87 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. Login to comment
88 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). Login to comment
89 In addition, the activity of CFTR-L1254A was not completely saturated at 10 mM ATP. Login to comment
90 These results suggest that the L1254A mutation may have impaired the interaction with ATP. Login to comment
98 (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. Login to comment
105 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). Login to comment
107 Thus, Ap5A reduced the Hill coefficient to ,1 in wild-type and L1254A CFTR suggesting conformational changes associated with negative cooperativity. Login to comment
108 CFTR-L1254A interacts with AMP and other nucleotides We tested several other agents to learn how the L1254A mutation affected function. Login to comment
111 We found that, as with wild-type channels, AMP stimulated and ADP inhibited current from the L1254A mutant (Fig. 6, A and B). Login to comment
112 These results suggest that the L1254A mutation did not completely disrupt the AMP-binding site in CFTR. Login to comment
113 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). Login to comment
117 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. Login to comment
118 These results suggest that the L1254A mutation did not completely disrupt the area around ATP-binding Site 2. Login to comment
119 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. Login to comment
123 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). Login to comment
124 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. Login to comment
126 Adding Ap5A alone yielded a failure to activate CFTR-L1254A in five other experiments. Login to comment
127 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. Login to comment
131 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. Login to comment
132 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. Login to comment
134 stabilized the open state and predicted that Ap5A would increase the burst duration of CFTR-L1254A. Login to comment
135 In excised, inside-out patches of membrane, CFTR-L1254A showed reduced channel activity compared to wild-type CFTR. Login to comment
137 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. Login to comment
139 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 . Login to comment
143 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). Login to comment
144 Several observations suggest that the AMP-binding site remained available in CFTR-L1254A. Login to comment
145 First, Ap5A altered CFTR-L1254A current. Login to comment
146 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. Login to comment
149 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. Login to comment
153 Second, AMP stimulated CFTR-L1254A current in the presence of ATP. Login to comment
155 Third, ADP inhibited CFTR-L1254A current. Login to comment
157 The data also indicate that the L1254A variant altered the interaction with ATP. Login to comment
160 Studies of NBD crystal structures from related ABC proteins are also consistent with the conclusion that the L1254A mutation altered the interaction with ATP. Login to comment
164 Our finding that CFTR-L1254A had a prolonged burst duration is consistent with a reduced enzymatic activity associated with mutating those residues. Login to comment
165 How did Ap5A stimulate CFTR-L1254A current? Login to comment
166 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. Login to comment
168 2), The L1254A mutation partially disrupts ATP binding at Site 2. Login to comment
172 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). Login to comment
175 Why would the L1254A mutation reduce the affinity for ATP but allow Ap5A to bind? Login to comment
177 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. Login to comment
178 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. Login to comment
183 N ¼ 5 for both wild-type CFTR and CFTR-L1254A. Login to comment
185 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). Login to comment
188 Although L1254A is not a mutation that causes disease, it does reduce CFTR function. Login to comment

PMID: 23921386 [PubMed] Randak CO et al: "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."

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. Login to comment