ABCC7 p.Lys978Pro
| 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] 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
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).
X
ABCC7 p.Lys978Pro 23620589:104:148
status: NEW105 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.
X
ABCC7 p.Lys978Pro 23620589:105:152
status: NEW155 Fig. 5, C and D, show that the AMP-PNP-activated currents mediated by K978P-CFTR were strongly but slowly inhibited by diamide/GSH (inactivation time constant b0e;1 min).
X
ABCC7 p.Lys978Pro 23620589:155:70
status: NEW157 K978P-CFTR channels lacking Cys-1344 were insensitive to diamide/GSH (Fig. 5D, supplemental Fig. S2), as we observed previously for channels without the GOF mutation (27).
X
ABCC7 p.Lys978Pro 23620589:157:0
status: NEW158 The slow rate of K978P-CFTR inactivation by diamide/GSH is consistent with the idea that AMP-PNP binds tightly at the NBD dimer interface and restricts access of the reactive glutathione to Cys-1344 (27).
X
ABCC7 p.Lys978Pro 23620589:158:17
status: NEW169 A and B, representative macropatch records show AMP-PNP activation of K978P-CFTR (A) and K190C/K978C-CFTR (B).
X
ABCC7 p.Lys978Pro 23620589:169:70
status: NEW220 D, mean inhibition by diamide/glutathione of the AMP-PNP activated currents for K978P-CFTR (n afd; 5) and K978P/C1344A- CFTR (n afd; 3) is shown.
X
ABCC7 p.Lys978Pro 23620589:220:80
status: NEWX
ABCC7 p.Lys978Pro 23620589:220:109
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).
X
ABCC7 p.Lys978Pro 23620589:259:108
status: NEW