ABCC7 p.Glu54Ala
| Predicted by SNAP2: | A: N (53%), C: N (57%), D: N (53%), F: D (63%), G: D (63%), H: N (72%), I: D (59%), K: D (63%), L: D (59%), M: D (53%), N: D (53%), P: D (71%), Q: N (87%), R: D (66%), S: N (66%), T: D (53%), V: N (53%), W: D (66%), Y: D (59%), |
| Predicted by PROVEAN: | A: D, C: D, D: N, F: D, G: D, H: D, I: D, K: N, 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] Cysteine substitutions reveal dual functions of th... J Biol Chem. 2001 Sep 21;276(38):35660-8. Epub 2001 Jul 23. Fu J, Kirk KL
Cysteine substitutions reveal dual functions of the amino-terminal tail in cystic fibrosis transmembrane conductance regulator channel gating.
J Biol Chem. 2001 Sep 21;276(38):35660-8. Epub 2001 Jul 23., 2001-09-21 [PMID:11468285]
Abstract [show]
Previously, we observed that the cystic fibrosis transmembrane conductance regulator (CFTR) channel openings are destabilized by replacing several acidic residues in the amino-terminal tail with alanines (Naren, A. P., Cormet-Boyaka, E., Fu, J., Villain, M., Blalock, J. E., Quick, M. W., and Kirk, K. L. (1999) Science 286, 544-548). Here we determined whether this effect is due to the loss of negative charge at these sites and whether the amino-terminal tail also modulates other aspects of channel gating. We introduced cysteines at two of these positions (E54C/D58C) and tested a series of methanethiosulfonate (MTS) reagents for their effects on the gating properties of these cysteine mutants in intact Xenopus oocytes and excised membrane patches. Covalent modification of these sites with either neutral (MMTS) or charged (2-carboxyethylmethanethiosulfonate (MTSCE) and 2-(trimethylammonium)ethylmethanethiosulfonate (MTSET)) reagents markedly inhibited channel open probability primarily by reducing the rate of channel opening. The MTS reagents had negligible effects on the gating of the wild type channel or a corresponding double alanine mutant (E54A/D58A) under the same conditions. The inhibition of the opening rate of the E54C/D58C mutant channel by MMTS could be reversed by the reducing agent dithiothreitol (200 microm) or by elevating the bath ATP concentration above that required to activate maximally the wild type channel (>1 mm). Interestingly, the three MTS reagents had qualitatively different effects on the duration of channel openings (i.e. channel closing rate), namely the duration of openings was negligibly changed by the neutral MMTS, decreased by the positively charged MTSET, and increased by the negatively charged MTSCE. Our results indicate that the CFTR amino tail modulates both the rates of channel opening and channel closing and that the negative charges at residues 54 and 58 are important for controlling the duration of channel openings.
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No. Sentence Comment
4 The MTS reagents had negligible effects on the gating of the wild type channel or a corresponding double alanine mutant (E54A/D58A) under the same conditions.
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ABCC7 p.Glu54Ala 11468285:4:121
status: NEW40 This inhibition of opening rate was observed at concentrations of MTS reagents that had no effect on the gating of the wild type channel or of a corresponding alanine mutant (E54A/D58A).
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ABCC7 p.Glu54Ala 11468285:40:175
status: NEW89 In addition, like the corresponding double alanine mutant (E54A/D58A) (13), the double cysteine mutant deactivated faster than wild type CFTR fol- lowing removal of the cAMP-activating mixture (Fig. 1C).
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ABCC7 p.Glu54Ala 11468285:89:59
status: NEW96 We also tested the effects of MMTS on the wild type channel (WT CFTR) and the double alanine mutant (E54A/D58A) to determine if the inhibition was specifically due to modification of the engineered cysteines at residues 54 and 58.
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ABCC7 p.Glu54Ala 11468285:96:101
status: NEW97 At this concentration MMTS had small effects (5-10% inhibition) on the macroscopic currents mediated by WT CFTR and E54A/ D58A in intact oocytes (Fig. 2, A and B) and had no effects on the channel activities of these constructs in excised patches (see below).
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ABCC7 p.Glu54Ala 11468285:97:116
status: NEW108 When added to the cytoplasmic face of excised patches, MMTS had little effect on the channel open probability and gating kinetics (opening rate, burst duration) of either wild type CFTR or the double alanine mutant (E54A/D58A) (Fig. 3, A and B).
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ABCC7 p.Glu54Ala 11468285:108:216
status: NEW138 More cRNA was injected for the mutants than for WT CFTR to achieve approximately the same absolute current levels following activation with the cAMP mixture (2 ng for E54C/D58C; 1 ng for E54C and E54A/D58A, and 0.5 ng for WT CFTR).
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ABCC7 p.Glu54Ala 11468285:138:196
status: NEW143 A and B, representative channel records showing negligible effects of 10 M MMTS on the gating of WT CFTR and E54A/D58A CFTR.
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ABCC7 p.Glu54Ala 11468285:143:117
status: NEW212 This inhibitory effect was not observed for the wild type channel or for a corresponding double alanine mutant (E54A/D58A), which argues against a nonspecific effect of these reagents on channel activity.
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ABCC7 p.Glu54Ala 11468285:212:112
status: NEW[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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None has been submitted yet.
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.Glu54Ala 11600681:18:67
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.Glu54Ala 11600681:58:32
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.Glu54Ala 11600681:113:224
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.Glu54Ala 11600681:119:42
status: NEW167 The N-tail mutations have little effect on bulk CFTR phosphorylation A, in vitro phosphorylation of wild-type (WT) CFTR and double (E54A, D58A) and triple N-tail mutants.
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ABCC7 p.Glu54Ala 11600681:167:132
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.Glu54Ala 11600681:228:166
status: NEW