ABCMdb

PMID: 23620589

Okeyo G, Wang W, Wei S, Kirk KL
Converting nonhydrolyzable nucleotides to strong cystic fibrosis transmembrane conductance regulator (CFTR) agonists by gain of function (GOF) mutations.
J Biol Chem. 2013 Jun 14;288(24):17122-33. doi: 10.1074/jbc.M112.442582. Epub 2013 Apr 25., [PubMed]

Sentences

No. Mutations Sentence Comment

9 ABCC7 p.Gly551Asp AMP-PNP or ATPॹS activation required both nucleotide binding domains (NBDs) and was disrupted by a cystic fibrosis mutation in NBD1 (G551D). Login to comment 13 ABCC7 p.Gly1349Asp A GOF mutation substantially rescued defective ATP-dependent gating of G1349D-CFTR, a cystic fibrosis NBD2 signature sequence mutant. Login to comment 14 ABCC7 p.Gly1349Asp Interestingly, the G1349D mutation strongly disrupted activation by AMP-PNP but not by ATPॹS, indicating that these analogs interact differently with the NBDs. Login to comment 81 ABCC7 p.Lys978Cys RESULTS A GOF Mutation Increases CFTR Activation by Poorly Hydrolyzable Nucleotides-Fig. 1 shows the strong activation of a previously characterized GOF mutant (K978C-CFTR) by AMP-PNP, ATPॹS, and GTPॹS in excised inside-out macropatches. Login to comment 83 ABCC7 p.Lys978Cys Previously we showed that the K978C substitution increased the single channel open probabilities (Po) of phosphorylated channels in the absence of nucleotide by at least 2 orders of magnitude (13). Login to comment 84 ABCC7 p.Lys978Cys This ligand-independent activity of K978C-CFTR is detected in excised macropatch recordings as a small current that persists after removal of bath ATP by a scavenger (hexokinase/glucose) and subsequent bath perfusion with ATP-free solution (Fig. 1A). Login to comment 87 ABCC7 p.Lys978Cys In contrast, these analogs strongly increased the currents mediated by K978C-CFTR, although not to the level of saturating ATP (Fig. 1, A, C, and F and supplemental Fig. S2). Login to comment 94 ABCC7 p.Lys978Cys Fig. 2 shows activation of the K978C GOF mutant by a saturating dose of ATPॹS (1.5 mM) at the single channel level. Login to comment 99 ABCC7 p.Lys978Cys ATPॹS-activated K978C-CFTR channels opened and closed dynamically (i.e. were not "locked open") with a broad distribution of open durations that ranged from b0d;100 ms to occasional openings that lasted 10-20 s (Fig. 2B). Login to comment 104 ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys ABCC7 p.Lys978Pro Accordingly, we compared the activation by AMP-PNP of several previously described GOF CFTR mutants with increasing degrees of unliganded activity (K978P, K978C, and K190C/K978C; see Fig. 3). Login to comment 105 ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys ABCC7 p.Lys978Pro All three GOF mutants were more strongly activated by AMP-PNP than WT-CFTR, with the lowest and greatest relative activation observed for the weakest (K978P; Fig. 3, A, C, and D) and strongest (K190C/K978C; Fig. 3, B, C, and D) GOF mutant. Login to comment 110 ABCC7 p.Lys978Cys In contrast, the K978C-CFTR currents that were activated by ATPॹS or AMP-PNP deactivated very slowly upon the removal of these agonists (Fig. 4, A-D). Login to comment 112 ABCC7 p.Lys978Cys Poorly hydrolyzable nucleotides strongly activate the K978C-CFTR GOF mutant. Login to comment 113 ABCC7 p.Lys978Cys A and B, representative excised inside-out macropatch records compare activation of K978C-CFTR (A) and WT-CFTR (B) by the indicated concentrations of AMP-PNP. Login to comment 118 ABCC7 p.Lys978Cys Hexokinase/glucose was re-added after activation of K978C-CFTR by AMP-PNP followed by the addition of the CFTR-specific inhibitor, CFTRinh-172 (10 òe;M). Login to comment 119 ABCC7 p.Lys978Cys The lower macroscopic control current for K978C-CFTR (PKA plus ATP) in panel A is due to somewhat lower expression of this GOF mutant compared with WT-CFTR and not to lower channelactivity.Infact,thismutanthadasubstantiallygreatersinglechannelPo thanWT-CFTRundercontrolconditionsandafterATPremoval(Ref.13andFig. Login to comment 121 ABCC7 p.Lys978Cys C and D, representative excised inside-out macropatch records compare activation of K978C-CFTR (C) and WT-CFTR (D) by ATPॹS. Login to comment 128 ABCC7 p.Lys978Cys Wild type channels also deactivated fairly slowly after ATPॹS removal but considerably faster than K978C-CFTR (Fig. 4, C and D). Login to comment 130 ABCC7 p.Lys978Cys In this regard, we also observed that ATP-activated K978C-CFTR channels deactivated much slower than WT-CFTR when ATP was removed either by adding the hexokinase/glucose scavenger to the bath (13) or by bath perfusion (Fig. 4F). Login to comment 131 ABCC7 p.Lys978Cys The deactivation time courses for ATPॹS-activated K978C-CFTR channels (b0e;100s) were considerably longer than the mean open times observed in the single channel experiments in Fig. 2 (b0d;5s). Login to comment 132 ABCC7 p.Lys978Cys This disparity argues that K978C channels open and close dynamically even when ATPॹS is tightly bound. Login to comment 133 ABCC7 p.Lys978Cys To explore this point further, we tracked the gating of K978C and wild type channels after ATPॹS removal in patches containing sufficiently few channels to permit detection of unitary events. Login to comment 134 ABCC7 p.Lys978Cys K978C-CFTR channels continued to open and close up to several min after ATPॹS removal (Fig. 4E). Login to comment 136 ABCC7 p.Lys978Cys The slow deactivation observed especially for K978C-CFTR channels implies that ATPॹS and AMP-PNP bind tightly, presumably at the NBD dimer interface (see below). Login to comment 140 ABCC7 p.Lys978Cys Previously we reported that truncated channels that lack NBD2 but possess a GOF mutation (K978C/èc;1198-CFTR) exhibit detectable FIGURE 2. Login to comment 141 ABCC7 p.Lys978Cys ATPॹS markedly increases the single channel open probability of K978C-CFTR. Login to comment 142 ABCC7 p.Lys978Cys A, shown is a single channel record for excised inside-out patch containing one K978C-CFTR channel under control conditions (1.5 mM MgATP), after ATP removal with hexokinase/glucose, and after the addition of 1.5 mM ATPॹS.Channelswereprephosphorylatedwith110units/mlPKAfollowedbyPKIaddition.Holdingpotentialwasafa;60mV.Openingsaredownward.Measured Po values for each condition in this record are indicated. Login to comment 145 ABCC7 p.Lys978Cys C, mean data (afe;S.E.; n afd; 3 patches) show that ATPॹS greatly increased the Po and single channel opening rate of K978C-CFTR. Login to comment 151 ABCC7 p.Lys978Cys It should be noted that these truncated channels are not maximally active under the conditions of this experiment; i.e. previously we observed that K978C/èc;1198-CFTR channels are stimulated substantially by a compound that activates CFTR currents by an unknown mechanism (curcumin; Refs. 13 and 24; see also Fig. 5, E and F). Login to comment 155 ABCC7 p.Lys978Pro Fig. 5, C and D, show that the AMP-PNP-activated currents mediated by K978P-CFTR were strongly but slowly inhibited by diamide/GSH (inactivation time constant b0e;1 min). Login to comment 157 ABCC7 p.Lys978Pro K978P-CFTR channels lacking Cys-1344 were insensitive to diamide/GSH (Fig. 5D, supplemental Fig. S2), as we observed previously for channels without the GOF mutation (27). Login to comment 158 ABCC7 p.Lys978Pro The slow rate of K978P-CFTR inactivation by diamide/GSH is consistent with the idea that AMP-PNP binds tightly at the NBD dimer interface and restricts access of the reactive glutathione to Cys-1344 (27). Login to comment 160 ABCC7 p.Gly551Asp The most common CF gating mutation (G551D) abolishes ATP-dependent channel opening by disrupting the signature sequence in NBD1 (12). Login to comment 162 ABCC7 p.Gly551Asp ABCC7 p.Lys978Cys ABCC7 p.Lys978Ser In an earlier study we found that GOF mutations at residue 978 (K978C or K978S) promoted a substantial ATP-independent activity for the G551D mutant that could be augmented further by curcumin (13). Login to comment 163 ABCC7 p.Gly551Asp ABCC7 p.Lys978Cys Here we observed that K978C/G551D-CFTR channels could not be activated by ATPॹs (Fig. 5E) and instead were slightly but reproducibly inhibited by this nucleotide. Login to comment 165 ABCC7 p.Gly551Asp The G551D mutation also inhibited activation by AMP-PNP, although for this nucleotide a very small but reproducible stimulation was detected (Fig. 5F). Login to comment 166 ABCC7 p.Gly1349Asp The analogous mutation in the NBD2 signature sequence (G1349D) causes milder CF disease apparently because it incompletely disrupts channel gating. Login to comment 167 ABCC7 p.Gly1349Asp G1349D-CFTR channels retain ATP-dependent activity but with a maximal single chan- FIGURE 3. Login to comment 169 ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys ABCC7 p.Lys978Pro A and B, representative macropatch records show AMP-PNP activation of K978P-CFTR (A) and K190C/K978C-CFTR (B). Login to comment 171 ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys Note the previously described voltage-dependent rectification of K190C/K978C-CFTR currents (13). Login to comment 174 ABCC7 p.Lys978Cys WT and K978C data are from Fig. 1F. Login to comment 180 ABCC7 p.Gly1349Asp As reported by others (12), we detected relatively small ATP-dependent control currents for G1349D-CFTR channels in excised macropatches. Login to comment 182 ABCC7 p.Gly1349Asp ABCC7 p.Lys978Cys Introduction of one of the GOF mutations into this CF mutant (K978C/G1349D-CFTR) substantially rescued its activity as evidenced by much higher ATP-dependent control currents and much lower relative activation by the potentiators (Fig. 6, B and C). Login to comment 183 ABCC7 p.Gly1349Asp ABCC7 p.Lys978Cys K978C/G1349D-CFTR channels also exhibited detectable currents in the absence of any bath nucleotide as expected (Fig. 6D). Login to comment 184 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys Unlike K978C/G551D-CFTR channels, K978C/G1349D-CFTR channels remained sensitive to ATPॹS as did the G1349D mutant without the GOF substitution (Fig. 6, D-F). Login to comment 186 ABCC7 p.Gly1349Asp The G1349D results support two conFIGURE 4. Login to comment 188 ABCC7 p.Lys978Cys A, macropatch record shows slow deactivation of K978C-CFTR current after AMP-PNP washout. Login to comment 191 ABCC7 p.Lys978Cys B, shown is slow deactivation of K978C-CFTR current after ATPॹS washout. Login to comment 192 ABCC7 p.Lys978Cys C, WT-CFTR currents also deactivate fairly slowly after ATPॹS washout but faster than K978C-CFTR currents. Login to comment 194 ABCC7 p.Lys978Cys D, mean deactivation time constants (afe;S.E.) were estimated from single exponential fits of currents after ATPॹS washout for WT-CFTR and K978C-CFTR (n afd; 6 each). Login to comment 196 ABCC7 p.Lys978Cys By comparison, the mean deactivation time course for ATP-activated K978C-CFTR channels after washout of 1.5 mM ATP using the samerampprotocolwas51.0afe;3.3s(nafd;7).ThedeactivationtimecourseforATP-activatedwildtypechannelsafterATPremovalwastoorapidtoresolveusing this ramp protocol (i.e. WT-CFTR channels deactivate faster than the 10-s ramp period after ATP removal). Login to comment 197 ABCC7 p.Lys978Cys E, micropatch experiments at a constant holding potential (afa;60 mV) show that ATPॹS-activated K978C-CFTR channels (top) and WT-CFTR channels (bottom) continue to open and close many seconds after removingATPॹSbybathperfusion(initiatedatthearrow).TheWT-CFTRpatchwasobtainedusingalargertippipettetooptimizedetectionofATPॹS-activated channels. Login to comment 199 ABCC7 p.Lys978Cys Time to full deactivation for K978C-CFTR channels after ATPॹS removal in these micropatch experiments ranged from 19 to 246 s. Login to comment 200 ABCC7 p.Lys978Cys F, control micropatch experiments (also at afa;60 mV) show more rapid deactivation of WT-CFTR channels and K978C-CFTRchannelsafterremovalofATPbybathperfusion.Thesepatcheswereobtainedusingsmallertippipettestodetectunitarycurrentsinthepresence of 3 mM ATP. Login to comment 201 ABCC7 p.Lys978Cys The slower deactivation of K978C-CFTR channels after ATP removal is consistent with the slower macroscopic deactivation time course reported previously (Ref. 13; see also "Discussion"). Login to comment 203 ABCC7 p.Gly1349Asp First, the defective ATP-dependent gating of G1349D-CFTR channels can be rescued substantially by a GOF mutation located in the cytosolic loops well away from the NBDs. Login to comment 205 ABCC7 p.Gly1349Asp The P-N-P linkage in the latter may reduce its ability to accommodate to distortions in the nucleotide binding pockets and dimer interface that are induced by the G1349D mutation. Login to comment 210 ABCC7 p.Lys978Cys Fourth, poorly hydrolyzable nucleotides are weaker CFTR activators than ATP even when they apparently bind tightly to the NBDs, as was the case for the K978C GOF mutant. Login to comment 217 ABCC7 p.Lys978Cys Perturbation of the NBD dimer interface inhibits K978C-CFTR activation by AMP-PNP and ATPॹS. Login to comment 219 ABCC7 p.Lys978Cys B, no activation of the K978C/èc;1198 NBD2 deletion mutant by AMP-PNP added to final concentrations (in mM) of 0.1, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, and 3.0 is shown. Note that the control current mediated by this construct is entirely ATP-independent (e.g. no effect of hexokinase(Hex)/glucose).C,diamide(Dia)/glutathione(20òe;M each)slowlyinhibitstheK978P-CFTRcurrentactivatedby2mM AMP-PNP.Subsequentaddition of 5 mM DTT reversed this inhibition. Login to comment 220 ABCC7 p.Cys1344Ala ABCC7 p.Lys978Pro ABCC7 p.Lys978Pro D, mean inhibition by diamide/glutathione of the AMP-PNP activated currents for K978P-CFTR (n afd; 5) and K978P/C1344A- CFTR (n afd; 3) is shown. Login to comment 222 ABCC7 p.Gly551Asp ABCC7 p.Lys978Cys E, no activation of K978C/G551D-CFTR by 1.5 mM ATPॹS is shown. Note subsequent strong activation by 30 òe;M curcumin. Login to comment 224 ABCC7 p.Gly551Asp ABCC7 p.Lys978Cys F, very small activation of K978C/G551D-CFTR by 2 mM AMP-PNP is shown. Login to comment 226 ABCC7 p.Lys978Cys were activated by these analogs precluded performing quantitative titrations like we performed for the K978C GOF mutant. Login to comment 228 ABCC7 p.Lys978Cys Our results also indicate that, at least for the K978C GOF mutant, these analogs are less effective than ATP at promoting channel opening at saturating concentrations when they apparently bind tightly at the NBD dimer interface. Login to comment 231 ABCC7 p.Lys978Cys In agreement with this, ATPॹS-activated K978C-CFTR channels opened and closed on a much faster time scale (i.e. they were not locked open) than they deactivated upon ligand removal. Login to comment 233 ABCC7 p.Lys978Cys K978C channels also were observed to open and close several minutes after removing the ATPॹS (Fig. 4), which supports the view that the opening and closing of ATPॹS-activated channels is not tightly coupled to ATPॹS binding and unbinding (although interpreting these nonsteady-state single channel experiments is complicated by the fact that we cannot distinguish di-liganded channels from mono-liganded channels that have unbound one ligand molecule; see also below). Login to comment 236 ABCC7 p.Gly1349Asp ABCC7 p.Lys978Cys Rescue of G1349D-CFTR gating by K978C and differential activation of this NBD2 mutant by ATPॹS and AMP-PNP. Login to comment 237 ABCC7 p.Gly1349Asp A, a representative macropatch record shows low control current for G1349D-CFTR (1.5 mM ATP) and large activation by serial addition of 10 òe;M 5-nitro-2-(3-phenylpropylamino) benzamide (NPPB-AM) and 30 òe;M curcumin. Login to comment 238 ABCC7 p.Gly1349Asp ABCC7 p.Lys978Cys B, a corresponding macropatch record for K978C/G1349D-CFTR shows large control current and relatively small activation by NPPB-AM and curcumin. Login to comment 239 ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Lys978Cys C, shown are mean control currents (1.5 mM ATP) and mean -fold activation by the combination of curcumin and NPPB-AM for G1349D-CFTR and K978C/G1349D-CFTR (n afd; 6-17; **, p b0d; 0.01 compared with G1349D). Login to comment 240 ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Lys978Cys D and E, representative macropatch records show much smaller activation of K978C/G1349D-CFTR or G1349D-CFTR current by 2 mM AMP-PNP versus 1.5 mM ATPॹS. Hex, hexokinase. Login to comment 241 ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp F, mean relative activation of G1349D-CFTRandK978C/G1349D-CFTRbyAMP-PNPandATPॹS.Percentstimulationwascalculatedbynormalizingtheincreaseincurrentabovebaseline(i.e. current in the absence of nucleotide) to the control current before ATP removal by hexokinase/glucose. Login to comment 259 ABCC7 p.Lys978Cys ABCC7 p.Lys978Ser ABCC7 p.Lys978Pro In this regard, the substitutions at position Lys-978 that had the strongest GOF effects (K978C, K978S, and K978P; Ref. 13) also are predicted to have the greatest disruptive effects on the presumed helical structure of cytosolic loop3 and TM9 based on secondary structure predictions (results not shown). Login to comment 265 ABCC7 p.Lys978Cys The slower deactivation (and presumably slower unbinding) after ATPॹS removal that we observed for the K978C GOF mutant relative to wild type CFTR (Fig. 4) is consistent with the latter mechanism. Login to comment 269 ABCC7 p.Gly551Asp Activation by either required both NBDs and was reduced (AMP-PNP) or eliminated (ATPॹS) by the G551D signature sequence mutation in NBD1. Login to comment 271 ABCC7 p.Lys978Cys In addition, ATPॹS was effective at much lower concentrations, and unlike the case for AMP-PNP, the ATPॹS titration curve for K978C-CFTR activation could be fit only by a two-binding site model with different affinities. Login to comment 282 ABCC7 p.Lys978Cys The notion that AMP-PNP might be functionally effective primarily at site 1 in the absence of ATP would be consistent with the stronger effect of the NBD2 signature sequence mutation (nearest site 1) on AMP-PNP activation of K978C-CFTR. Login to comment 285 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp GOF Mutations Rescue G551D and G1349D CF Channels but in Different Ways-The ABC signature sequences line the two ATP binding pockets and play important roles in ABC transporter function and CFTR channel gating. Login to comment 287 ABCC7 p.Gly551Asp The G551D mutation in the NBD1 signature sequence abolishes ATP-dependent CFTR gating and causes severe CF. Login to comment 288 ABCC7 p.Gly1349Asp The analogous mutation in NBD2 (G1349D) causes less severe disease because it incompletely inhibits ATP-dependent gating (12). Login to comment 289 ABCC7 p.Gly551Asp ABCC7 p.Lys978Cys Previously we showed that the activity of the severely defective G551D mutant was increased substantially by GOF mutations such as K978C (13). Login to comment 290 ABCC7 p.Gly551Asp But this elevated activity was due entirely to an increase in ATP-independent (unliganded) activity; we observed no rescue of ATP-dependent gating of the G551D mutant nor did we observe activation by ATPॹS here. Login to comment 291 ABCC7 p.Gly1349Asp ABCC7 p.Lys978Cys In contrast, we observed a more authentic rescue for the G1349D- K978C construct in the form of much greater ATP-dependent currents and lower relative activation by potentiators. Login to comment 292 ABCC7 p.Lys978Cys This construct also exhibited larger ATP-independent currents, as expected for channels with the K978C GOF mutation. Login to comment 293 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp This more authentic rescue of the G1349D channel presumably relates to the incomplete loss of ATP-dependent gating caused by this signature sequence mutation versus the complete abolition of ATP dependence by the corresponding G551D mutation (12). Login to comment 294 ABCC7 p.Gly1349Asp ABCC7 p.Lys978Cys G1349D-CFTR rescue by the K978C GOF mutation is analogous to its enhancement of CFTR activation by ATPॹS or AMP-PNP. Login to comment 297 ABCC7 p.Gly1349Asp In the case of G1349D-CFTR, the signature sequence mutation in NBD2 may compromise the efficacy of ATP to open the channel. Login to comment