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. 2020 Nov 1;319(5):L770-L785.
doi: 10.1152/ajplung.00262.2019. Epub 2020 Sep 2.

G551D mutation impairs PKA-dependent activation of CFTR channel that can be restored by novel GOF mutations

Wei Wang  1   2 , Lianwu Fu  1   2 , Zhiyong Liu  2 , Hui Wen  2 , Andras Rab  3 , Jeong S Hong  3 , Kevin L Kirk  1   2 , Steven M Rowe  1   2   4   5
Affiliations

G551D mutation impairs PKA-dependent activation of CFTR channel that can be restored by novel GOF mutations

Wei Wang et al. Am J Physiol Lung Cell Mol Physiol. .

Abstract

G551D is a major disease-associated gating mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an ATP- and phosphorylation-dependent chloride channel. G551D causes severe cystic fibrosis (CF) disease by disrupting ATP-dependent channel opening; however, whether G551D affects phosphorylation-dependent channel activation is unclear. Here, we use macropatch recording and Ussing chamber approaches to demonstrate that G551D impacts on phosphorylation-dependent activation of CFTR, and PKA-mediated phosphorylation regulates the interaction between the x-loop in nucleotide-binding domain 2 (NBD2) and cytosolic loop (CL) 1. We show that G551D not only disrupts ATP-dependent channel opening but also impairs phosphorylation-dependent channel activation by largely reducing PKA sensitivity consistent with the reciprocal relationship between channel opening/gating, ligand binding, and phosphorylation. Furthermore, we identified two novel GOF mutations: D1341R in the x-loop near the ATP-binding cassette signature motif in NBD2 and D173R in CL1, each of which strongly increased PKA sensitivity both in the wild-type (WT) background and when introduced into G551D-CFTR. When D1341R was combined with a second GOF mutation (e.g., K978C in CL3), we find that the double GOF mutation maximally increased G551D channel activity such that VX-770 had no further effect. We further show that a double charge-reversal mutation of D1341R/D173R-CFTR exhibited similar PKA sensitivity when compared with WT-CFTR. Together, our results suggest that charge repulsion between D173 and D1341 of WT-CFTR normally inhibits channel activation at low PKA activity by reducing PKA sensitivity, and negative allostery by the G551D is coupled to reduced PKA sensitivity of CFTR that can be restored by second GOF mutations.

Keywords: CFTR; G551D mutation; phosphorylation; x-loop.

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Conflict of interest statement

No conflicts of interest, financial or otherwise, are declared by the authors.

Figures

Fig. 1.
Fig. 1.
G551D-CFTR impairs protein kinase A (PKA)-dependent activation. Macroscopic currents from inside-out excised patches from HEK293T cells were evoked by a ramp protocol of ±80 mV. Zero current level is indicated by the dotted line. Control condition was 143 U/mL protein kinase A (PKA) and 1.5 mM Mg-ATP, unless indicated. Two CFTR modulators, the potentiator VX-770 (200 nM) and activator curcumin (30 µM), PKA, and a CFTR channel inhibitor, CFTRInh-172 (20 μM) were added to the bath where indicated. A: CFTR modulators (VX-770: 200 nM, curcumin: 30 µM) strongly activated G551D-CFTR. B: high doses of PKA are required to increase G551D-CFTR channel activity. C: PKA dose dependently increases G551D-CFTR channel activity in the presence of ATPγs. A macropatch record shows slow activation of G551D-CFTR after adding multiple doses of PKA, whereas ATPγs had no effect on channel activity before adding PKA (n = 6). D: protein kinase A inhibitor (PKI) prevents further increase in channel activity that was activated by PKA and ATPγs. Inset: an enlarged section between zero time of recording and before adding VX-770 (n = 2). E: PKA dose dependently increases channel activity of G551D-CFTR in the presence of a low dose ATP (25 µM, n = 2). F: PKA titration of WT-CFTR illustrating PKA sensitivity. G: comparison of normalized PKA dose-response curve between WT- and G551D-CFTR. The current was normalized to the currents measured after maximal PKA activation. Curves are best fits to the Hill equation (V = Vmax [S]n/(K1/2) + [S]s); K1/2 = 25.4 ± 3.4 and 561.3 ± 89.2 for WT (n = 10) and G551D (n = 6), respectively. G551D mutant largely shifted EC50 of PKA to the right as compared with WT-CFTR. t test: ***P < 0.001. CFTR, cystic fibrosis transmembrane conductance regulator; WT, wild-type.
Fig. 2.
Fig. 2.
High doses of PKA increase channel open probability (Po) and opening rate of G551D-CFTR. A: multichannel record for inside-out patch excised from FRT cell stably expressed G551D-CFTR. Control condition was identical to Fig. 1, A and B. Additional PKA and 200 nM VX-770 were added where indicated. The holding potential: −60 mV. B: current traces from indicated sections (a, b, and c) in A. C: comparison of mean Po (left) and opening rate (right) between control and high dose of PKA as indicated by circle (control), square (PKA: 286 U/mL), and triangle (PKA: 572 U/mL). Bar graphs indicate means ± SE t test: *P < 0.05 and **P < 0.01. D: multichannel record for inside-out patch excised from FRT cell stably expressed WT-CFTR. Experimental conditions were identical to Fig. 2A. F: current traces from indicated sections (a, b, and c) in E. Comparison of mean Po (left) and opening rate (right) between control and high dose of PKA as indicated by circle (control), square (PKA: 286 U/mL), and triangle (PKA 572 U/mL). NS, not significant; CFTR, cystic fibrosis transmembrane conductance regulator; FRT, Fisher rat thyroid; PKA, protein kinase A; WT, wild-type.
Fig. 3.
Fig. 3.
Charge-reversal mutations of D1341R and D173R at interface of x-loop of NBD2 and cytosolic loop1 increase PKA sensitivity. A: human CFTR structure at phosphorylated and ATP-bound conformation (left) (49). Right displays enlarged local region around D1341 (magenta) and D173 (orange). B–D: Macroscopic currents of WT-, D1341R-, and D173R-CFTR from HEK293 cells were evoked by a ramp protocol under control condition as the same in Fig. 1. Protein kinase A inhibitor (PKI, 3 U/mL) was used to prevent further phosphorylation. E and F: PKA dose-dependent activation of D1341R and D173R mutants. Note that both D1341R and D173R require much less PKA for channel activation. G: mean data of percentage of inhibition by PKI for WT, D1341R, D173R, and K978C-CFTR (K978C data from ref 45) by PKI (left bar graph) and percentage of full activation at PKA dose of 3 U/mL (right bar graph). Data are presented as means ± SE. t test: ***P < 0.001. H: Western blot analysis of expression of WT and mutant CFTR as defined by C band (top). The relative expression levels of D1341R- and D173R-CFTR were similar to WT-CFTR as measured by densities (bottom, see materials and methods). CFTR, cystic fibrosis transmembrane conductance regulator; PKA, protein kinase A; WT, wild-type.
Fig. 4.
Fig. 4.
D1341R and D173R modestly increase ATP-independent channel activity and ATP sensitivity. Macroscopic currents in HEK 293T cells were first evoked by a ramp protocol under control condition (Fig. 1, A and B). To examine ATP-independent channel activity, bath ATP was eliminated by hexokinase plus glucose followed by bath perfusion with ATP-free solution. A and B: representative ATP titration for D1341R- and D173R-CFTR. C: mean normalized ATP-dose response curves for WT (circle), K978C (square), D1341R (diamond), and D173R (triangle). Curves are best fits to the Hill equation. D: scatter plot showing ATP-independent currents normalized to PKI control currents (right) and K1/2 of ATP dose response (left) for WT (open circle, n = 10–14), K978C (open square, n = 6–11, data from ref 45), D1341R (open triangle, n = 5), and D173R (open diamond, n = 9). Mean data were indicated by vertical bold line in WT- and mutant-CFTR. Data are presented as means ± SE. Data of mutant-CFTR were compared with WT-CFTR. t test: ***P < 0.001, **P < 0.01, and *P < 0.05. CFTR, cystic fibrosis transmembrane conductance regulator; PKI, protein kinase A inhibitor; WT, wild-type.
Fig. 5.
Fig. 5.
A double charge-reversal mutation of D1341R/D173R-CFTR exhibits similar PKI, ATP scavenge, and PKA sensitivity to that of WT-CFTR. A and B: macroscopic current records from WT- and D1341R/D173R-CFTR in HEK 293T cells. Hexokinase/glucose and additional washout without ATP were applied for eliminating bath ATP. Note that the inhibition of currents of D1341R/D173R double mutant by PKI is similar to WT-CFTR. Like WT-CFTR, the residual currents of D1341R/D173R after removal of ATP is negligible. C: PKA titration of D1341R/D173R. Inset: PKA dose-response curve for mean data (n = 6) of PKA titration with a K1/2 of 37.7 ± 10.1, which is similar to 25.4 ± 3.4 of WT-CFTR in Fig. 1D. PKA titration curve was best fit to the Hill equation as indicated in Fig. 1. CFTR, cystic fibrosis transmembrane conductance regulator; PKA, protein kinase A; PKI, protein kinase A inhibitor; WT, wild-type.
Fig. 6.
Fig. 6.
D1341R and D173R mutants rescue channel activity of G551D-CFTR. Macroscopic currents in HEK 293T cells were activated and significant basal currents were observed under control condition (as in Fig. 1, A and B) before adding high doses of PKA. A and B: G551D/D1341R- and G551D/D173R-CFTR. C: PKA dose-response curves of G551D/D1341R- and G551D/D173R-CFTR. The current was normalized to the currents measured after maximal PKA-activated currents. Curves are best fits to the Hill equation as in Fig. 1. K1/2 = 283.7 ± 44 and 272.2 ± 66.1 for G551D/D1341R (circle, n = 4) and G551D/D173R-CFTR (square, n = 5), respectively. D: Western blot analysis of expression of WT and mutant CFTR as defined by C band (top). The relative expression levels of G551D/D1341R- and G551D/D173R-CFTR were similar to WT- and G551D-CFTR as measured by densities (bottom). CFTR, cystic fibrosis transmembrane conductance regulator; PKA, protein kinase A; WT, wild-type.
Fig. 7.
Fig. 7.
Charge-reversal mutations of D1341R and D173R increase forskolin sensitivity of WT and G551D-CFTR across intact FRT epithelial monolayers. A: representatives Isc tracings of forskolin titration for WT and G551D-CFTR obtained under voltage-clamp conditions. B: mean normalized forskolin dose-response curve for WT (n = 4) and G551D-CFTR (n = 4). Paired t test: **P < 0.01. C: D1341R and D173R increase forskolin sensitivity of WT background CFTR. D: mean normalized forskolin dose-response curve for WT (n = 4) and D1341R (n = 4) and D173R-CFTR (n = 4). t test: **P < 0.01. E: D1341R and D173R increase epithelial currents of G551D mutant by increasing forskolin sensitivity. F: mean normalized forskolin dose-response curve for G551D (n = 4), G551D/D1341R (n = 4), and G551D/D173R (n = 4). Paired t test: **P < 0.05. G: mean EC50 for WT and indicated mutant construct. Data are presented as means ± SE. Mean data were compared with G551D by paired t test. **P < 0.01 and *P < 0.05. H: Western blot analysis of relative expression of WT and mutant CFTR in FRT cells. Representative of three different experiments. CFTR, cystic fibrosis transmembrane conductance regulator; FRT, Fisher rat thyroid; WT, wild-type.
Fig. 8.
Fig. 8.
A double GOF mutation of K978C and D1341R maximally activate G551D-CFTR. A: macroscopic record from K978C/D1341R-CFTR that exhibits high channel activation at low dose of PKA. B: K978C/D1341R-CFTR largely activated in the absence of PKA. C: mean percentage of maximal activation of K978C/D1341R-CFTR in the absence or presence of 3 U/mL PKA. t test: **P < 0.01. D: G551D/K978C/D1341R fully activated under control condition. Note that VX-770 had almost no effect on the channel activity of G551D/K978C/D1341R. E: G551D/K978C/D1341R requires less of PKA (2 U/mL) for its maximal activation. F: G551D/K978C/D1341R-mediated channel activity is ATP-independent. Note that hexokinase plus glucose and additional wash had little effect on currents. CFTR, cystic fibrosis transmembrane conductance regulator; WT, wild-type.
Fig. 9.
Fig. 9.
Ser737 of G551D is less amenable to phosphorylation compared with wild-type CFTR. A: comparison of phosphorylation status of Ser737 between WT and G551D-CFTR at two time points (5 and 30 min) after forskolin (10 µM) treatment of FRT cells. UNC570 antibody (epitope: aa731–742, phosphor aa: 737) was used to recognize unphosphorylated protein (n = 4). B: control phosphorylation insensitive antibody (UNC596) that recognizes a part of NBD2 (epitope: aa1204–1211), but not the R domain, was used to show total CFTR amount in each sample. CFTR, cystic fibrosis transmembrane conductance regulator; FRT, Fisher rat thyroid; WT, wild-type.
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