ABCC7 p.Lys978Ser
| ClinVar: |
c.2932A>T
,
p.Lys978*
D
, Likely pathogenic
|
| Predicted by SNAP2: | A: D (71%), C: D (75%), D: D (91%), E: D (85%), F: D (85%), G: D (80%), H: D (71%), I: D (75%), L: D (80%), M: D (63%), N: D (71%), P: D (91%), Q: D (75%), R: D (66%), S: D (66%), T: D (75%), V: D (80%), W: D (91%), Y: D (80%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: N, F: D, G: D, H: D, I: D, L: D, M: D, N: N, P: D, Q: N, R: N, S: N, T: D, V: D, W: D, Y: D, |
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[hide] ATP-independent CFTR channel gating and allosteric... Proc Natl Acad Sci U S A. 2010 Feb 23;107(8):3888-93. Epub 2010 Feb 3. 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 [PMID:20133716]
Abstract [show]
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel, an ATP binding cassette (ABC) transporter. CFTR gating is linked to ATP binding and dimerization of its two nucleotide binding domains (NBDs). Channel activation also requires phosphorylation of the R domain by poorly understood mechanisms. Unlike conventional ligand-gated channels, CFTR is an ATPase for which ligand (ATP) release typically involves nucleotide hydrolysis. The extent to which CFTR gating conforms to classic allosteric schemes of ligand activation is unclear. Here, we describe point mutations in the CFTR cytosolic loops that markedly increase ATP-independent (constitutive) channel activity. This finding is consistent with an allosteric gating mechanism in which ligand shifts the equilibrium between inactive and active states but is not essential for channel opening. Constitutive mutations mapped to the putative symmetry axis of CFTR based on the crystal structures of related ABC transporters, a common theme for activating mutations in ligand-gated channels. Furthermore, the ATP sensitivity of channel activation was strongly enhanced by these constitutive mutations, as predicted for an allosteric mechanism (reciprocity between protein activation and ligand occupancy). 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. Importantly, constitutive mutants that opened without ATP or NBD2 still required R domain phosphorylation for optimal activity. Our results confirm that (i) CFTR gating exhibits features of protein allostery that are shared with conventional ligand-gated channels and (ii) the R domain modulates CFTR activity independent of ATP-induced NBD dimerization.
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No. Sentence 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).
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ABCC7 p.Lys978Ser 20133716:113:32
status: NEW117 Introducing the K978C or K978S mutation strongly enhanced the basal activities of G551D-CFTR and Δ1198-CFTR Fig. 2.
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ABCC7 p.Lys978Ser 20133716:117:25
status: NEW130 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.
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ABCC7 p.Lys978Ser 20133716:130:50
status: NEW138 (C-E) High control currents for G551D and Δ1198-CFTR channels containing K978C or K978S mutations.
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ABCC7 p.Lys978Ser 20133716:138:88
status: NEW146 The currents mediated by K978S/G551D and K978C/G551D were statistically greater than the G551D currents (P < 0.05, unpaired t test).
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ABCC7 p.Lys978Ser 20133716:146:25
status: NEW[hide] CFTR inhibition by glibenclamide requires a positi... Biochim Biophys Acta. 2007 Oct;1768(10):2438-46. Epub 2007 May 21. Melin P, Hosy E, Vivaudou M, Becq F
CFTR inhibition by glibenclamide requires a positive charge in cytoplasmic loop three.
Biochim Biophys Acta. 2007 Oct;1768(10):2438-46. Epub 2007 May 21., [PMID:17582383]
Abstract [show]
The sulfonylurea glibenclamide is widely used as an open-channel blocker of the CFTR chloride channel. Here, we used site-directed mutagenesis to identify glibenclamide site of interaction: a positively charged residue K978, located in the cytoplasmic loop 3. Charge-neutralizing mutations K978A, K978Q, K978S abolished the inhibition of forskolin-activated CFTR chloride current by glibenclamide but not by CFTR(inh)-172. The charge-conservative mutation K978R did not alter glibenclamide sensitivity of CFTR current. Mutations of the neighbouring R975 (R975A, R975S, R975Q) did not affect electrophysiological and pharmacological properties of CFTR. No alteration of halide selectivity was observed with any of these CFTR mutant channels. This study identifies a novel potential inhibitor site within the CFTR molecule, and suggests a novel role of cytoplasmic loop three, within the second transmembrane domain of CFTR protein. This work is the first to report on the role of a residue in a cytoplasmic loop in the mechanism of action of the channel blocker glibenclamide.
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No. Sentence Comment
2 Charge-neutralizing mutations K978A, K978Q, K978S abolished the inhibition of forskolin-activated CFTR chloride current by glibenclamide but not by CFTRinh-172.
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ABCC7 p.Lys978Ser 17582383:2:44
status: NEW78 Effect of K978S mutation on the whole cell EGFP-CFTR chloride currents.
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ABCC7 p.Lys978Ser 17582383:78:10
status: NEW79 Representative traces of global currents recorded on HEK-293 transfected with EGFP-CFTR channels wild-type (left) and charge neutralizing mutant K978S (right) are shown in presence of Fsk 10 bc;M (A), after perfusion of glibenclamide 100 bc;M (B), and sub-sequential addition of CFTRinh-172 10 bc;M (C).
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ABCC7 p.Lys978Ser 17582383:79:145
status: NEW80 (D) Corresponding current-voltage relationships normalized by cell capacitance (n=6 for wt, n=10 for K978S).
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ABCC7 p.Lys978Ser 17582383:80:101
status: NEW81 K978S compared to CFTR-wt.
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ABCC7 p.Lys978Ser 17582383:81:0
status: NEW84 Similar effects in the presence of Fsk were recorded with cells expressing K978A, K978Q, K978R and K978S mutants CFTR.
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ABCC7 p.Lys978Ser 17582383:84:99
status: NEW85 Representative chloride currents recorded with K978S are shown in Fig. 2A (right).
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ABCC7 p.Lys978Ser 17582383:85:47
status: NEW87 Interestingly, perfusion of glibenclamide did not induce inhibition of forskolin-activated chloride currents in EGFP-CFTR-K978S expressing cells (Fig. 2B right).
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ABCC7 p.Lys978Ser 17582383:87:122
status: NEW88 The difference between the two I/V curves in the presence of Fsk versus Fsk+Glib, at all voltages for K978S channels, was not statistically different (PN0.05) using analysis of variance followed by a Bonferroni post hoc test (Fig. 2D right).
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ABCC7 p.Lys978Ser 17582383:88:102
status: NEW92 In contrast, K978S activity, after glibenclamide application, was fully inhibited by CFTRinh-172 (Fig. 3A, right).
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ABCC7 p.Lys978Ser 17582383:92:13
status: NEW93 From continuous whole cell recordings with voltage steps between -40 mV and +40 mV, we estimated that full inhibition of CFTR-wt current by glibenclamide was achieved in ~250 s (Fig. 3B left) whereas K978S activity remained remarkably stable as long as the sulfonylurea was present (Fig. 3B right).
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ABCC7 p.Lys978Ser 17582383:93:200
status: NEW94 The inhibition by CFTRinh-172 of CFTR-K978S was, on the contrary, almost immediate and very rapid as illustrated at the end of the recording, Fig. 3B (right).
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ABCC7 p.Lys978Ser 17582383:94:38
status: NEW95 Thus, the charge neutralizing mutant K978S is resistant to glibenclamide block, but remains sensitive to the blocker CFTRinh-172.
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ABCC7 p.Lys978Ser 17582383:95:37
status: NEW98 Fig. 4A presents the ratio Iglib/Ifsk, recorded at -100 mV, with 100 bc;M glibenclamide, for K978A, K978Q, K978R, K978S channels compared to CFTR-wt.
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ABCC7 p.Lys978Ser 17582383:98:117
status: NEW101 However, for K978S channels, the ratio at this voltage was significantly (Pb0.001) increased to 0.81&#b1;0.04 (n=4).
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ABCC7 p.Lys978Ser 17582383:101:13
status: NEW104 (A) Summary of mean current densities (pA/pF), recorded at -100 mV and +40 mV, in various conditions (indicated on graph) for HEK cells transfected with EGFP-CFTR-wt (left) or EGFP-CFTR-K978S (right).
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ABCC7 p.Lys978Ser 17582383:104:186
status: NEW105 Data are mean&#b1;S.E.M. of n experiments (n=7 for wt, n=10 for K978S).
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ABCC7 p.Lys978Ser 17582383:105:64
status: NEW107 Note that EGFP-CFTR-K978S channels were inhibited by CFTRinh-172 but not by glibenclamide.
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ABCC7 p.Lys978Ser 17582383:107:20
status: NEW119 For K978S channels, refractory to glibenclamide, the experimental reversal potentials were -43&#b1;1.5 mV (n=4) for bromide, -42.5&#b1; 1.5 mV (n=4) for chloride, and -29&#b1;5 mV (n=3) for iodide.
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ABCC7 p.Lys978Ser 17582383:119:4
status: NEW138 This was the case for the mutations that neutralized the charge of the side chain of the residue 978 (K978A, K978Q, K978S).
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ABCC7 p.Lys978Ser 17582383:138:116
status: NEW[hide] Converting nonhydrolyzable nucleotides to strong c... J Biol Chem. 2013 Jun 14;288(24):17122-33. doi: 10.1074/jbc.M112.442582. Epub 2013 Apr 25. 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., [PMID:23620589]
Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is the only ligand-gated ion channel that hydrolyzes its agonist, ATP. CFTR gating has been argued to be tightly coupled to its enzymatic activity, but channels do open occasionally in the absence of ATP and are reversibly activated (albeit weakly) by nonhydrolyzable nucleotides. Why the latter only weakly activates CFTR is not understood. Here we show that CFTR activation by adenosine 5'-O-(thiotriphosphate) (ATPgammaS), adenosine 5'-(beta,gamma-imino)triphosphate (AMP-PNP), and guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) is enhanced substantially by gain of function (GOF) mutations in the cytosolic loops that increase unliganded activity. This enhancement correlated with the base-line nucleotide-independent activity for several GOF mutations. AMP-PNP or ATPgammaS activation required both nucleotide binding domains (NBDs) and was disrupted by a cystic fibrosis mutation in NBD1 (G551D). GOF mutant channels deactivated very slowly upon AMP-PNP or ATPgammaS removal (taudeac approximately 100 s) implying tight binding between the two NBDs. Despite this apparently tight binding, neither AMP-PNP nor ATPgammaS activated even the strongest GOF mutant as strongly as ATP. ATPgammaS-activated wild type channels deactivated more rapidly, indicating that GOF mutations in the cytosolic loops reciprocally/allosterically affect nucleotide occupancy of the NBDs. A GOF mutation substantially rescued defective ATP-dependent gating of G1349D-CFTR, a cystic fibrosis NBD2 signature sequence mutant. Interestingly, the G1349D mutation strongly disrupted activation by AMP-PNP but not by ATPgammaS, indicating that these analogs interact differently with the NBDs. We conclude that poorly hydrolyzable nucleotides are less effective than ATP at opening CFTR channels even when they bind tightly to the NBDs but are converted to stronger agonists by GOF mutations.
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No. Sentence 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).
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ABCC7 p.Lys978Ser 23620589:162:73
status: NEW259 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).
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ABCC7 p.Lys978Ser 23620589:259:97
status: NEW