ABCC7 p.Arg303Cys
| 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, |
[switch to compact view]
Comments [show]
None has been submitted yet.
[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
119 However, modification of R303C is illustrated in Figs. 7 and 8.
X
ABCC7 p.Arg303Cys 17043152:119:25
status: NEW124 Similar rundown was also observed in R303C (Fig. 7, A and C; Fig. 8 A), however, the shape of the I-V curve changed during the rundown process, becoming more outwardly rectifying after addition of MTSES (Fig. 7, B and E; Fig. 9) and more inwardly rectifying after application of MTSET (Figs. 8 and 9).
X
ABCC7 p.Arg303Cys 17043152:124:37
status: NEW137 Data from the same patches as in A, recorded before (control) and 6 min after addition of MTSES, are scaled to show the development of outward rectification in R303C but not wild type.
X
ABCC7 p.Arg303Cys 17043152:137:160
status: NEW152 (A) Individual examples of the time course of MTSET-induced current rundown in wild type and R303C.
X
ABCC7 p.Arg303Cys 17043152:152:93
status: NEW[hide] Cysteine-independent inhibition of the CFTR chlori... Br J Pharmacol. 2009 Jul;157(6):1065-71. Epub 2009 May 19. Li MS, Demsey AF, Qi J, Linsdell P
Cysteine-independent inhibition of the CFTR chloride channel by the cysteine-reactive reagent sodium (2-sulphonatoethyl) methanethiosulphonate.
Br J Pharmacol. 2009 Jul;157(6):1065-71. Epub 2009 May 19., [PMID:19466983]
Abstract [show]
BACKGROUND AND PURPOSE: Methanethiosulphonate (MTS) reagents are used extensively to modify covalently cysteine side chains in ion channel structure-function studies. We have investigated the interaction between a widely used negatively charged MTS reagent, (2-sulphonatoethyl) methanethiosulphonate (MTSES), and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. EXPERIMENTAL APPROACH: Patch clamp recordings were used to study a 'cys-less' variant of human CFTR, in which all 18 endogenous cysteine residues have been removed by mutagenesis, expressed in mammalian cell lines. Use of excised inside-out membrane patches allowed MTS reagents to be applied to the cytoplasmic face of active channels. KEY RESULTS: Intracellular application of MTSES, but not the positively charged MTSET, inhibited the function of cys-less CFTR. Inhibition was voltage dependent, with a K(d) of 1.97 mmol x L(-1) at -80 mV increasing to 36 mmol x L(-1) at +80 mV. Inhibition was completely reversed on washout of MTSES, inconsistent with covalent modification of the channel protein. At the single channel level, MTSES caused a concentration-dependent reduction in unitary current amplitude. This inhibition was strengthened when extracellular Cl(-) concentration was decreased. CONCLUSIONS AND IMPLICATIONS: Our results indicate that MTSES inhibits the function of CFTR in a manner that is independent of its ability to modify cysteine residues covalently. Instead, we suggest that MTSES functions as an open channel blocker that enters the CFTR channel pore from its cytoplasmic end to physically occlude Cl(-) permeation. Given the very widespread use of MTS reagents in functional studies, our findings offer a broadly applicable caveat to the interpretation of results obtained from such studies.
Comments [show]
None has been submitted yet.
No. Sentence Comment
103 This could be misinterpreted as being the result of charge deposition in the permeation pathway due to covalent modification of a cysteine side chain. For example, we previously showed that intracellular application of a low concentration of MTSES (200 mmol·L-1 ) led to outward rectification of the macroscopic current-voltage relationship in a CFTR mutant (R303C) but not wild-type CFTR, which we proposed was due to the electrostatic effects of the addition of a negative charge close to the inner mouth of the pore by covalent modification of the introduced cysteine side chain (St. Aubin and Linsdell 2006).
X
ABCC7 p.Arg303Cys 19466983:103:364
status: NEW104 The change in macroscopic current-voltage relationship shape observed in MTSES-modified R303C-CFTR is, however, similar to that resulting from voltage-dependent block by MTSES (Figure 1A).
X
ABCC7 p.Arg303Cys 19466983:104:88
status: NEW105 Nevertheless, it seems unlikely that open channel block contributed to the reported effects of MTSES on R303C-CFTR, since (i) qualitatively opposite effects were observed with positively charged MTSET, and (ii) these effects were observed at MTSES concentrations of only 200 mmol·L-1 , a concentration which does not result in significant current blockage (Figure 1).
X
ABCC7 p.Arg303Cys 19466983:105:104
status: NEW[hide] Functional Architecture of the Cytoplasmic Entranc... J Biol Chem. 2015 Jun 19;290(25):15855-65. doi: 10.1074/jbc.M115.656181. Epub 2015 May 5. El Hiani Y, Linsdell P
Functional Architecture of the Cytoplasmic Entrance to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore.
J Biol Chem. 2015 Jun 19;290(25):15855-65. doi: 10.1074/jbc.M115.656181. Epub 2015 May 5., [PMID:25944907]
Abstract [show]
As an ion channel, the cystic fibrosis transmembrane conductance regulator must form a continuous pathway for the movement of Cl(-) and other anions between the cytoplasm and the extracellular solution. Both the structure and the function of the membrane-spanning part of this pathway are well defined. In contrast, the structure of the pathway that connects the cytoplasm to the membrane-spanning regions is unknown, and functional roles for different parts of the protein forming this pathway have not been described. We used patch clamp recording and substituted cysteine accessibility mutagenesis to identify positively charged amino acid side chains that attract cytoplasmic Cl(-) ions to the inner mouth of the pore. Our results indicate that the side chains of Lys-190, Arg-248, Arg-303, Lys-370, Lys-1041, and Arg-1048, located in different intracellular loops of the protein, play important roles in the electrostatic attraction of Cl(-) ions. Mutation and covalent modification of these residues have charge-dependent effects on the rate of Cl(-) permeation, demonstrating their functional role in maximization of Cl(-) flux. Other nearby positively charged side chains were not involved in electrostatic interactions with Cl(-). The location of these Cl(-)-attractive residues suggests that cytoplasmic Cl(-) ions enter the pore via a lateral portal located between the cytoplasmic extensions to the fourth and sixth transmembrane helices; a secondary, functionally less relevant portal might exist between the extensions to the 10th and 12th transmembrane helices. These results define the cytoplasmic mouth of the pore and show how it attracts Cl(-) ions from the cytoplasm.
Comments [show]
None has been submitted yet.
No. Sentence Comment
45 Altering the side chain charge at this position (either by mutagenesis or by covalent modification of R303C by charged cysteine-reactive methanethiosulfonate (MTS) reagents) suggested that the positive charge at this position played a key role in attracting Clafa; ions electrostatically from the cytoplasm to the inner mouth of the pore (19).
X
ABCC7 p.Arg303Cys 25944907:45:102
status: NEW103 A-D, examples of modification of four different mutants (A, R303C, used in this study as a positive control; B, K190C; C, K370C; D, R1048C) by MTSES (left panels) or MTSET (right panels).
X
ABCC7 p.Arg303Cys 25944907:103:60
status: NEW109 Application of MTSES (200 òe;M) following channel activation with PKA and ATP never caused an increase in macroscopic current amplitude but decreased current amplitude in K190C, R248C, R251C, R303C, K370C, K946C, R975C, K1041C, and R1048C (Figs. 2 and 3).
X
ABCC7 p.Arg303Cys 25944907:109:196
status: NEW110 The effect of MTSET on these MTSES-sensitive mutants was to increase (K190C and R303C), decrease (R248C, K946C, K1041C, and R1048C), or have no effect (R251C, K370C, and R975C) on macroscopic current amplitude (Figs. 2 and 3).
X
ABCC7 p.Arg303Cys 25944907:110:80
status: NEW114 The effects of MTS modification of side chains within the ICLs could reflect changes in Clafa; conductance (as suggested previously for R303C (19)) or in open probability (as shown previously for K946C and R975C (11)) or a combination of the two.
X
ABCC7 p.Arg303Cys 25944907:114:139
status: NEW116 Results with R303C, K946C, and R975C, which as described above are expected to affect predominantly Clafa; conductance (R303C) or gating (K946C and R975C), suggest that use of PPi in this way can effectively separate effects on Clafa; conductance from those on gating.
X
ABCC7 p.Arg303Cys 25944907:116:13
status: NEWX
ABCC7 p.Arg303Cys 25944907:116:123
status: NEW117 Thus, the effects of MTSES and MTSET on R303C were preserved following PPi treatment, consistent with a gating-independent effect on Clafa; conductance (Figs. 4 and 5).
X
ABCC7 p.Arg303Cys 25944907:117:40
status: NEW123 This implies that modification with positively charged MTSET caused an increase in Clafa; conductance in each of K190C, R248C, R303C, K370C, and R1048C, an effect that may potentially be masked by inhibitory effects on channel open probability in normally gating channels.
X
ABCC7 p.Arg303Cys 25944907:123:130
status: NEW132 Error bars represent the means afe; S.E. from three to five patches. Cytoplasmic Entrance to the CFTR Channel Pore JUNE 19, 2015ߦVOLUME 290ߦNUMBER 25 JOURNAL OF BIOLOGICAL CHEMISTRY 15859 rent amplitude in each of K190C, R248C, R303C, K370C, K1041C, and R1048C.
X
ABCC7 p.Arg303Cys 25944907:132:244
status: NEW141 Cytoplasmic Entrance to the CFTR Channel Pore 15860 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290ߦNUMBER 25ߦJUNE 19, 2015 at SEMMELWEIS UNIV OF MEDICINE on December 4, amplitudes in unmodified channels was Cys-less b; K946C b03; R975C b0e; K370C b0e; R251C b0e; K1041C b03; R248C b0e; R1048C b0e; R303C b0e; K190C (Fig. 7A).
X
ABCC7 p.Arg303Cys 25944907:141:331
status: NEW142 Among channels showing MTSES-sensitive current amplitudes, the order following modification was K1041C b0e; R1048C b0e; K370C b0e; R248C b0e; R303C b0e; K190C (Fig. 6, B and C, and 7B).
X
ABCC7 p.Arg303Cys 25944907:142:154
status: NEW155 Single channel recording indicated directly that for each of K190C, R248C, R303C, K370C, K1041C, and R1048C Clafa; conductance was reduced relative to Cys-less (Figs. 6 and 7A), Clafa; conductance was further reduced by modification by negatively charged MTSES (Figs. 6 and 7B), and Clafa; conductance was restored to near wild-type (Cys-less) values by modification by positively charged MTSET (Figs. 6 and 7C).
X
ABCC7 p.Arg303Cys 25944907:155:75
status: NEW166 In contrast, the effects of MTS modification of PPi-treated channels in K190C, R248C, R303C, K370C, and R1048C (Fig. 5) closely mirrored the charge-dependent effects of MTS modification on single channel current amplitude (Figs. 6 and 7).
X
ABCC7 p.Arg303Cys 25944907:166:86
status: NEW173 We believe that the functional importance of Lys-190, Arg-248, Arg-303, and Lys-370 is likely greater than that of Lys-1041 and Arg-1048 because of the greater reduction in single channel current amplitude following introduction of a negative charge by MTSES modification in K190C, R248C, R303C, and K370C FIGURE 6.
X
ABCC7 p.Arg303Cys 25944907:173:289
status: NEW181 Single channel current amplitude was very low in MTSES- modified K190C, R248C, R303C, and K370C channels (b0d;25% of Cys-less current amplitude; Figs. 6, B and C, and 7B), suggesting that most permeating Clafa; ions must pass close to these residues.
X
ABCC7 p.Arg303Cys 25944907:181:79
status: NEW