ABCC7 p.Arg303Glu
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.
Comments [show]
None has been submitted yet.
No. Sentence Comment
7 However, the very low conductance of both R303E and R352E-CFTR could be greatly increased by elevating intracellular Cl-concentration.
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ABCC7 p.Arg303Glu 17043152:7:42
status: NEW43 In contrast to wild-type CFTR, mutants R303E and, to a lesser extent, R352E showed outward rectification of the I-V relationship with symmetrical 154 mM Cl- solutions.
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ABCC7 p.Arg303Glu 17043152:43:39
status: NEW63 In contrast, both R303E and R352E were associated with strong outward rectification (Fig. 2 A), which is quantified in Fig. 2 B.
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ABCC7 p.Arg303Glu 17043152:63:18
status: NEW71 As shown in Fig. 3 B, macroscopic I-V rectification was indeed highly sensitive to symmetrical Cl-concentration in both R303E and R303Q, suggesting that increasing the Cl-concentration partially reverses the effects of removing these positive charges.
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ABCC7 p.Arg303Glu 17043152:71:120
status: NEW75 (B) The degree of rectification in R303E, R303Q, and R352Q is dependent on the Cl- concentra- tion.
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ABCC7 p.Arg303Glu 17043152:75:35
status: NEW95 (B) Mean single channel current-voltage relationships constructed from such recordings for wild type (■), R303E (●) and R352E (○).
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ABCC7 p.Arg303Glu 17043152:95:113
status: NEW100 Single channel currents carried by R303E and R352E under the same symmetrical Cl- solutions used in Fig. 2 are shown in Fig. 5 A.
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ABCC7 p.Arg303Glu 17043152:100:35
status: NEW103 For example, at -80 mV, single channel current amplitude was reduced by 89 ± 0% (n = 4) in R303E and by 75 ± 2% (n = 3) in R352E, while at +80 mV, unitary currents were reduced by 30 ± 1% (n = 4) in R303E and by 25 ± 1% (n = 4) in R352E (Fig. 5 B).
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ABCC7 p.Arg303Glu 17043152:103:96
status: NEWX
ABCC7 p.Arg303Glu 17043152:103:214
status: NEW104 The Cl-dependence of I-V shape in R303E and R352E is consistent with removal of positive surface charges that normally act to increase the Cl-concentration at the inner mouth of the pore.
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ABCC7 p.Arg303Glu 17043152:104:34
status: NEW114 (A) Example single channel currents recorded from wild type, R303E, and R352E at different intracellular Cl- concentrations (154 or 304 mM, as indicated), at a membrane potential of -20 mV. For each trace, the line to the left represents the current level when the channel is closed.
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ABCC7 p.Arg303Glu 17043152:114:61
status: NEW126 The results of these experiments are summarized in Fig. 9. Modification of the introduced cysteine residue by negatively charged MTSES leads to pronounced outward rectification, similar to that seen in R303E (Fig. 2 B) and consistent with the deposition of a negative charge at the inner mouth of the pore, while modification by positively charged MTSET resulted in a wild type-like, moderately inwardly rectified I-V relationship.
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ABCC7 p.Arg303Glu 17043152:126:202
status: NEW149 Under these conditions, current carried not only by wild-type CFTR, but also by each of the channel mutants R303E, R352E (Fig. 10), R80E, R242E, R933E, R1102E, and R352Q (not depicted) showed reversal potentials that were not significantly different from the calculated Cl- equilibrium potential (+33.4 mV).
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ABCC7 p.Arg303Glu 17043152:149:108
status: NEW184 Furthermore, rectification in R303Q and R303E appeared more sensitive to the intracellular Cl-concentration than in R352Q and R352E (Fig. 3 B).
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ABCC7 p.Arg303Glu 17043152:184:40
status: NEW196 Unfortunately we were unable to obtain single channel recordings at Cl- concentrations higher than 304 mM; however, the dramatic increase in conductance seen in both R303E and R352E when intracellular [Cl-] was increased from 154 to 304 mM (Fig. 6) suggests that the conductance of these mutant channels is far from saturated at this elevated Cl-concentration, consistent with a large increase in Km.
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ABCC7 p.Arg303Glu 17043152:196:166
status: NEW199 Nevertheless, unitary currents carried by Cl- influx were also reduced in both R303E and R352E (Fig. 5).
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ABCC7 p.Arg303Glu 17043152:199:79
status: NEW208 R134Q is associated with outward rectification (Fig. 10, A and B); however, this effect shows a complex Cl-dependence that, unlike that of R303E, R303Q, and R352Q (Fig. 3 B), is not consistent with a simple surface charge effect. Furthermore, R134Q shows an extremely small apparent unitary conductance (Fig. 10 C).
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ABCC7 p.Arg303Glu 17043152:208:139
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.
Comments [show]
None has been submitted yet.
No. Sentence Comment
97 The greatest weakening of inhibition was observed in the side chain charge-reversing R303E mutant, whereas the Fig. 4.
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ABCC7 p.Arg303Glu 17293558:97:85
status: NEW