ABCMdb

PMID: 20133716

Wang W, Wu J, Bernard K, Li G, Wang G, Bevensee MO, Kirk KL
ATP-independent CFTR channel gating and allosteric modulation by phosphorylation.
Proc Natl Acad Sci U S A. 2010 Feb 23;107(8):3888-93. Epub 2010 Feb 3., 2010-02-23 [PubMed]

Sentences

No. Mutations Sentence Comment

9 ABCC7 p.Gly551Asp Introducing constitutive mutations into CFTR channels that cannot open in response to ATP (i.e., the G551D CF mutant and an NBD2-deletion mutant) substantially rescued their activities. Login to comment 47 ABCC7 p.Lys978Cys 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 ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys 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 ABCC7 p.Lys978Cys 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 ABCC7 p.Lys978Cys The K978C mutant also had a higher Po in the presence of saturating ATP as compared with WT-CFTR. Login to comment 58 ABCC7 p.Lys978Cys (B) K978C-CFTR macroscopic current across inside-out membrane patch excised from HEK-293T cell [ramp protocol (±80 mV); Methods]. Login to comment 71 ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys (F) Very large ATP-independent current for K190C/K978C-CFTR. Login to comment 75 ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys 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 ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys Numbers of patches analyzed: three (WT, +ATP), six (WT, -ATP), six (K978C, +ATP), and five (K978C, -ATP). Login to comment 89 ABCC7 p.Lys190Glu Interestingly, some of the K190 substitutions (particularly K190E) exhibited voltage-dependent current rectification (Fig. 1F, Inset, and Fig. S6), which might indicate that this position is relatively close to the inner pore vestibule. Login to comment 96 ABCC7 p.Lys978Cys 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 ABCC7 p.Lys978Cys 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 ABCC7 p.Lys978Cys 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 ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys 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 ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys 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 ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys 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 ABCC7 p.Gly551Asp ABCC7 p.Lys978Cys ABCC7 p.Lys978Ser 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 114 ABCC7 p.Gly551Asp The G551D mutant has negligible activity because of disruption of the ABC signature sequence in NBD1, which lines one of the two ATP binding pockets (28). Login to comment 117 ABCC7 p.Gly551Asp ABCC7 p.Lys978Cys ABCC7 p.Lys978Ser Introducing the K978C or K978S mutation strongly enhanced the basal activities of G551D-CFTR and Δ1198-CFTR Fig. 2. Login to comment 119 ABCC7 p.Lys978Cys (A) Slower deactivation of the K978C constitutive mutant following ATP removal by hexokinase/ glucose addition. Login to comment 121 ABCC7 p.Lys978Cys (B) ATP titration curves for K978C-CFTR and WT-CFTR. Login to comment 123 ABCC7 p.Lys978Cys Each symbol is the mean ± SEM for six (K978C) and eight (WT) experiments. Login to comment 124 ABCC7 p.Lys978Cys 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 ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys (C) Titration of ADP inhibition of K190C/K978C-CFTR current in the absence of ATP. Login to comment 128 ABCC7 p.Gly551Asp ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys 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 130 ABCC7 p.Gly551Asp ABCC7 p.Lys978Ser The relative degree of curcumin activation of the K978S/C combination mutants was much lower than for the original G551D and Δ1198-CFTR constructs (Fig. 3E), consistent with the substantial elevation of basal channel activity by these substitutions. Login to comment 131 ABCC7 p.Gly551Asp Whole-cell patch-clamp experiments were performed to define quantitatively the degree to which G551D-CFTR channel function within intact cells was rescued by these constitutive mutations. Login to comment 132 ABCC7 p.Gly551Asp Macroscopic currents in transfected HEK-293T cells were nor- malizedtocellcapacitanceandcomparedwithcurrentsmediatedby WT-CFTR and the original G551D construct. Login to comment 136 ABCC7 p.Gly551Asp (A) G551D-CFTR channels exhibit low control currents in the presence of PKA/ATP but can be activated by curcumin (30 μM) in excised membrane patches (conditions are shown in Fig. 1). Login to comment 138 ABCC7 p.Gly551Asp ABCC7 p.Lys978Cys ABCC7 p.Lys978Ser (C-E) High control currents for G551D and Δ1198-CFTR channels containing K978C or K978S mutations. Login to comment 141 ABCC7 p.Gly551Asp (F) Immunoblots and surface biotinylation results showing similar expression levels and surface localization of the indicated G551D constructs in transfected HEK-293T cells (SI Methods). Login to comment 144 ABCC7 p.Gly551Asp (G) Mean whole-cell currents normalized to cell capacitance for the indicated G551D constructs. Login to comment 146 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Lys978Cys ABCC7 p.Lys978Ser 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 147 ABCC7 p.Gly551Asp mediated by G551D-CFTR were virtually undetectable. Login to comment 148 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Conversely,thecurrentsmediatedbyK978C/G551D-CFTRandK978S/ G551D-CFTR were 30-40% of WT levels (Fig. 3G), a greater than 20-fold increase in the macroscopic activity of the G551D mutant. Login to comment 153 ABCC7 p.Gly551Asp ABCC7 p.Lys978Cys 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 ABCC7 p.Lys978Cys 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 ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys 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 ABCC7 p.Lys978Cys First, the K978C loop mutation strongly increased the rate of activation at low doses of PKA (Fig. 4 F-H). Login to comment 167 ABCC7 p.Lys978Cys 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 ABCC7 p.Gly551Asp ABCC7 p.Lys978Cys (A) Repre- sentativewhole-cellcurrent record showing strong stimulation of K978C/ G551D-CFTR by a forskolin-containing cAMP-activating mixture. Login to comment 183 ABCC7 p.Lys978Cys (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 ABCC7 p.Lys978Cys (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 ABCC7 p.Lys978Cys (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 ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys (E) K190C/K978C/ΔR/Δ1198 channels are maximallyactiveunderbaselineconditions. Login to comment 189 ABCC7 p.Lys978Cys (F and G) Representative records of PKA activation of K978C-CFTR and WT-CFTR, respectively. Login to comment 190 ABCC7 p.Lys978Cys (H) Mean time course data for activation by 3 U/mL PKA (K978C) or 3-9 U/mL (WT). Login to comment 194 ABCC7 p.Lys978Cys 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 195 ABCC7 p.Gly551Asp This result is consistent with previous arguments that G551D-CFTR channels exhibit low levels of ATP-independent but PKA-sensitive activity (12) and that curcumin activation of Δ1198-CFTR channels also depends on prior PKA phosphorylation (22). Login to comment 211 ABCC7 p.Cys832Ala ABCC7 p.Lys978Cys 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 213 ABCC7 p.Ser660Ala ΔR-S660A-CFTR (29) was provided by Michael Welsh (University of Iowa, Iowa City, IA).Cells transfected with cysteine-free or ΔR constructs were cultured at 26-28 °C for 24-48 h before patch clamping to increase expression of these mutants, which mature less efficiently than WT-CFTR (16, 17, 30). Login to comment 214 ABCC7 p.Val510Ala We also introduced the V510A substitution into the cysteine-free mutants, which is known to promote their maturation (35). Login to comment 241 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Bompadre SG, Sohma Y, Li M, Hwang T-C (2007) G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects. Login to comment 252 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Bompadre SG, Sohma Y, Li M, Hwang T-C (2007) G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects. Login to comment