ABCMdb

PMID: 17178710

Wang W, Bernard K, Li G, Kirk KL
Curcumin opens cystic fibrosis transmembrane conductance regulator channels by a novel mechanism that requires neither ATP binding nor dimerization of the nucleotide-binding domains.
J Biol Chem. 2007 Feb 16;282(7):4533-44. Epub 2006 Dec 18., 2007-02-16 [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCC7 p.Gly551Asp ABCC7 p.Trp1282* Consequently, this compound potently activated CF mutant channels that are defective for the normal ATP-dependent mode of gating (e.g. G551D and W1282X), including channels that lack NBD2. Login to comment 44 ABCC7 p.Gly551Asp ABCC7 p.Trp1282* CFTR constructs that are robustly activated by curcumin include two of the more common CF mutants, G551D (mutation in NBD1 (21)) and W1282X (nonsense mutation deleting most of NBD2 (22)). Login to comment 50 ABCC7 p.Ser660Ala ABCC7 p.Ser660Ala The ⌬R/S660A-CFTR mutant was provided by M. Welsh (University of Iowa) (23), subcloned into the pCDNA3 vector, and truncated by PCR mutagenesis to make ⌬1198/⌬R/S660A-CFTR. Login to comment 53 ABCC7 p.Trp1282* HeLa cells stably transfected with W1282X-CFTR were provided by J. P. Clancy at the University of Alabama at Birmingham. Login to comment 80 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp RESULTS ATP-independent Activation of G551D-CFTR and Wild Type CFTR Channels by Curcumin-In a previous study we observed that the G551D regulation mutant is resistant to activation by a class of CFTR agonist that strongly activates ⌬F508-CFTR and wild type channels (5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) analogs; Ref. 19). Login to comment 81 ABCC7 p.Gly551Asp G551D-CFTR channels normally exhibit very low single channel open probabilities (Po) because this mutation disrupts the ABC signature sequence in NBD1, which lines the ATP-binding pocket at the NBD dimer interface in other ABC transporters (9-11). Login to comment 82 ABCC7 p.Gly551Asp Fig. 1A shows that G551D-CFTR channels are strongly stimulated by curcumin. Login to comment 86 ABCC7 p.Gly551Asp As noted previously, the neutral NPPB derivative (NPPB-AM) only weakly activates G551D-CFTR channels at a dose that maximally activates the wild type channel (10 ␮M; Ref. 19). Login to comment 87 ABCC7 p.Gly551Asp Conversely, curcumin exhibited a strong and dose-dependent activation of the G551D-CFTR currents (see also single channel data in Fig. 3). Login to comment 91 ABCC7 p.Gly551Asp Because the G551D-CFTR mutant is strongly disrupted for the normal ATP-dependent mode of channel gating, we tested whether curcumin also could activate this mutant in the absence of bath ATP. Login to comment 92 ABCC7 p.Gly551Asp Fig. 1B shows that curcumin strongly stimulates G551D-CFTR currents after removing bath ATP and by adding hexokinase/glucose to enzymatically eliminate residual ATP. Login to comment 97 ABCC7 p.Gly551Asp Thus, both wild type channels and G551D-CFTR channels can be activated by curcumin under these conditions. Login to comment 105 ABCC7 p.Gly551Asp Curcumin Activates CFTR Channels That Lack NBD2-The finding that curcumin promotes the opening of G551D-CFTR channels and wild type channels in the absence of ATP raised the possibility that this compound acts at a step downstream of ATP binding and possibly NBD dimerization. Login to comment 107 ABCC7 p.Gly551Asp To our initial surprise we found that CFTR channels that lack the entire NBD2 as well as the COOH-terminal tail (⌬1198-CFTR) express at high levels in HEK-293T cells where they localize to the cell surface as efficiently as wild type or G551D-CFTR channels (see schematic of constructs in Fig. 2A and surface biotinylation results in Fig. 2B). Login to comment 109 ABCC7 p.Gly551Asp Like for G551D-CFTR channels, however, the currents mediated by ⌬1198-CFTR are markedly stimulated by curcumin (Fig. 2C). Login to comment 114 ABCC7 p.Trp1282* The activation of ⌬1198-CFTR channels by curcumin did not require bath ATP; in fact, ATP substantially reduced the curcumin response for this construct (see Fig. 4 and W1282X-CFTR below). Login to comment 117 ABCC7 p.Gly551Asp This feature was not unique to the activation of ⌬1198-CFTR currents, i.e. persistently activated currents were observed for every CFTR construct that was tested including G551D and wild type CFTR (e.g. Fig. 1A). Login to comment 124 ABCC7 p.Gly551Asp A, curcumin strongly stimulates G551D-CFTR in a dose-dependent fashion. Login to comment 128 ABCC7 p.Gly551Asp B, curcumin also activates G551D-CFTR channels in the absence of ATP. Login to comment 143 ABCC7 p.Lys95Glu D, curcumin also stimulates K95E/⌬1198-CFTR currents, which showstrongoutwardrectification.Inset,I-Vcurvesforcontrol(curve1),afteraddingcurcumin(curve2),afterre-addingcurcuminwithnoATP(curve3),andafter curcuminwashout(curve4).E,curcumintitrationfor⌬1198-CFTRchannelsintheabsenceofbathATP.Inset,meantitrationdatafittoasingleMichaelis-Menten function(EC50 ϭ20.8Ϯ5.4␮M,nϭ4).F,curcuminalsoactivatesW1282X-CFTRchannels.Alloftherecordsarerepresentativeofatleastfourexperimentsexcept D (n ϭ 2). Login to comment 144 ABCC7 p.Lys95Glu ATP-independent CFTR Channel Gating Because the ⌬1198-CFTR construct is new to the CFTR field, we validated that the currents that are mediated by this construct are authentic CFTR currents in two ways: (i) by performing unitary currents recordings, which confirmed that this NBD2 deletion construct exhibits the appropriate single channel conductance (6-8 pS) and linear I-V behavior expected for CFTR channels (Fig. 3) (5, 6) and (ii) by introducing a point mutation (K95E) in this deletion construct that had been shown by Linsdell (30) to induce strong outward rectification of the currents mediated by the full-length channel. Login to comment 145 ABCC7 p.Lys95Glu Fig. 2D shows that the currents mediated by K95E/⌬1198-CFTR exhibit strong outward rectification, as expected, and that these rectifying currents are robustly activated by curcumin. Login to comment 146 ABCC7 p.Trp1282* We next determined whether W1282X-CFTR channels also can be activated by curcumin. Login to comment 147 ABCC7 p.Trp1282* W1282X is a fairly common nonsense mutation in certain CF populations including Ashkenazi Jews, for whom it is the most frequent CF mutation (22). Login to comment 149 ABCC7 p.Trp1282* W1282X-CFTR channels are targeted to the cell surface, albeit less efficiently than ⌬1198-CFTR channels (see biotinylation results in Fig. 2B). Login to comment 150 ABCC7 p.Trp1282* Like ⌬1198-CFTR channels, W1282X-CFTR channels exhibit low (but detectable activity) in excised patches either in the absence or presence of bath ATP (Fig. 2F). Login to comment 151 ABCC7 p.Trp1282* W1282X-CFTR channels were also robustly stimulated by curcumin under either condition (Fig. 2F), although the effect of curcumin is more pronounced in the absence of bath ATP (see also Fig. 4). Login to comment 152 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Trp1282* Gating Properties of Curcumin-activated Channels; Curcumin Increases the Opening Rates of G551D-CFTR Channels and the NBD2 Deletion Mutants-To explore how curcumin influences the gating properties of these mutant constructs, we tested its effects on G551D-CFTR, ⌬1198-CFTR, and W1282X-CFTR channels in excised micropatches containing small numbers of channels (less than eight). Login to comment 156 ABCC7 p.Gly551Asp ABCC7 p.Trp1282* Individual records for G551D-CFTR, ⌬1198-CFTR, and W1282X-CFTR channels are shown in Fig. 3 (A, B, and D). Login to comment 157 ABCC7 p.Gly551Asp Mean data for the stimulation of ⌬1198-CFTR channels by 5 ␮M curcumin are summarized in Fig. 3C. Not surprisingly, the most obvious effect of curcumin was to increase the opening rates of G551D-CFTR and the NBD2 deletion constructs, which otherwise open at extremely low rates. Login to comment 165 ABCC7 p.Trp1282* Curcumin Activation of ⌬1198-CFTR Channels Is Inhibited by ATP Binding to NBD1-Fig. 4 shows another striking feature of the effect of curcumin on the NBD2 deletion constructs (both ⌬1198-CFTR and W1282X-CFTR), namely strong inhibition of this activating effect by bath ATP. Login to comment 174 ABCC7 p.Ala462Phe Fig. 4 (D and E) shows that the inhibitory effect of bath ATP on the curcumin response was eliminated by introducing a mutation in the Walker A sequence (A462F) in NBD1. Login to comment 176 ABCC7 p.Ala462Phe The lack of effect of ATP on the curcumin activation of the A462F/⌬1198-CFTR construct FIGURE 3. Login to comment 179 ABCC7 p.Gly551Asp A, G551D-CFTR. Login to comment 183 ABCC7 p.Trp1282* D, W1282X-CFTR;-60mV.E,representativepatchshowingdynamicsofopeningandclosingof⌬1198-CFTRchannelsduringthereversibleandpersistentphases of curcumin activation. Login to comment 187 ABCC7 p.Gly551Asp It should be noted that ATP did not obviously blunt the curcumin activation of channels that possess NBD2 (e.g. G551D-CFTR and wild type channels; results not shown). Login to comment 195 ABCC7 p.Ala462Phe D, bath ATP does not inhibit A462F/⌬1198-CFTR channels. Login to comment 196 ABCC7 p.Ala462Phe E, mean data comparing the effects of bath ATP (1.5 mM) on ⌬1198-CFTR (n ϭ 16) and A462F/⌬1198-CFTR (n ϭ 10) activation by 30 ␮M curcumin. Login to comment 198 ABCC7 p.Ala462Phe ABCC7 p.Ala462Phe Note that the absolute currents mediated by A462F/⌬1198-CFTR are lower because the A462F mutation partially disrupts ER processing and cell surface localization (33).3 All of the records are representative of at least three experiments. Login to comment 204 ABCC7 p.Ser660Ala ABCC7 p.Ser660Ala D, curcumin activates ⌬1198/⌬R-S660A-CFTR channels in the absence of ATP and PKA. Login to comment 208 ABCC7 p.Gly551Asp ABCC7 p.Trp1282* A similar PKA dependence of the curcumin effect was observed for G551D-CFTR, W1282X-CFTR, and wild type channels (results not shown). Login to comment 211 ABCC7 p.Ser660Ala ABCC7 p.Ser660Ala We also tested the effect of curcumin on channels that lack a large portion of the R domain in addition to NBD2 (⌬1198/⌬R/S660A), because previous work has shown that ⌬R/S660A-CFTR channels exhibit constitutive activity in the absence of added kinase (23). Login to comment 214 ABCC7 p.Gly551Asp ABCC7 p.Trp1282* DISCUSSION Our data indicate that curcumin strongly activates mutant CFTR channels that normally have very low activities because of defects in ATP binding and/or NBD heterodimerization (e.g. G551D-CFTR and W1282X-CFTR channels). Login to comment 216 ABCC7 p.Trp1282* That the stimulatory effect of curcumin does not require heterodimerization of the two NBDs is best exemplified by its potent activation of channels that lack all (⌬1198-CFTR) or part (W1282X-CFTR) of NBD2. Login to comment 222 ABCC7 p.Gly551Asp Curcumin Activation Is Phosphorylation-dependent-Curcumin activates the NBD2 deletion constructs or G551D-CFTR channels in the absence of active PKA in the bath or following deletion of most of the R domain. Login to comment 244 ABCC7 p.Trp1282* NBD1 Is a Plausible Interaction Site for Curcumin-ATP strongly inhibits the activation of channels that lack NBD2 (⌬1198-CFTR and W1282X-CFTR) by curcumin. Login to comment 246 ABCC7 p.Ala462Phe Introducing a mutation in the Walker A motif that was shown previously to disrupt ATP binding to NBD1 (A462F (33)) severely blunted the inhibitory effect of ATP on ⌬1198-CFTR channel activation by curcumin. Login to comment 253 ABCC7 p.Gly551Asp ABCC7 p.Trp1282* Tight ATP binding to NBD1 is less likely for mutants that are disrupted for ATP binding and/or the dimerization of the two NBDs (e.g. G551D, W1282X, and ⌬1198-CFTR), given that dimerization would be expected to stabilize ATP binding to NBD1 (9-11). Login to comment 256 ABCC7 p.Gly551Asp Interestingly, the activation of G551D-CFTR channels by curcumin is not blunted by ATP (results not shown). Login to comment 257 ABCC7 p.Gly551Asp Presumably this difference is due to the presence of the second NBD in the G551D mutant that also modulates channel gating and that may inhibit the curcumin response in the absence of ATP (e.g. by reducing curcumin access to its binding site). Login to comment 266 ABCC7 p.Gly551Asp If so, then curcumin (or more potent analogs) might have value for treating CF patients with mutations that primarily disrupt the normal ATP-dependent mode of channel regulation (e.g. G551D). Login to comment 267 ABCC7 p.Trp1282* ABCC7 p.Ser660Ala Acknowledgments-We thank J. P. Clancy for the HeLa cells stably transfected with W1282X-CFTR and M. J. Welsh for the ⌬R/S660A-CFTR construct. Login to comment