ABCMdb

PMID: 18805924

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., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Leu1254Ala In this study, we report that Ap5A increased activity of CFTR with an L1254A mutation. Login to comment 75 ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala Surprisingly, the L1254A mutation reversed the effect of Ap5A, so that Ap5A stimulated CFTR current. Login to comment 85 ABCC7 p.Leu1254Ala When added alone to phosphorylated wild-type CFTR (18) or CFTR-L1254A (Fig. 4), Ap5A did not generate activity. Login to comment 86 ABCC7 p.Leu1254Ala ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala In addition, the activity of CFTR-L1254A was not completely saturated at 10 mM ATP. Login to comment 90 ABCC7 p.Leu1254Ala These results suggest that the L1254A mutation may have impaired the interaction with ATP. Login to comment 98 ABCC7 p.Leu1254Ala ABCC7 p.Leu1254Ala (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 ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala These results suggest that the L1254A mutation did not completely disrupt the AMP-binding site in CFTR. Login to comment 113 ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala ABCC7 p.Leu1254Ala ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala These results suggest that the L1254A mutation did not completely disrupt the area around ATP-binding Site 2. Login to comment 119 ABCC7 p.Leu1254Ala ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala Adding Ap5A alone yielded a failure to activate CFTR-L1254A in five other experiments. Login to comment 127 ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala stabilized the open state and predicted that Ap5A would increase the burst duration of CFTR-L1254A. Login to comment 135 ABCC7 p.Leu1254Ala In excised, inside-out patches of membrane, CFTR-L1254A showed reduced channel activity compared to wild-type CFTR. Login to comment 137 ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala Several observations suggest that the AMP-binding site remained available in CFTR-L1254A. Login to comment 145 ABCC7 p.Leu1254Ala First, Ap5A altered CFTR-L1254A current. Login to comment 146 ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala ABCC7 p.Leu1254Ala ABCC7 p.Leu1254Ala ABCC7 p.Leu1254Ala ABCC7 p.Leu1254Ala ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala Second, AMP stimulated CFTR-L1254A current in the presence of ATP. Login to comment 155 ABCC7 p.Leu1254Ala Third, ADP inhibited CFTR-L1254A current. Login to comment 157 ABCC7 p.Leu1254Ala The data also indicate that the L1254A variant altered the interaction with ATP. Login to comment 160 ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala How did Ap5A stimulate CFTR-L1254A current? Login to comment 166 ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala 2), The L1254A mutation partially disrupts ATP binding at Site 2. Login to comment 171 ABCC7 p.Lys1250Ala ABCC7 p.Asp1370Asn For example, the K1250A and D1370N mutations also have a reduced opening rate, prolonged burst duration, and increased ATP EC50 (11,12,14,15,29,32,39,43). Login to comment 172 ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala Why would the L1254A mutation reduce the affinity for ATP but allow Ap5A to bind? Login to comment 177 ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala ABCC7 p.Leu1254Ala N ¼ 5 for both wild-type CFTR and CFTR-L1254A. Login to comment 185 ABCC7 p.Leu1254Ala 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 ABCC7 p.Leu1254Ala Although L1254A is not a mutation that causes disease, it does reduce CFTR function. Login to comment