ABCC7 p.Glu267Cys
| Predicted by SNAP2: | A: N (61%), C: D (63%), D: N (78%), F: D (80%), G: D (59%), H: D (75%), I: D (63%), K: D (59%), L: D (63%), M: D (53%), N: N (57%), P: D (71%), Q: N (66%), R: D (66%), S: N (66%), T: N (61%), V: D (53%), W: D (85%), Y: D (75%), |
| Predicted by PROVEAN: | A: N, C: D, D: N, F: D, G: N, H: N, I: D, K: N, L: D, M: D, N: N, P: N, Q: N, R: N, S: N, T: N, V: D, W: D, Y: D, |
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[hide] An electrostatic interaction at the tetrahelix bun... J Biol Chem. 2014 Oct 31;289(44):30364-78. doi: 10.1074/jbc.M114.595710. Epub 2014 Sep 4. Wang W, Roessler BC, Kirk KL
An electrostatic interaction at the tetrahelix bundle promotes phosphorylation-dependent cystic fibrosis transmembrane conductance regulator (CFTR) channel opening.
J Biol Chem. 2014 Oct 31;289(44):30364-78. doi: 10.1074/jbc.M114.595710. Epub 2014 Sep 4., [PMID:25190805]
Abstract [show]
The CFTR channel is an essential mediator of electrolyte transport across epithelial tissues. CFTR opening is promoted by ATP binding and dimerization of its two nucleotide binding domains (NBDs). Phosphorylation of its R domain (e.g. by PKA) is also required for channel activity. The CFTR structure is unsolved but homology models of the CFTR closed and open states have been produced based on the crystal structures of evolutionarily related ABC transporters. These models predict the formation of a tetrahelix bundle of intracellular loops (ICLs) during channel opening. Here we provide evidence that residues E267 in ICL2 and K1060 in ICL4 electrostatically interact at the interface of this predicted bundle to promote CFTR opening. Mutations or a thiol modifier that introduced like charges at these two positions substantially inhibited ATP-dependent channel opening. ATP-dependent activity was rescued by introducing a second site gain of function (GOF) mutation that was previously shown to promote ATP-dependent and ATP-independent opening (K978C). Conversely, the ATP-independent activity of the K978C GOF mutant was inhibited by charge- reversal mutations at positions 267 or 1060 either in the presence or absence of NBD2. The latter result indicates that this electrostatic interaction also promotes unliganded channel opening in the absence of ATP binding and NBD dimerization. Charge-reversal mutations at either position markedly reduced the PKA sensitivity of channel activation implying strong allosteric coupling between bundle formation and R domain phosphorylation. These findings support important roles of the tetrahelix bundle and the E267-K1060 electrostatic interaction in phosphorylation-dependent CFTR gating.
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No. Sentence Comment
127 As we showed in Fig. 2 the single E267C and K1060C mutations reduced CFTR channel activity especially after PKI addition but not to the extent of the charge-reversal substitutions.
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ABCC7 p.Glu267Cys 25190805:127:34
status: NEW128 Subsequent exposure of E267C-CFTR to MTSET rapidly and markedly inhibited the post-PKI current which was reversed by bath addition of DTT (Fig. 4A).
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ABCC7 p.Glu267Cys 25190805:128:23
status: NEW131 To test this idea we exposed E267C-CFTR channels to the poorly hydrolyzable AMP-PNP in the continued presence of ATP prior to MTSET addition (Fig. 4D).
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ABCC7 p.Glu267Cys 25190805:131:29
status: NEW133 E267C-CFTR channels also were strongly activated by AMP-PNP but remained sensitive to subsequent treatment with MTSET followed by DTT (Fig. 4D).
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ABCC7 p.Glu267Cys 25190805:133:0
status: NEW136 These findings support the idea that the accessibility of E267C to thiol modification is reduced in the open channel conformation(s) as would be expected if this position is buried in a helix bundle upon channel opening.
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ABCC7 p.Glu267Cys 25190805:136:58
status: NEW212 A positively charged thiol modifier strongly inhibits E267C-CFTR currents in an activity-dependent manner but modestly stimulates K1060C-CFTR currents.
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ABCC7 p.Glu267Cys 25190805:212:54
status: NEW213 A, E267C-CFTR macropatch record showing strong and rapid inhibition of this cysteine mutant by bath addition of 30 òe;M MTSET and reversal of this inhibition by 5 mM DTT.
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ABCC7 p.Glu267Cys 25190805:213:3
status: NEW220 D, E267C-CFTR macropatch record showing that MTSET inhibition was very slow when added after the current was stimulated with 2 mM AMP-PNP.
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ABCC7 p.Glu267Cys 25190805:220:3
status: NEW235 In addition, the rate of inhibition of the E267C mutant by this reagent was slowed markedly by AMP-PNP, a poorly hydrolysable ATP analog that prolongs open channel bursts and stabilizes the NBD dimer.
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ABCC7 p.Glu267Cys 25190805:235:43
status: NEW