ABCC7 p.Arg303Lys
| Predicted by SNAP2: | A: D (85%), C: D (85%), D: D (95%), E: D (91%), F: D (91%), G: D (91%), H: D (85%), I: D (91%), K: D (80%), L: D (80%), M: D (85%), N: D (80%), P: D (95%), Q: D (80%), S: D (80%), T: D (85%), V: D (91%), W: D (91%), Y: D (91%), |
| Predicted by PROVEAN: | A: N, C: D, D: N, E: N, F: D, G: D, H: N, I: D, K: N, L: D, M: N, N: N, P: D, Q: N, S: N, T: N, V: D, W: D, Y: N, |
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[hide] Positive charges at the intracellular mouth of the... J Gen Physiol. 2006 Nov;128(5):535-45. Epub 2006 Oct 16. Aubin CN, Linsdell P
Positive charges at the intracellular mouth of the pore regulate anion conduction in the CFTR chloride channel.
J Gen Physiol. 2006 Nov;128(5):535-45. Epub 2006 Oct 16., [PMID:17043152]
Abstract [show]
Many different ion channel pores are thought to have charged amino acid residues clustered around their entrances. The so-called surface charges contributed by these residues can play important roles in attracting oppositely charged ions from the bulk solution on one side of the membrane, increasing effective local counterion concentration and favoring rapid ion movement through the channel. Here we use site-directed mutagenesis to identify arginine residues contributing important surface charges in the intracellular mouth of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel pore. While wild-type CFTR was associated with a linear current-voltage relationship with symmetrical solutions, strong outward rectification was observed after mutagenesis of two arginine residues (R303 and R352) located near the intracellular ends of the fifth and sixth transmembrane regions. Current rectification was dependent on the charge present at these positions, consistent with an electrostatic effect. Furthermore, mutagenesis-induced rectification was more pronounced at lower Cl(-) concentrations, suggesting that these mutants had a reduced ability to concentrate Cl(-) ions near the inner pore mouth. R303 and R352 mutants exhibited reduced single channel conductance, especially at negative membrane potentials, that was dependent on the charge of the amino acid residue present at these positions. However, the very low conductance of both R303E and R352E-CFTR could be greatly increased by elevating intracellular Cl(-) concentration. Modification of an introduced cysteine residue at position 303 by charged methanethiosulfonate reagents reproduced charge-dependent effects on current rectification. Mutagenesis of arginine residues in the second and tenth transmembrane regions also altered channel permeation properties, however these effects were not consistent with changes in channel surface charges. These results suggest that positively charged arginine residues act to concentrate Cl(-) ions at the inner mouth of the CFTR pore, and that this contributes to maximization of the rate of Cl(-) ion permeation through the pore.
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No. Sentence Comment
67 Charge-conservative R303K and R352K mutations were associated with wild type like linear I-V relationships (Fig. 3 A).
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ABCC7 p.Arg303Lys 17043152:67:20
status: NEW[hide] Identification of a second blocker binding site at... Mol Pharmacol. 2007 May;71(5):1360-8. Epub 2007 Feb 9. St Aubin CN, Zhou JJ, Linsdell P
Identification of a second blocker binding site at the cytoplasmic mouth of the cystic fibrosis transmembrane conductance regulator chloride channel pore.
Mol Pharmacol. 2007 May;71(5):1360-8. Epub 2007 Feb 9., [PMID:17293558]
Abstract [show]
Chloride transport by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is inhibited by a broad range of substances that bind within a wide inner vestibule in the pore and physically occlude Cl(-) permeation. Binding of many of these so-called open-channel blockers involves electrostatic interactions with a positively charged lysine residue (Lys95) located in the pore. Here, we use site-directed mutagenesis to identify a second blocker binding site located at the cytoplasmic mouth of the pore. Mutagenesis of a positively charged arginine at the cytoplasmic mouth of the pore, Arg303, leads to significant weakening of the blocking effects of suramin, a large negatively charged organic molecule. Apparent suramin affinity is correlated with the side chain charge at this position, consistent with an electrostatic interaction. In contrast, block by suramin is unaffected by mutagenesis of Lys95, suggesting that it does not approach close to this important pore-forming lysine residue. We propose that the CFTR pore inner vestibule contains two distinct blocker binding sites. Relatively small organic anions enter deeply into the pore to interact with Lys95, causing an open-channel block that is sensitive to both the membrane potential and the extracellular Cl(-) concentration. Larger anionic molecules can become lodged in the cytoplasmic mouth of the pore where they interact with Arg303, causing a distinct type of open-channel block that is insensitive to membrane potential or extracellular Cl(-) ions. The pore may narrow significantly between the locations of these two blocker binding sites.
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No. Sentence Comment
109 charge-conserving R303K mutant did not significantly affect the Kd value for suramin inhibition (Fig. 6).
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ABCC7 p.Arg303Lys 17293558:109:18
status: NEW[hide] Molecular mechanism of arachidonic acid inhibition... Eur J Pharmacol. 2007 Jun 1;563(1-3):88-91. Epub 2007 Mar 3. Zhou JJ, Linsdell P
Molecular mechanism of arachidonic acid inhibition of the CFTR chloride channel.
Eur J Pharmacol. 2007 Jun 1;563(1-3):88-91. Epub 2007 Mar 3., [PMID:17397825]
Abstract [show]
Arachidonic acid inhibits the activity of a number of different Cl- channels, however its molecular mechanism of action is not known. Here we show that inhibition of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels by arachidonic acid is weakened following mutagenesis of two positively charged pore-lining amino acids. Charge-neutralizing mutants K95Q and R303Q both increased the Kd for inhibition from approximately 3.5 microM in wild type to approximately 17 microM. At both sites, the effects of mutagenesis were dependent of the charge of the substituted side chain. We suggest that arachidonic acid interacts electrostatically with positively charged amino acid side chains in the cytoplasmic vestibule of the CFTR channel pore to block Cl- permeation.
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No. Sentence Comment
38 Thus, while the charge conservative K95R and R303K mutations were not associated with a significant increase in Kd (PN0.3, two-tailed t-test) all mutations which neutralized or reversed either of these two positive charges were associated with a significant increase in Kd (Fig. 2).
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ABCC7 p.Arg303Lys 17397825:38:45
status: NEW