ABCMdb

ABCC7 p.Lys978Cys

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Publications

PMID: 20133716 [PubMed] Wang W et al: "ATP-independent CFTR channel gating and allosteric modulation by phosphorylation."

No. Sentence Comment

47 Cysteine modification experiments indicated that K978C is accessible to thiol-reactive compounds [e.g., the positively charged methanethiosulfonate reagent (MTSET)], which reversibly in- hibitedtheATP-independentcurrentsmediatedbythismutant(Fig. S2). Login to comment
48 For micropatches that contained sufficiently few K978C channels to resolve unitary currents, we observed that (i) the unitary currents (i.e., single-channel conductances) exhibited by the K978C construct were similar to those for WT-CFTR (Figs. S2 and S3); (ii) theK978Cconstructgateddynamicallyintheabsenceorpresenceof bath ATP (opened and closed spontaneously, albeit with somewhat longer openings than are typically observed for WT-CFTR (Figs. S2 and S3); and (iii) the primary effect of MTSET was to inhibit single-channel open probability (Po) by inhibiting the channel opening rate (Fig. S2). Login to comment
49 Table 1 compares the Pos, equilibrium gating constants, and Gibbs free energy differences for channel opening (ΔGos) for WT-CFTR channels and K978C-CFTR channels measured in the presenceand absenceofATP(examplerecords provided inFig.S3). Login to comment
51 The K978C mutant also had a higher Po in the presence of saturating ATP as compared with WT-CFTR. Login to comment
58 (B) K978C-CFTR macroscopic current across inside-out membrane patch excised from HEK-293T cell [ramp protocol (±80 mV); Methods]. Login to comment
71 (F) Very large ATP-independent current for K190C/K978C-CFTR. Login to comment
75 Impact of K978C mutation on open probability (Po), equilibrium gating constant (Keq), and Gibbs free energy difference for channel opening (ΔG) in the presence and absence of 1.5 mM ATP +ATP -ATP Po Keq ΔG, kJ/mol Po Keq ΔG, kJ/mol WT 0.38 ± 0.01 0.61 ± 0.02 1.2 ± 0.1 2.7 × 10-4 2.7 × 10-4 21 ± 0.8 ±8 × 10-5 ±8 × 10-5 K978C 0.84 ± 0.03* 6.53 ± 1.57* -4.3 ± 0.6 0.09 ± 0.01* 0.10 ± 0.02* 5.8 ± 0.5* Keq = Po/(1 - Po) and ΔG = -RT ln Keq where R is gas constant and T is temperature. Login to comment
77 Numbers of patches analyzed: three (WT, +ATP), six (WT, -ATP), six (K978C, +ATP), and five (K978C, -ATP). Login to comment
96 This initial observation was followed up by performing ATP titrations for the K978C-CFTR constitutive mutant and for WT-CFTR, which showed a nearly 10-fold decrease in the EC50 for ATP activation of the constitutive mutant (Fig. 2B and Fig. S1C). Login to comment
97 This large increase in ATP sensitivity of the K978C mutant is consistent with the predicted reciprocity between channel opening and ligand occupancy. Login to comment
100 The latter mechanism would be consistent with the elevated Po of the K978C mutant at saturating ATP (Fig. S3 and Table 1) and the slower deactivation of this construct following ATP removal. Login to comment
104 We were clued that ADP inhibits the ATP-independent activity of the constitutive mutants (e.g., K190C/K978C-CFTR channels) by the finding that their currents were lowest when hexokinase/glucose was added to induce current deactivation in the presence of 1.5 mM ATP (instead of perfusing ATP from the bath before adding the enzyme). Login to comment
106 Fig. S1D shows that the currents mediated by the K190C/K978C constitutive mutant increased on subsequent bath perfusion to remove the enzyme and all nucleotides and were reversibly inhibited by about 50% by adding back ADP alone. Login to comment
108 The constitutive activity of the K190C/K978C mutant was not inhibited by ADP when these loop mutations were introduced into a deletion construct that lacks NBD2 (Fig. S7 and Fig. 3). Login to comment
113 Fig. 3 shows that the K978C and K978S mutations markedly increased the macroscopic currents mediated by G551D-CFTR (the most common CF regulation mutant) and by Δ1198-CFTR (a deletion construct that lacks NBD2 and the carboxy terminal tail). Login to comment
117 Introducing the K978C or K978S mutation strongly enhanced the basal activities of G551D-CFTR and Δ1198-CFTR Fig. 2. Login to comment
119 (A) Slower deactivation of the K978C constitutive mutant following ATP removal by hexokinase/ glucose addition. Login to comment
121 (B) ATP titration curves for K978C-CFTR and WT-CFTR. Login to comment
123 Each symbol is the mean ± SEM for six (K978C) and eight (WT) experiments. Login to comment
124 Data were fit to the Michaelis-Menten equation; Km = 8.1 ± 1.4 μM and 65.2 ± 10.4 μM for K978C and WT, respectively. Login to comment
125 (C) Titration of ADP inhibition of K190C/K978C-CFTR current in the absence of ATP. Login to comment
128 The large basal currents mediated by K978C,S/ G551D and K978C,S/Δ1198 were ATP-independent but inhibited by CFTRinh-172 or glibenclamide. Login to comment
138 (C-E) High control currents for G551D and Δ1198-CFTR channels containing K978C or K978S mutations. Login to comment
146 The currents mediated by K978S/G551D and K978C/G551D were statistically greater than the G551D currents (P < 0.05, unpaired t test). Login to comment
153 In our whole-cell patch-clamp analysis of the K978C,S/G551D constructs, we observed that these mutants were strongly stimulated by a cAMP-activating mixture (Fig. 4A). Login to comment
155 To confirm and extend this point, we tested the PKA dependence of the activity of one of the constitutive mutants that lacks NBD2 (K978C/Δ1198) in excised membrane patches. Login to comment
160 We also introduced the double constitutive loop mutant (K190C/K978C) into a construct lacking both the R domain and NBD2 (Fig. 4E). Login to comment
164 First, the K978C loop mutation strongly increased the rate of activation at low doses of PKA (Fig. 4 F-H). Login to comment
167 This reciprocity is conceptually analogous to the observed reci- procitybetween constitutive activation bythe K978C mutation and the corresponding increase in ATP sensitivity (Fig. 2B). Login to comment
181 (A) Repre- sentativewhole-cellcurrent record showing strong stimulation of K978C/ G551D-CFTR by a forskolin-containing cAMP-activating mixture. Login to comment
183 (B) Representative current record showing PKA (110 U/ mL) activation of K978C/ Δ1198-CFTR in excised patch (1.5 mM ATP present initially). Login to comment
185 (C) No PKA stimulation of the current mediated by a corresponding construct lacking a large portion of the R domain, K978C/ΔR/Δ1198 [lacking residues 700-835 of the R domain (29)]. Login to comment
186 (D) Mean data showing relative activation of K978C/Δ1198-CFTR currents by PKA with or without the R domain (±SEMs, n = 7 and 13). Login to comment
187 (E) K190C/K978C/ΔR/Δ1198 channels are maximallyactiveunderbaselineconditions. Login to comment
189 (F and G) Representative records of PKA activation of K978C-CFTR and WT-CFTR, respectively. Login to comment
190 (H) Mean time course data for activation by 3 U/mL PKA (K978C) or 3-9 U/mL (WT). Login to comment
194 This conclusion is best supported by our finding that the activity of an NBD2-deletion construct that cannot be stimulated by ATP alone (K978C/Δ1198) is nonetheless strongly dependent on R domain phosphorylation. Login to comment
211 For the MTS (methanethiosulfonate) experiments (Fig. S2), the cysteine substitutions (e.g., K978C) were introduced into the C832A background because modification of C832 can affect CFTR currents (34). Login to comment

PMID: 20952391 [PubMed] Wang G et al: "State-dependent regulation of cystic fibrosis transmembrane conductance regulator (CFTR) gating by a high affinity Fe3+ bridge between the regulatory domain and cytoplasmic loop 3."

No. Sentence Comment

7 Similarly, a K978C mutation from cytoplasmic loop 3 (CL3), which promotes ATP-independent channel opening, greatly weakened inhibition by Fe3؉ no matter whether NBD2 was present or not. Login to comment
25 It has been demonstrated that K978C/P/S in CL3 promotes channel activity without ATP (17). Login to comment
97 Once the channel was open, Fe3ϩ could not exert any effect on hCFTR activity. Supporting this notion, a K978C mutant from CL3 was also insensitive to Fe3ϩ for a limited time because of a high open probability of 0.84 (17). Login to comment
98 As shown in Fig. 2B, the K978C mutant channel exhibited spontaneous activity when excised from the HEK-293T cell but the resulting current ran down by an unknown mechanism. Login to comment
100 A similar result was also found in a K978C-⌬1198 construct (Fig. 2C). Login to comment
114 Once K978C reduced the activation energy for channel opening, Fe3ϩ had no influence on channel activity. Login to comment
149 Macroscopic currents across inside-out membrane patches excised from transfected HEK-293T cells expressing the hCFTR construct (A) and the K978C mutant (B). Login to comment
151 The arrows indicate the final concentrations. C, fractional inhibition of the current by Fe3ϩ for mutants K978C and K978C/ ⌬1198 (n ϭ 3-9). Login to comment
215 Once a K978C- or AMP-PNP-induced decrease in the activation energy overcomes an energy barrier caused by the interfacial Fe3ϩ bridge, channel activity will no longer be modulated by the metal bridge (Fig. 8B). Login to comment

PMID: 21059651 [PubMed] Wang G et al: "The inhibition mechanism of non-phosphorylated Ser768 in the regulatory domain of cystic fibrosis transmembrane conductance regulator."

No. Sentence Comment

22 Our recent study also demonstrated that a K190C/S mutation from CL1 enhances ATP-independent channel opening induced by a K978C/P/S mutation from CL3 (17). Login to comment
185 More importantly, H950R/S768R completely removed PKA dependence of channel activity (Fig. 7, A and D) no matter whether K978C, which promotes the channel opening without ATP, was inserted and accelerated channel activation by ATP (Fig. 7B) or not. Login to comment
255 PKA-dependent activity of His950 and Ser768 mutants with different H-bond donors and acceptors. A and B, activation of H950R/S768R (A) and H950R/S768R/K978C (B) before and after ATP (1. Login to comment

PMID: 21296873 [PubMed] Kirk KL et al: "A unified view of cystic fibrosis transmembrane conductance regulator (CFTR) gating: combining the allosterism of a ligand-gated channel with the enzymatic activity of an ATP-binding cassette (ABC) transporter."

No. Sentence Comment

134 The strongest evidence for the latter is the observation that CFTR channels that lack NBD2 but possess one of the constitutive loop mutations (e.g. CFTR-K978C/⌬1198) are strongly stimulated by PKA phosphorylation of the R domain even 4 Regarding Fig. 4A, it is important to note that we do not consider the open pore conformation at the TMDs (whatever that looks like structurally) to be strictly demanded by a tight NBD1-NBD2 dimer, nor do we consider the closed pore conformation to be demanded by the absence of the NBD1-NBD2 dimer. Login to comment

PMID: 21965669 [PubMed] Wang W et al: "Thermally unstable gating of the most common cystic fibrosis mutant channel (DeltaF508): "rescue" by suppressor mutations in nucleotide binding domain 1 and by constitutive mutations in the cytosolic loops."

No. Sentence Comment

7 Here, we determined whether these various rescue protocols induce a ⌬F508-CFTR conformation that is thermally stable in excised membrane patches. We also tested the impact of constitutive cytosolic loop mutations that increase ATP-independent channel activity (K978C and K190C/K978C) on ⌬F508-CFTR function. Login to comment
52 In a recent study we showed that two cytosolic loop mutations (e.g. K190C and K978C) promote ATP-independent CFTR channel activity, allosterically increase ATP and PKA sensitivity, and also significantly restore the function of mutant CFTR channels that cannot be activated by ATP binding or NBD dimerization (e.g. G551D and ⌬1198, which lacks NBD2) (47). Login to comment
57 The cytosolic loop mutations (K978C and K190C/K978C) do not improve ⌬F508-CFTR processing but do increase the ATP-independent channel activity of ⌬F508 channels that are delivered to the cell surface by incubation at low temperature. Login to comment
64 K978C and K190C/K978C mutations, which promote ATP-independent channel activity (47), were introduced into the ⌬F508-CFTR construct. Login to comment
162 Constitutive Loop Mutations Increase ATP-independent Channel Activity of ⌬F508-CFTR, Which Is More Stable at Physiologic Temperature-Previously, we showed that several mutations in intracellular loop1 and loop3 (e.g. K190C and K978C) increase the ATP-independent channel activities of several CFTR constructs, including mutant forms that cannot be activated by ATP (e.g. G551D and ⌬1198-CFTR, an NBD2 deletion mutant) (47). Login to comment
164 Fig. 6A shows that neither K978C nor K190C/K978C improved the maturation of ⌬F508-CFTR when cells were grown either at 37 °C or 27 °C, as determined by immunoblotting. Login to comment
169 Fig. 7A shows that K978C/⌬F508-CFTR exhibited a partial decrease of current when the bath temperature was elevated to 36.5 °C, but on average 30-40% of this current remained after warming the bath (see Fig. 7D). Login to comment
170 Interestingly, for approximately 50% of the patches the current slightly recovered when the temperature returned to room temperature, indicating that some of the current decrease for K978C/⌬F508-CFTR was reversible. Login to comment
171 These results indicate that K978C partially protects ⌬F508 channel activity from thermal inactivation. Login to comment
172 We then added hexokinase/glucose to remove ATP to determine whether the remaining current is ATP-independent. As shown in Fig. 7A, most of the current that remained following warming the bath was insensitive to removal of ATP, indicating that the ATP-independent channel activity of K978C/⌬F508-CFTR is far more stable at 36.5 °C compared with that of ⌬F508-CFTR alone (Fig. 1B). Login to comment
173 To follow up this result we examined the thermal stability of the K190C/K978C/⌬F508 construct that has a greater relative ATP-independent channel activity. Login to comment
174 Fig. 7B shows that the K190C/K978C double mutation greatly protected the channel activity from thermal inactivation with nearly all of the remaining current following warming the bath being ATP-independent. As a control we also tested the thermal stability of the ATP-independent channel activity of G551D-CFTR channels with the K978C mutation. Login to comment
175 Fig. 7C shows that raising the temperature did not inactivate the K978C/G551D-CFTR current, indicating that the ATP-independent channel activity of K978C/G551D is thermally stable. Login to comment
185 Error bars, S.E. Thermal Instability of ⌬F508-CFTR Gating DECEMBER 9, 2011•VOLUME 286•NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 41943 the ATP-independent channel activity of K978C/⌬F508-CFTR and to a greater extent K190C/K978C/⌬F508-CFTR was resistant to temperature-induced inactivation. Login to comment
188 To examine this idea further we recorded the unitary currents mediated by single K978C/⌬F508-CFTR channels in micropatches obtained with small tip pipettes. Login to comment
190 Fig. 8E shows that the amplitudes of the singlechannelcurrentsforK978C/⌬F508afterincubationat36 °C were similar to those for WT- and K978C-CFTR (47), indicating that the conformation of the pore for K978C/⌬F508 is not obviously compromised at physiologic temperature. Login to comment
195 We found that constitutive mutations in cytosolic loop1 and loop3 (i.e. K978C and K190C/K978C) increased ATP-independent channel activity of low temperature-rescued ⌬F508-CFTR. Login to comment
199 FIGURE6.Cytosolicloopmutations(K978CandK190C/K978C)increaseATP-independentchannelactivityoflowtemperature-recued⌬F508-CFTR.A, immunoblot showing that loop mutations do not improve maturation of ⌬F508 at 27 °C. B-D, ATP-dependent channel activities of ⌬F508, K978C/⌬F508, and K190C/K978C/⌬F508 revealed by adding hexokinase/glucose to scavenge ATP. Login to comment
201 E, mean fractional ATP-independent currents for ⌬F508 (n ϭ 6), K978C/⌬F508 (n ϭ 5), and K190C/K978C/⌬F508-CFTR (n ϭ 5). Login to comment
218 K978C/⌬F508 and K190C/K978C/⌬F508-CFTR are more stable at physiologic temperature. Login to comment
219 A, effect of raising temperature on K978C/⌬F508. Login to comment
221 B, effect of raising temperature on K190C/K978C/⌬F508-CFTR, which is more thermally stable. Login to comment
222 C, effect of raising temperature on K978C/G551D-CFTR. Login to comment
223 D, mean fractional current decrease for ⌬F508 (data from Fig. 1D), K978C/⌬F508, K190C/K978C/⌬F508, and K978C/ G551D. Login to comment
237 A and B, effect of raising temperature on ATP-independent currents of K978C/⌬F508- and K190C/K978C/⌬F508-CFTR. Login to comment
239 C, mean fractional loss of ATP-independent current for K978C/⌬F508 and K190C/ K978C/⌬F508-CFTR (n ϭ 4 for each construct). Login to comment
240 D, effect of raising temperature on unitary currents of K978C/⌬F508. Login to comment
242 Effect of raising bath temperature on K978C/⌬F508 current was monitored at the macroscopic level (ramp protocol), and then the unitary currents were recorded in the gap-free mode after reduction to room temperature. Login to comment

PMID: 24876383 [PubMed] Wei S et al: "Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps."

No. Sentence Comment

109 Fig. 1A also shows the location of a previously characterized GOF mutation (K978C) that locates near the base of TM9 in the outer TM collar (16). Login to comment
172 Additive GOF Effects of the P355A Mutation and a Mutation in the Outer TM Collar-We previously reported another class of GOF mutation that locates to the base of TM9 (Lys-978) in the putative outer TM collar that surrounds the principal pore-lining TMs (see Fig. 1A for predicted location of Lys-978 in CFTR structural models) (16). Login to comment
173 We reasoned that, given the different locations of the two classes of GOF mutations (pore versus outer collar), a double mutant (P355A/K978C) may exhibit an additive GOF effect. Login to comment
175 GOF mutations in the pore-lining TM6 (P355A) and in the outer TM collar (K978C) have additive effects on CFTR channel activity. Login to comment
176 A, macroscopic current record showing the relatively large ATP-independent current and robust activation by 2 mM AMP-PNP for the P355A/K978C double mutant.Fortherampprotocol,conditionswerethesameasforFigs.2-4.BandC,meanfractionalATP-freecurrentandrelativeAMP-PNPactivationnormalized to the control current at 1.5 mM ATP for the indicated single and double mutants. Login to comment
191 G, immunoblot of P355A-èc;1198, P355A-G551D, and P355A/K978C double mutants transiently expressed in HEK-293T cells. Login to comment
197 The double mutant (P355A/K978C-CFTR) exhibited relatively high ATP-independent currents in excised macropatches (b03;35% of the ATP control current) and strong activation by AMP-PNP that approached that by ATP (b03;90% of the ATP control current). Login to comment
199 It is also evident from the single mutant data in Fig. 5, B and C, that the K978C mutation had a somewhat stronger GOF effect than the P355A mutation. Login to comment
207 Introducing the P355A mutation increased the apparent ATP affinity of the Y1219G mutant (leftward shift in Fig. 6B). Login to comment
208 The K978C mutation in the outer TM collar (TM9) also increased the apparent ATP affinity of the Y1219G mutant and to a greater degree than the Pro-355 substitution, as would be expected if the former is a stronger GOF mutation (see also Fig. 5). Login to comment
210 These results also indicate that both classes of GOF mutations (Pro-355 in TM6; K978C in the outer TM collar) can compensate for a partial defect in ATP binding by allosteric coupling between the TMs and the NBDs (i.e. by "allosteric rescue" of the ATP binding defect). Login to comment
220 Curves, best fits to Hill equation with K values of 1522, 683, and 180 òe;M and Hill coefficients of 1.69, 1.93, and 1.72 for Y1219G (n afd; 6 patches), P355A/Y1219G (n afd; 5), and K978C/Y1219G (n afd; 4), respectively. Login to comment
284 GOF effects of the P355A and K978C mutations were additive, implying that they influence channel gating by different mechanisms. Login to comment
308 Both the P355A and K978C GOF mutations increased the ATP sensitivity of the Y1219G mutant with the K978C substitution restoring the ATP sensitivity of channel gating to nearly wild type levels. Login to comment
108 Fig. 1A also shows the location of a previously characterized GOF mutation (K978C) that locates near the base of TM9 in the outer TM collar (16). Login to comment
174 GOF mutations in the pore-lining TM6 (P355A) and in the outer TM collar (K978C) have additive effects on CFTR channel activity. Login to comment
190 G, immunoblot of P355A-èc;1198, P355A-G551D, and P355A/K978C double mutants transiently expressed in HEK-293T cells. Login to comment
196 The double mutant (P355A/K978C-CFTR) exhibited relatively high ATP-independent currents in excised macropatches (b03;35% of the ATP control current) and strong activation by AMP-PNP that approached that by ATP (b03;90% of the ATP control current). Login to comment
198 It is also evident from the single mutant data in Fig. 5, B and C, that the K978C mutation had a somewhat stronger GOF effect than the P355A mutation. Login to comment
209 These results also indicate that both classes of GOF mutations (Pro-355 in TM6; K978C in the outer TM collar) can compensate for a partial defect in ATP binding by allosteric coupling between the TMs and the NBDs (i.e. by "allosteric rescue" of the ATP binding defect). Login to comment
219 Curves, best fits to Hill equation with K values of 1522, 683, and 180 òe;M and Hill coefficients of 1.69, 1.93, and 1.72 for Y1219G (n afd; 6 patches), P355A/Y1219G (n afd; 5), and K978C/Y1219G (n afd; 4), respectively. Login to comment
283 GOF effects of the P355A and K978C mutations were additive, implying that they influence channel gating by different mechanisms. Login to comment
307 Both the P355A and K978C GOF mutations increased the ATP sensitivity of the Y1219G mutant with the K978C substitution restoring the ATP sensitivity of channel gating to nearly wild type levels. Login to comment

PMID: 26606940 [PubMed] Wei S et al: "Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels."

No. Sentence Comment

201 To test this idea, the extracellular F337S mutation was combined with the previously characterized K978C mutation, which locates tothe cytosolic side below TM9 (14, 15). Login to comment
202 The K978C substitution increases ATP-free channel activity and enhances the ATP and PKA sensitivities of CFTR activation similar to that shown here for the F337S mutation (14). Login to comment
204 Removing bath ATP in the standard excised macropatch protocol decreased the currents mediated by F337S/K978C-CFTR by less than 20% (Fig. 10A, B). Login to comment
207 More strikingly, F337S/K978C-CFTR appeared to be nearly maximally active in the absence of both PKA and ATP (Fig. 10C-F). Login to comment
208 Substantial F337S/K978C-CFTR-mediated currents could be detected for macropatches that were excised in the absence of both PKA and ATP in Figure 6. Login to comment
220 In support of this interpretation, the Po of F337S/K978C-CFTR estimatedfrom multichannel records in theabsence ofbothPKAandATP(examplerecordinFig.10D)ranged from 0.34 to 0.95 with a mean Po (6SEM) of 0.53 6 0.08 (n = 7 patches;estimated using the conventional Clampfit protocol; see Methods). Login to comment
276 The F337S/K978C double mutant is nearly fully active in the absence of both exogenous PKA and ATP. Login to comment
277 A) Representative macroscopic current record showing that ATP removal by scavenger addition followed by bath perfusion with an ATP-free solution only modestly decreases the activities of F337S/K978C-CFTR channels. Conditions were identical to Fig. 4. Login to comment
282 C) Representative macroscopic current record showing that F337S/K978C-CFTR channels are nearly maximally active in excised patches in the absence of both ATP and exogenous PKA. Login to comment
284 D) Unitary current recording at a single holding potential for an excised patch containing 3 F337S/K978C-CFTR channels. Login to comment
288 E) Gap-free record at a single holding potential for an excised patch containing 80-100 F337S/K978C-CFTR channels showing substantial activity in the absence of bath PKA and ATP. Login to comment
289 F) Stationary noise plot for F337S/K978C-CFTR channels in the absence of PKA and ATP. Login to comment
329 Engineering a superactive CFTR The F337S/K978C double mutant has the highest single-channel activity in the absence of exogenous PKA and ATP of any CFTR construct that we have characterized to date. Login to comment
332 We anticipated that the F337S and K978C mutations would have additive GOF effects on ATP-free CFTR activity because they locate to opposite sides of the pore where they presumably impact CFTR structure in different ways. Login to comment
200 To test this idea, the extracellular F337S mutation was combined with the previously characterized K978C mutation, which locates tothe cytosolic side below TM9 (14, 15). Login to comment
203 Removing bath ATP in the standard excised macropatch protocol decreased the currents mediated by F337S/K978C-CFTR by less than 20% (Fig. 10A, B). Login to comment
206 More strikingly, F337S/K978C-CFTR appeared to be nearly maximally active in the absence of both PKA and ATP (Fig. 10C-F). Login to comment
219 In support of this interpretation, the Po of F337S/K978C-CFTR estimatedfrom multichannel records in theabsence ofbothPKAandATP(examplerecordinFig.10D)ranged from 0.34 to 0.95 with a mean Po (6SEM) of 0.53 6 0.08 (n = 7 patches;estimated using the conventional Clampfit protocol; see Methods). Login to comment
275 The F337S/K978C double mutant is nearly fully active in the absence of both exogenous PKA and ATP. Login to comment
281 C) Representative macroscopic current record showing that F337S/K978C-CFTR channels are nearly maximally active in excised patches in the absence of both ATP and exogenous PKA. Login to comment
283 D) Unitary current recording at a single holding potential for an excised patch containing 3 F337S/K978C-CFTR channels. Login to comment
287 E) Gap-free record at a single holding potential for an excised patch containing 80-100 F337S/K978C-CFTR channels showing substantial activity in the absence of bath PKA and ATP. Login to comment
328 Engineering a superactive CFTR The F337S/K978C double mutant has the highest single-channel activity in the absence of exogenous PKA and ATP of any CFTR construct that we have characterized to date. Login to comment
331 We anticipated that the F337S and K978C mutations would have additive GOF effects on ATP-free CFTR activity because they locate to opposite sides of the pore where they presumably impact CFTR structure in different ways. Login to comment

PMID: 23620589 [PubMed] Okeyo G et al: "Converting nonhydrolyzable nucleotides to strong cystic fibrosis transmembrane conductance regulator (CFTR) agonists by gain of function (GOF) mutations."

No. Sentence Comment

81 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 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 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 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 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 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 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 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 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 Poorly hydrolyzable nucleotides strongly activate the K978C-CFTR GOF mutant. Login to comment
113 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 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 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 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 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 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 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 This disparity argues that K978C channels open and close dynamically even when ATPॹS is tightly bound. Login to comment
133 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 K978C-CFTR channels continued to open and close up to several min after ATPॹS removal (Fig. 4E). Login to comment
136 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 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 ATPॹS markedly increases the single channel open probability of K978C-CFTR. Login to comment
142 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 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 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
162 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 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
169 A and B, representative macropatch records show AMP-PNP activation of K978P-CFTR (A) and K190C/K978C-CFTR (B). Login to comment
171 Note the previously described voltage-dependent rectification of K190C/K978C-CFTR currents (13). Login to comment
174 WT and K978C data are from Fig. 1F. Login to comment
182 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 K978C/G1349D-CFTR channels also exhibited detectable currents in the absence of any bath nucleotide as expected (Fig. 6D). Login to comment
184 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
188 A, macropatch record shows slow deactivation of K978C-CFTR current after AMP-PNP washout. Login to comment
191 B, shown is slow deactivation of K978C-CFTR current after ATPॹS washout. Login to comment
192 C, WT-CFTR currents also deactivate fairly slowly after ATPॹS washout but faster than K978C-CFTR currents. Login to comment
194 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 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 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 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 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 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
210 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 Perturbation of the NBD dimer interface inhibits K978C-CFTR activation by AMP-PNP and ATPॹS. Login to comment
219 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
222 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 F, very small activation of K978C/G551D-CFTR by 2 mM AMP-PNP is shown. Login to comment
226 were activated by these analogs precluded performing quantitative titrations like we performed for the K978C GOF mutant. Login to comment
228 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 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 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 Rescue of G1349D-CFTR gating by K978C and differential activation of this NBD2 mutant by ATPॹS and AMP-PNP. Login to comment
238 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 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 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
259 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 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
271 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 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
289 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
291 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 This construct also exhibited larger ATP-independent currents, as expected for channels with the K978C GOF mutation. Login to comment
294 G1349D-CFTR rescue by the K978C GOF mutation is analogous to its enhancement of CFTR activation by ATPॹS or AMP-PNP. Login to comment

PMID: 25190805 [PubMed] Wang W et al: "An electrostatic interaction at the tetrahelix bundle promotes phosphorylation-dependent cystic fibrosis transmembrane conductance regulator (CFTR) channel opening."

No. Sentence Comment

11 ATP-dependent activity was rescued by introducing a second site gain of function (GOF) mutation that was previously shown to promote ATP-dependent and ATP-independent opening (K978C). Login to comment
12 Conversely, the ATP-independent activity of the K978C GOF mutant was inhibited by charge-reversal mutations at positions 267 or 1060 either in the presence or absence of NBD2. Login to comment
141 Combining E267 and K1060 Charge-reversal Mutations with a Gain of Function (GOF) Mutation Reveals the Importance of the Tetrahelix Bundle in Mediating Channel Opening in the Absence of ATP Binding or NBD Dimerization-Previously we reported a class of GOF mutation in ICL3 near the base of TM9 that increases both ATP-dependent and ATP-independent channel activity (e.g. K978C (12)). Login to comment
144 Regarding the first issue, Fig. 6A shows that introducing the K978C substitution into the E267R mutant substantially restored ATP-dependent channel activity (compare with Fig. 2B). Login to comment
145 The E267R/K978C double mutant exhibited normalized control currents and potentiator responses in macropatch experiments that were similar to wild type levels (see legend for mean data). Login to comment
146 This rescue of the ATP-dependent activity of the E267 charge-reversal mutant is perhaps not surprising given that the K978C substitution reduces the free energy difference FIGURE 2. Login to comment
171 Regarding the second issue, Fig. 6, B-E show that the E267R and K1060E mutations reduced the ATP-free activity of the K978C GOF mutant. Login to comment
174 The latter interpretation is further supported by the results in Fig. 7, which show that the E267R mutation also reduced the ATP-free channel activity of K978C/èc;1198-CFTR, a truncation mutant lacking NBD2 but possessing the indicated GOF mutation. Login to comment
262 Disrupting the E267-K1060 Interaction Primarily Impacts Channel Opening, not Burst Duration or NBD Dimer Stability-The charge-reversal mutations at these two positions dramatically decreased the macroscopic currents and apparent single FIGURE 6. Interplay between the charge-reversal mutations at the bundle interface and a previously reported GOF mutation (K978C); evidence that the E267RandK1060EmutationsalsoinhibitATP-freechannelactivity.A,E267R/K978CdoublemutantbehavesmorelikewildtypechannelsinthepresenceofATP inmacropatchexperiments.ConditionswereidenticaltoFig.2A.Themeanpercentcontrolcurrentsofthisdoublemutantwhennormalizedtothemaximalcurrents measuredafterpotentiatoradditionwere94afe;5.4%and82.8afe;3.9%beforeandafterPKIaddition,respectively(nafd;5).Thesevaluesarenotsmaller(infact,somewhat larger) than those for WT-CFTR (see Fig. Login to comment
264 B-D, macroscopic records for the indicated K978C single and double mutants showing that the E267R and K1060E substitutions decreased the fractional currents remaining after ATP removal. Login to comment
266 E267R/K978C-CFTR was activated strongly by the subsequent addition of 2 mM AMP-PNP in the absence of ATP, as reported previously for the K978C GOF mutant (panels B, C and Ref. 47). Login to comment
280 A, control macroscopic record showing the substantial control current and correspondingly modest stimulation by potentiator addition for the previously described K978C/èc;1198 truncation mutant lacking NBD2. Login to comment
282 As reported earlier (12), the channel activity of this construct is ATP-independent and is attributable to the presence of the K978C GOF mutation. Login to comment
289 **, p b0d; 0.01 compared with K978C/èc;1198 by unpaired t test. N is indicated in parentheses. CFTR Gating Mechanism 30374 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289ߦNUMBER 44ߦOCTOBER 31, 2014 at SEMMELWEIS UNIV OF MEDICINE on December 4, tion between inactive and active conformations in response to ATP binding and NBD dimerization. Login to comment
290 The E267-K1060 Interaction Also Promotes CFTR Channel Opening in the Absence of ATP Binding or NBD Dimerization- Combining a previously reported GOF mutation (K978C) with the E267R or K1060E bundle mutations revealed that the latter mutations also inhibit ATP-free channel activity both in the presence and absence of NBD2 (i.e. for the èc;1198-CFTR truncation construct). Login to comment
291 The K978C substitution did restore nearly normal ATP-dependent activity of the E267R mutant as evidenced by high control currents in the presence of ATP and PKA and small relative activation by potentiators. Login to comment
293 Apparently the favorable effect of the K978C mutation on the energetics of channel opening is sufficient to overcome the negative impact of the bundle mutation on gating when ATP binding and NBD dimerization can occur. Login to comment
294 Conversely, each of the E267R and K1060E mutations substantially reduced the fractional ATP-independent currents exhibited by the full-length K978C-CFTR construct and also reduced the control currents and increased the relative stimulation by potentiators for the NBD2-deletion construct bearing the GOF mutation (K978C/ èc;1198-CFTR). Login to comment
298 According to this view, GOF mutations such as K978C enhance the channel activity of the èc;1198-CFTR construct lacking NBD2 by allosterically promoting the E267-K1060 interaction at the bundle interface. Login to comment
301 The latter mechanism is supported by our earlier observation that PKA phosphorylation of the R domain markedly increases the channel activity of the NBD2 deletion construct, K978C/èc;1198-CFTR (12). Login to comment
326 CFTR Gating Mechanism 30376 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289ߦNUMBER 44ߦOCTOBER 31, 2014 at SEMMELWEIS UNIV OF MEDICINE on December 4, observed that the K978C GOF mutation in ICL3 near the base of TM9 increased the PKA sensitivity of channel activation even though this residue is nowhere near a PKA phosphorylation site in the polypeptide sequence (12). Login to comment

PMID: 25944907 [PubMed] El Hiani Y et al: "Functional Architecture of the Cytoplasmic Entrance to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore."

No. Sentence Comment

112 We did not observe any ATPand PKA-dependent, CFTRinh-172-sensitive macroscopic currents in inside-out patches associated with R153C, H949C, or K978C (Fig. 3), most likely due to lack of channel expression in the membrane (11). Login to comment