ABCMdb

PMID: 25277268

Broadbent SD, Ramjeesingh M, Bear CE, Argent BE, Linsdell P, Gray MA
The cystic fibrosis transmembrane conductance regulator is an extracellular chloride sensor.
Pflugers Arch. 2015 Aug;467(8):1783-94. doi: 10.1007/s00424-014-1618-8. Epub 2014 Oct 4., [PubMed]

Sentences

No. Mutations Sentence Comment

81 ABCC7 p.Arg899Gln Figure 2b, c shows that removing the positive charge at position 899 (R899Q) completely abolished [Cl- ]o sensing by CFTR (high Cl- stimulation; 4.3&#b1; 6.6 %, n=7), whereas all the other ECL charge neutralising mutants had no significant effect on the response (Fig. 2c). Login to comment 82 ABCC7 p.Arg899Glu ABCC7 p.Arg899Lys Furthermore, introducing a lysine residue in place of arginine (R899K) maintained the response to [Cl- ]o (Fig. 2c) while mutating R899 to the negatively charged glutamic acid (R899E) did not, clearly showing that the positive charge at position 899 in ECL4 of CFTR is essential for [Cl- ]o sensing. Login to comment 83 ABCC7 p.Arg899Gln Typical I-V plots obtained in this series of experiments for WT, R899Q and the vector control are shown in Fig. 2b. Login to comment 92 ABCC7 p.Glu1371Gln We then examined whether the ATP hydrolysis-deficient CFTR mutant, E1371Q [38], could sense [Cl- ]o with the expectation that it would not. Login to comment 93 ABCC7 p.Glu1371Gln Like WT CFTR exposed to AMP-PNP, the E1371Q mutant channels display extended open channel bursts in the presence of ATP [16, 23, 40]. Login to comment 94 ABCC7 p.Glu1371Gln In our experiments, E1371Q CFTR expressed in HEK cells was constitutively active and large currents were detected without prior stimulation with FSK (Fig. 3d (i) (first set of fWCR current traces), and 3e), which agrees with previous work performed in baby hamster kidney (BHK) cells [27, 42, 46]. Login to comment 95 ABCC7 p.Glu1371Gln The reason for this constitutive activity of E1371Q is unclear and it contrasts with the behaviour of wild type CFTR and other mutants (except DeltaR-CFTR) previously investigated in our laboratories using the BHK and HEK cell expression systems [46]. Login to comment 96 ABCC7 p.Glu1371Gln Exposing E1371Q CFTR (without FSK) to an increase in [Cl- ]o caused only a small stimulation of channel activity (21.9&#b1;14.1 %, n=6; Fig. 3f). Login to comment 97 ABCC7 p.Glu1371Gln Surprisingly in view of the WT CFTR data with AMP-PNP, if E1371Q CFTR channels were first phosphorylated with FSK (which itself had no significant effect on the size of the whole cell currents (Fig. 3d (ii) (second set of fWCR traces)) and Table 1), [Cl- ]o sensing was restored (174.5&#b1;56.2 % stimulation, n=8; Fig. 3d, e and f). Login to comment 98 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln To confirm the role of phosphorylation in [Cl- ]o sensing, we reduced the endogenous phosphorylation of E1371Q channels by removing ATP/GTP from the intracellular solution prior to stimulation with FSK, which significantly reduced basal E1371Q CFTR currents (No ATP/GTP: -186&#b1; 122 pA/pF n=8 vs ATP/GTP: -1,259&#b1;433 pA/pF, n=8, P<0.001). Login to comment 99 ABCC7 p.Glu1371Gln Under these conditions, the E1371Q CFTR channels failed to respond to changes in [Cl- ]o even in the presence of FSK (Fig. 3f), consistent with the idea that only phosphorylated channels are gated by [Cl- ]o. Login to comment 100 ABCC7 p.Glu1371Gln Since E1371Q CFTR is hydrolysis-deficient [38], we expected that [Cl- ]o sensing by the mutant would not be affected by AMP-PNP. Login to comment 101 ABCC7 p.Glu1371Gln However, unexpectedly, we found that the ability of FSK-stimulated E1371Q CFTR channels to respond to changes in [Cl- ]o was markedly reduced by the non-hydrolysable ATP analogue, just like WT CFTR (Fig. 3f). Login to comment 102 ABCC7 p.Glu1371Gln ABCC7 p.Arg899Gln ABCC7 p.Arg899Gln To check that the restoration of [Cl- ]o sensing by E1371Q CFTR after phosphorylation (Fig. 3d) was due to an C A B WT R899Q VEC WT (i) (ii) (iii) (iv) (i) (ii) (iii) (iv) R899Q Fig. 2 Arginine residue 889 in extracellular loop 4 of CFTR is essential for [Cl- ]o sensing. Login to comment 103 ABCC7 p.Arg899Gln ABCC7 p.Arg899Gln a Representative fWCR current recordings measured between &#b1;100 mV in 20 mV steps from HEK cells transfected with wild type (WT) CFTR and R899Q CFTR, as indicated. The current traces are from the top down: (i) unstimulated in 155.5 mM [Cl- ]o, (ii) forskolin (FSK)-stimulated in 155.5 mM [Cl- ]o, (iii) FSK-stimulated in 35.5 mM [Cl- ]o and (iv) FSK-stimulated in 155.5 mM [Cl- ]o. Dotted line to the right of the current traces indicates zero current level. b Representative IV plots for the Vector Control (VEC), WT and R899Q CFTR, for unstimulated in 155.5 mM [Cl- ]o (black squares); FSK-stimulated in 155.5 mM [Cl- ]o (black rhombus); FSK-stimulated in 35.5 mM [Cl- ]o (white circles) and FSK-stimulated in 155.5 mM [Cl- ]o washoff (white triangles). Login to comment 110 ABCC7 p.Glu1371Gln ABCC7 p.Arg899Gln **p<0.01 compared to WT CFTR interaction of Cl-with the extracellular domain of CFTR, we also studied the double-mutant R899Q-E1371Q. Login to comment 111 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln Figure 3f shows that [Cl- ]o sensing by the double mutant in the presence of FSK was significantly reduced compared to E1371Q CFTR under the same conditions, confirming that phosphorylated E1371Q CFTR channels respond to changes in [Cl- ]o via R899 in ECL4. Login to comment 112 ABCC7 p.Arg334Gln ABCC7 p.Lys335Ala ABCC7 p.Lys329Ala ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly ABCC7 p.Arg899Glu ABCC7 p.Arg117Gln ABCC7 p.Arg104Gln ABCC7 p.Arg1128Gln ABCC7 p.Arg899Gln ABCC7 p.Arg899Gln ABCC7 p.Arg899Gln ABCC7 p.Arg899Gln ABCC7 p.Lys114Cys ABCC7 p.Lys892Gln ABCC7 p.Arg899Lys To explore the role of phosphorylation further, we studied the effect of deleting the R domain from CFTR (residues 634-836) [12, 7], which removes all the major PKA/PKC Table 1 Summary of the FSK stimulation of whole cell currents and Erev shifts observed with the CFTR constructs used in this study CFTR Construct n FSK Stimulation (%&#b1;SEM) Erev shift (mV&#b1;SEM) WT (50 bc;M ATP) 5 180&#b1;96 15.0&#b1;3.6 WT (100 bc;M ATP) 6 12,000&#b1;6,000 15.2&#b1;3.0 WT (300 bc;M ATP) 8 1,200&#b1;600 17.0&#b1;3.0 WT (1 mM ATP) 24 13,000&#b1;6,000 23.7&#b1;1.8 WT (1.3 mM ATP) 9 1,400&#b1;900 16.7&#b1;2.6 WT (2 mM ATP) 24 6,100&#b1;5,300 16.7&#b1;1.6 WT (5 mM ATP) 7 1,600&#b1;1,000 20.1&#b1;4.4 WT (50 bc;M ATP + 50 bc;M P-ATP) 7 224&#b1;130 15.3&#b1;1.0 WT + Genistein 4 7,600&#b1;5,200 26.1&#b1;5.4 WT + AMP-PNP 5 2,800&#b1;2,500 21.8&#b1;5.5 WT (3 mM MgCl2) 7 28,000&#b1;17,000 18.3&#b1;3.1 R104Q 5 4,600&#b1;1,600 28.6&#b1;4.7 K114C 5 12,000&#b1;6,700 29.2&#b1;3.0 R117Q 4 33,000&#b1;20,000 30.1&#b1;3.4 K329A 5 13,000&#b1;10,000 33.7&#b1;2.1 R334Q 9 13,000&#b1;6,700 27.3&#b1;2.9 K335A 5 3,200&#b1;1,500 20.8&#b1;7.1 W401G 7 2,600&#b1;1,800 18.5&#b1;4.8 Delta-R (No Stim) 5 - 25.1&#b1;2.7 Delta-R (No FSK, Genistein) 5 140&#b1;13 22.7&#b1;3.0 Delta-R (FSK, No Genistein) 4 89&#b1;14 15.6&#b1;6.0 Delta-R (FSK + Genistein) 6 639&#b1;432 25.1&#b1;4.9 Delta-R-E1371S (No FSK) 9 - 21.4&#b1;4.8 Delta-R-E1371S (FSK) 4 2,600&#b1;1,400 15.3&#b1;4.7 K892Q 7 16,000&#b1;9,500 36.8&#b1;4.8 R899E 4 1,200&#b1;400 25.0&#b1;2.7 R899K 4 1,600&#b1;900 26.6&#b1;2.9 R899Q 7 5,400&#b1;2,800 30.0&#b1;1.3 R899Q + AMP-PNP 4 72,000&#b1;50,000 15.2&#b1;2.8 R899Q-E1371Q (No FSK) 4 - 18.4&#b1;5.9 R899Q-E1371Q (FSK) 6 107&#b1;48 15.6&#b1;3.0 R1128Q 6 14,000&#b1;6,100 41.1&#b1;4.2 Y1219G 6 3,200&#b1;2,500 19.2&#b1;3.3 E1371Q (No FSK) 6 - 25.5&#b1;3.5 E1371Q (FSK) 8 -28&#b1;9 22.3&#b1;4.0 E1371Q (FSK, No ATP, No GTP) 8 270&#b1;130 19.4&#b1;4.5 E1371Q + AMP-PNP (No FSK) 4 - 24.7&#b1;6.5 E1371Q + AMP-PNP (FSK) 8 180&#b1;170 17.4&#b1;4.0 Vector Control 4 15&#b1;38 - FSK stimulation was calculated as the percentage increase in current density at -60 mV from the Erev, after 5-min exposure to 10 bc;M FSK. Login to comment 120 ABCC7 p.Glu1371Gln Based on our previous result that the phosphorylated, hydrolysis-deficient, E1371Q mutant could sense [Cl- ]o (Fig. 3f), we predicted that preventing ATP hydrolysis at ATP binding site 2 of DeltaR-CFTR would have no effect on [Cl- ]o sensing. Login to comment 121 ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Gln We tested this prediction by using DeltaR-CFTR with the E1371S mutation, which like E1371Q CFTR is hydrolysis defective [8] and found, unexpectedly, that the double-mutant CFTR channels did not respond to changes in [Cl- ]o (external Cl- stimulation; no FSK/genistein: 0.0&#b1;7.0 %, n=9; with FSK/genistein: 16.7&#b1; 7.7 %, n=4) (Fig. 4c-e). Login to comment 122 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln Role of ATP binding and ATPase activity of the NBDs in [Cl- ]o sensing by CFTR Figures 3 and 4 suggest that the role of ATP hydrolysis in [Cl- ]o sensing by CFTR is complex; e.g. AMP-PNP abolishes WT + AMP-PNP 4 nA 100 ms (i) (ii) (iii) (iv) E1371Q (FSK) 100 ms 30 nA (i) (ii) (iii) (iv) E1371Q (FSK) WT + AMP-PNP A E D C B F Fig. 3 Gating of CFTR by [Cl- ]o requires ATP hydrolysis and phosphorylation. Login to comment 125 ABCC7 p.Glu1371Gln b, d Representative fWCR current recordings measured between &#b1;100 mV in 20 mV steps from HEK cells transfected with WT CFTR plus AMP-PNP and E1371Q CFTR, as indicated. The current traces are from the top down: (i) unstimulated in 155.5 mM [Cl- ]o, (ii) forskolin (FSK)-stimulated in 155.5 mM [Cl- ]o, (iii) FSK-stimulated in 35.5 mM [Cl- ]o and (iv) FSK-stimulated in 155.5 mM [Cl- ]o. Dotted line to the right of the current traces indicates zero current level. Login to comment 126 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Arg899Gln ABCC7 p.Arg899Gln c, e Representative I-V plots for the data presented in b and d. f Percentage stimulation of either basal or FSK-activated currents by [Cl- ]o for WT CFTR (n=24), the ECL4 mutant R899Q, the hydrolysis-deficient mutant E1371Q and the double-mutant R899Q-E1371Q (see Fig. 1) under different conditions as indicated (n=4-8). Login to comment 128 ABCC7 p.Glu1371Gln *p<0.05, **p<0.01, ***p<0.001 between indicated datasets [Cl- ]o sensing by WT CFTR, whereas the E1371Q mutation has no effect. Login to comment 131 ABCC7 p.Trp401Gly Note that both of these mutations substantially reduce CFTR channel activity through mechanisms that are not secondary to changes in ATP hydrolysis rate [49], and in the case of W401G, not due to an apparent change in ATP binding affinity. Login to comment 132 ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly Sub-panels a, b and e of Fig. 5 show that the FSK-stimulated W401G CFTR mutant exhibited significantly reduced [Cl- ]o sensing compared to WT CFTR, whereas the FSK-stimulated Y1219G CFTR mutant did not (Fig. 5c-e). Login to comment 133 ABCC7 p.Trp401Gly We then reasoned that if events downstream of ATP binding to site 1, and/or NBD dimerisation, were altered by changes in [Cl- ]o (as the W401G data suggested), then the effect of [Cl- ]o on CFTR activity should be sensitive to the concentration of cytosolic ATP. Login to comment 140 ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser Figure 6 shows that when total [Cl- ] was 3 nA 100 ms DeltaR (No Stim) (i) (ii) (iii) DeltaR (No Stim) DeltaR-E1371S (No Stim) A B D C E 3 nA 100 ms DeltaR-E1371S (No Stim) (i) (ii) (iii) Fig. 4 Role of the R domain in [Cl- ]o sensing by CFTR. Login to comment 141 ABCC7 p.Glu1371Ser a, c Representative fWCR current recordings measured between &#b1;100 mV in 20 mV steps from HEK cells transfected with deltaR-CFTR or deltaR-E1371S CFTR as indicated. Login to comment 143 ABCC7 p.Glu1371Gln The current traces are from the top down: (i) unstimulated in 155.5 mM [Cl- ]o, (ii) unstimulated in 35.5 mM [Cl- ]o and (iii) unstimulated in 155.5 mM [Cl- ]o. Dotted line to the right of the current traces indicates zero current level. b, d Representative I-V plots for the data presented in a and c. e Percentage current stimulation by [Cl- ]o for WT CFTR (n=24) and for DeltaR and DeltaR-E1371Q mutants (see Fig. 1) under unstimulated (No Stim) or after exposure to a combination of forskolin and genistein (FSK/Genistein). Login to comment 149 ABCC7 p.Trp401Gly ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Gly Although neutralization of the positive charge at R899, as well as charge reversal, eliminated [Cl- ]o sensing by CFTR (Fig. 2), we have no direct evidence that Cl-ions W401G Y1219G 100 ms 5 nA W401G (i) (ii) (iii) (iv) 100 ms 4 nA (i) (ii) (iii) (iv) Y1219G A B E C D F Fig. 5 ATP binding to site 1, but not site 2, underlies [Cl- ]o sensing by CFTR. Login to comment 150 ABCC7 p.Trp401Gly ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Gly a, c Representative fWCR current recordings measured between &#b1;100 mVin 20 mV steps from HEK cells transfected with W401G CFTR or Y1219G CFTR, as indicated. The current traces are from the top down: (i) unstimulated in 155.5 mM [Cl- ]o, (ii) forskolin (FSK)-stimulated in 155.5 mM [Cl- ]o, (iii) FSK-stimulated in 35.5 mM [Cl- ]o and (iv) FSK-stimulated in 155.5 mM [Cl- ]o. Dotted line to the right of the current traces indicates zero current level. b, d Representative I-V plots for the data presented in a and c. e Percentage current stimulation by [Cl- ]o for WT CFTR (n=24) and for W401G (NBD1) and Y1219G (NBD2) mutants (see Fig. 1) (n=7-8). Login to comment 162 ABCC7 p.Arg334Gln ABCC7 p.Lys335Ala ABCC7 p.Arg117Gln ABCC7 p.Arg104Gln In support of this conclusion, the stimulating effect of [Cl- ]o cannot be due to the ion entering the pore, since mutations that do affect CFTR Cl-conductance (R104Q, R117Q, R334Q, K335A) have no effect on [Cl- ]o stimulation (Fig. 2), and the one site that is important for sensing (R899) does not affect Cl-conductance [45, 47]. Login to comment 163 ABCC7 p.Arg899Gln It is conceivable that the R899Q/E mutations cause abnormal folding of CFTR, which results in a change in the interaction between Cl- and some other residue in the protein. Login to comment 166 ABCC7 p.Glu1371Gln First, cAMP/PKA-dependent phosphorylation is required for the response in WTand E1371Q CFTR channels. Login to comment 172 ABCC7 p.Glu1371Gln Secondly, in phosphorylated CFTR channels, impairing ATP hydrolysis using the E1371Q mutation did not prevent [Cl- ]o sensing. This result implies that chloride sensing does not lead to a change in channel gating simply via a change in ATPase activity. Login to comment 174 ABCC7 p.Glu1371Gln The fact that [Cl- ]o sensing is impaired in phosphorylated E1371Q CFTR channels when AMP-PNP is present, is probably explained by AMP-PNP substituting for ATP in its actions. Login to comment 175 ABCC7 p.Glu1371Gln As Fig. 5f shows, raising cytosolic [ATP] to 2 mM, or above, abolished sensing. This is why AMP-PNP effects are mimicked by ATP, but not by the E1371Q mutation. Login to comment 176 ABCC7 p.Glu1371Gln In this context, it is important to remember that although the E1371Q mutation substantially reduces ATP hydrolysis (~70 %, [38]), the channel is still gated by ATP, even in a DeltaR background [12, 7, 8]. Login to comment 177 ABCC7 p.Glu1371Ser The one caveat to this interpretation is that [Cl- ]o sensing by DeltaR-CFTR was abolished when ATP hydrolysis at NBD2 was blocked by the E1371S mutation. Login to comment 179 ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly Thirdly, the fact that [Cl- ]o sensing is abolished by the W401G mutation in NBD1, but not by the corresponding NBD2 mutant, Y1219G (Fig. 5e), suggests that ATP binding at NBD1, and not NBD2, is mainly responsible for transducing the [Cl- ]o-dependent gating changes. Login to comment 185 ABCC7 p.Arg899Gln In addition, whether substitution of arginine for glutamine at position 899 in ECL4 leads to a change in ATPase activity of the CFTR is an important area for future research. Login to comment