ABCC7 p.Asp47Ala
| Predicted by SNAP2: | A: N (53%), C: D (71%), E: N (72%), F: D (85%), G: D (80%), H: D (80%), I: D (85%), K: D (53%), L: D (85%), M: D (85%), N: D (75%), P: D (91%), Q: N (61%), R: D (63%), S: N (72%), T: D (71%), V: D (85%), W: D (91%), Y: D (85%), |
| Predicted by PROVEAN: | A: D, C: D, E: N, F: D, G: N, H: N, I: D, K: N, L: D, M: D, N: N, P: D, Q: N, R: N, S: N, T: N, V: D, W: D, Y: D, |
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[hide] A cluster of negative charges at the amino termina... J Physiol. 2001 Oct 15;536(Pt 2):459-70. Fu J, Ji HL, Naren AP, Kirk KL
A cluster of negative charges at the amino terminal tail of CFTR regulates ATP-dependent channel gating.
J Physiol. 2001 Oct 15;536(Pt 2):459-70., 2001-10-15 [PMID:11600681]
Abstract [show]
1. The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is activated by protein kinase A (PKA) phosphorylation of its R domain and by ATP binding at its nucleotide-binding domains (NBDs). Here we investigated the functional role of a cluster of acidic residues in the amino terminal tail (N-tail) that also modulate CFTR channel gating by an unknown mechanism. 2. A disease-associated mutant that lacks one of these acidic residues (D58N CFTR) exhibited lower macroscopic currents in Xenopus oocytes and faster deactivation following washout of a cAMP -activating cocktail than wild-type CFTR. 3. In excised membrane patches D58N CFTR exhibited a two-fold reduction in single channel open probability due primarily to shortened open channel bursts. 4. Replacing this and two nearby acidic residues with alanines (D47A, E54A, D58A) also reduced channel activity, but had negligible effects on bulk PKA phosphorylation or on the ATP dependence of channel activation. 5. Conversely, the N-tail triple mutant exhibited a markedly inhibited response to AMP-PNP, a poorly hydrolysable ATP analogue that can nearly lock open the wild-type channel. The N-tail mutant had both a slower response to AMP-PNP (activation half-time of 140 +/- 20 s vs. 21 +/- 4 s for wild type) and a lower steady-state open probability following AMP-PNP addition (0.68 +/- 0.08 vs. 0.92 +/- 0.03 for wild type). 6. Introducing the N-tail mutations into K1250A CFTR, an NBD2 hydrolysis mutant that normally exhibits very long open channel bursts, destabilized the activity of this mutant as evidenced by decreased macroscopic currents and shortened open channel bursts. 7. We propose that this cluster of acidic residues modulates the stability of CFTR channel openings at a step that is downstream of ATP binding and upstream of ATP hydrolysis, probably at NBD2.
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No. Sentence Comment
18 Replacing this and two nearby acidic residues with alanines (D47A, E54A, D58A) also reduced channel activity, but had negligible effects on bulk PKA phosphorylation or on the ATP dependence of channel activation.
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ABCC7 p.Asp47Ala 11600681:18:61
status: NEW58 The N-tail triple mutant (D47A, E54A, D58A) was prepared by using the Stratagene site-directed mutagenesis kit.
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ABCC7 p.Asp47Ala 11600681:58:26
status: NEW113 The currents mediated by D58N CFTR were somewhat greater than CFTR channel regulation by the amino tailJ. Physiol. 536.2 those observed for a triple mutant in which three acidic residues were replaced with alanines (D47A, E54A, D58A).
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ABCC7 p.Asp47Ala 11600681:113:218
status: NEW119 D58N CFTR and N-tail triple mutant (D47A, E54A, D58A) exhibit lower macroscopic currents and faster deactivation than wild-type CFTR A, schematic diagram of CFTR topology (left) and helical wheel plot of N-tail region of interest (right) showing locations of the mutations analysed in this study.
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ABCC7 p.Asp47Ala 11600681:119:36
status: NEW228 To further examine the mechanism by which the N-tail modulates ATP-dependent channel gating, we tested the responses of the wild-type and N-tail triple mutant (D47A, E54A, D58A) to ATP and AMP-PNP in single channel studies.
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ABCC7 p.Asp47Ala 11600681:228:160
status: NEW