ABCC7 p.Arg347Glu

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PMID: 16442101 [PubMed] Frelet A et al: "Insight in eukaryotic ABC transporter function by mutation analysis."
No. Sentence Comment
400 R347E had little or no effect on the halide permeability or conductance sequences [18].
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ABCC7 p.Arg347Glu 16442101:400:0
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PMID: 10026154 [PubMed] Cotten JF et al: "Cystic fibrosis-associated mutations at arginine 347 alter the pore architecture of CFTR. Evidence for disruption of a salt bridge."
No. Sentence Comment
1 To better understand the function of Arg-347 and to learn how mutations at this site disrupt channel activity, we mutated Arg-347 to Asp, Cys, Glu, His, Leu, or Lys and examined single-channel function.
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ABCC7 p.Arg347Glu 10026154:1:122
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5 To test this, we mutated anionic residues (Asp-924, Asp-993, and Glu-1104) to Arg in the context of either R347E or R347D mutations.
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ABCC7 p.Arg347Glu 10026154:5:107
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24 To better understand the role of Arg-347 in CFTR structure and function, we examined the effect of mutating Arg-347 to cysteine, aspartic acid, glutamic acid, lysine, and leucine on CFTR conductance.
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ABCC7 p.Arg347Glu 10026154:24:108
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25 We examined the cytosolic pH (pHc)-dependent behavior of CFTR-R347H and that of the other residue 347 mutants both with (R347C, R347D, R347E, and R347K) and without (R347L) a pHc-titratable residue.
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ABCC7 p.Arg347Glu 10026154:25:135
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86 Visual inspection suggested that the lifetimes of OL and OB states were also influenced by the nature of the residue at position 347: R347E and R347H tended to have longer dwell times in the OL and OB states, whereas R347L, R347C, and R347D tended to display shorter dwell times.
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ABCC7 p.Arg347Glu 10026154:86:134
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91 Single-channel Conductance of Residue 347 Mutants-To determine whether the residue at position 347 affects single-channel conductance and not merely the conductance state of the channel, we examined the I-V relationship and slope conductance of the mutants with slower pHc-dependent kinetics, R347H and R347E, as well as R347K.
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ABCC7 p.Arg347Glu 10026154:91:303
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94 The single-channel conductance at pHc 6.0 of wild-type CFTR, R347K, and the OB state of R347E and R347H were all very similar (in pS): 7.7 Ϯ 0.4, 8.3 Ϯ 0.6, 7.4 Ϯ 0.4, and 6.9 Ϯ 0.2, respectively (n ϭ 3 for each).
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ABCC7 p.Arg347Glu 10026154:94:88
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95 The single-channel conductance of the OL states of R347E and R347H at pHc 6.0 were also very similar (in pS): 1.5 Ϯ 0.1 and 1.6 Ϯ 0.1, respectively (n ϭ 3 and 4 for each).
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ABCC7 p.Arg347Glu 10026154:95:51
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102 The first explanation seems unlikely because the reciprocal lifetime of the OL state which represents the "on" rate for the proton is very slow (e.g. it is 6 ϫ 107 M -1 s-1 for R347E).
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ABCC7 p.Arg347Glu 10026154:102:183
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107 Dwell-time Analysis of OL and OB States-We performed a dwell-time analysis of the lifetimes of the OL and OB states of R347E and R347H to enable more quantitative comparisons between them and to better understand their pHc dependence.
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ABCC7 p.Arg347Glu 10026154:107:119
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113 The observable pK (0 mV) for the equilibrium between OL and OB of R347E and R347H were 6.4 and 6.3, respectively. The faster kinetics of R347D, R347C, and R347L made dwell-time analysis for these mutants less reliable.
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ABCC7 p.Arg347Glu 10026154:113:66
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119 Single-channel I-V relationships for R347E (OL and OB states), R347H (OL and OB states), R347K, R347D/D924R, and wild-type CFTR at pHc 6.0. n ϭ 2-4 at each data point.
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ABCC7 p.Arg347Glu 10026154:119:37
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122 As a control for the variance analysis, we examined the R347E mutant on which we had also done dwell-time analysis (Fig. 3A); as expected, Fig. 3B shows that the variance of R347E goes through a maximum between pHc 6.5 and 5.5.
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ABCC7 p.Arg347Glu 10026154:122:56
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ABCC7 p.Arg347Glu 10026154:122:174
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124 Fig. 4A shows qualitatively that both conductance states of R347E were voltage-dependent.
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ABCC7 p.Arg347Glu 10026154:124:60
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125 Fig. 4B shows quantitatively that at pHc 6.0 the OL and OB states for R347E and R347H were both influenced by the transmembrane voltage.
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ABCC7 p.Arg347Glu 10026154:125:70
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128 The degree of voltage dependence was similar for both mutants despite the charge differences at residue 347 and yielded a ␪z of 0.25 and 0.21 for R347E and R347H, respectively. The voltage dependence was asymmetrically disposed between the rate of entry into the OB state and the rate of exit from OB (Fig. 4B).
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ABCC7 p.Arg347Glu 10026154:128:153
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130 The rate of exit from the OB state (␶B -1 ) was more voltage-dependent than the rate of exit from the OL state (␶L -1 ) for both mutants (␦ ϭ 0.8 versus 1 - ␦ ϭ 0.2 for R347E and ␦ ϭ 0.7 versus 1 - ␦ ϭ 0.3 for R347H).
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ABCC7 p.Arg347Glu 10026154:130:209
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134 A, dwell-time analysis in the OL and OB conductance states versus pHc for R347E and R347H.
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ABCC7 p.Arg347Glu 10026154:134:74
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136 B, open-channel current variance of the R347C, R347D, R347L, and R347E mutants versus pHc.
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ABCC7 p.Arg347Glu 10026154:136:65
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142 A, current records from excised, inside-out membrane patch containing R347E channel.
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ABCC7 p.Arg347Glu 10026154:142:70
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144 B, dwell times (pHc 6.0) in the OL state (closed symbols) or OB state (open symbols) versus voltage for R347E (left, circles) and R347H (right, squares).
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ABCC7 p.Arg347Glu 10026154:144:104
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147 arises from charge movement through a voltage field and since R347E and R347H displayed similar voltage dependences and carry different charges at position 347, residue 347 is not likely moving through a transmembrane potential during interchange between OL and OB states.
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ABCC7 p.Arg347Glu 10026154:147:62
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153 To identify the Arg-347 interaction partner, we replaced Arg-347 with an anionic residue (R347E or R347D) and introduced an arginine residue in the place of candidate partners in a salt bridge.
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ABCC7 p.Arg347Glu 10026154:153:90
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154 We studied the conductance properties of the following double mutants: R347D/D924R, R347D/D993R, and R347E/E1104R.
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ABCC7 p.Arg347Glu 10026154:154:101
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155 The R347D/D993R and R347E/E1104R mutants each had two conductance states with pHc-dependent behavior (Fig. 5).
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ABCC7 p.Arg347Glu 10026154:155:20
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157 Accordingly, for R347D/D993R and R347E/E1104R the current variance in the open state increased with decreasing pHc (Fig. 5B).
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ABCC7 p.Arg347Glu 10026154:157:33
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158 Qualitatively, the lifetimes of the OL and OB conductance states in the R347D/D993R and R347E/E1104R were similar to that of the R347D and R347E mutants, respectively. The amplitude of the OL state was larger for both of these double mutants as compared with the single mutants (Figs.
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ABCC7 p.Arg347Glu 10026154:158:88
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ABCC7 p.Arg347Glu 10026154:158:139
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160 We also observed an infrequent, additional small conductance state in the R347E/E1104 mutant (see amplitude histogram in Fig. 5A); this is likely due to the E1104R mutation itself.
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ABCC7 p.Arg347Glu 10026154:160:74
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171 Additionally, the single-channel slope conductances of R347H and R347E were the same in both OB and OL states.
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ABCC7 p.Arg347Glu 10026154:171:65
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179 A, single-channel current tracings from excised, inside-out membrane patches containing R347E/E1104R, R347D/D924R, and R347D/D993R.
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ABCC7 p.Arg347Glu 10026154:179:88
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181 B, current variance of R347E/E1104R, R347D/D924R, and R347D/D993R at the indicated pHc was collected as in Fig. 3.
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ABCC7 p.Arg347Glu 10026154:181:23
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PMID: 10385235 [PubMed] Walsh KB et al: "Structural and ionic determinants of 5-nitro-2-(3-phenylprophyl-amino)-benzoic acid block of the CFTR chloride channel."
No. Sentence Comment
5 4 NPPB inhibition of CFTR currents in oocytes expressing the mutants K335E and R347E also occurred in a voltage-dependent manner.
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ABCC7 p.Arg347Glu 10385235:5:79
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6 However, the Kds for NPPB block were increased to 371 and 1573 mM, for the K335E and R347E mutants, respectively.
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ABCC7 p.Arg347Glu 10385235:6:85
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26 ), and the K335E and R347E mutants obtained from Dr K Kunzelmann (Albert-Ludwigs University, Freiburg, Germany).
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ABCC7 p.Arg347Glu 10385235:26:21
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76 E€ect of NPPB on K335E and R347E CFTR mutants The pKa of NPPB is close to 4.5 (Wangemann et al., 1986; Walsh & Wang, 1998), and thus the drug molecules are predominately charged (499%) in the ND-96 solution at pH 7.5.
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ABCC7 p.Arg347Glu 10385235:76:32
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77 Since the negatively charged drug may interact with positively charged amino acid residues in the pore of the CFTR channel, we examined the e€ect of NPPB on the mutants K335E and R347E.
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ABCC7 p.Arg347Glu 10385235:77:184
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90 The K335E channel displayed a more linear I/V relationship (Figure 5) and the R347E channel a more outward-rectifying I/V relationship (Figure 6) than that measured with the wild-type channel (Figure 3).
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ABCC7 p.Arg347Glu 10385235:90:78
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92 NPPB was less e€ective in blocking the CFTR currents in oocytes expressing the K335E and R347E channels.
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ABCC7 p.Arg347Glu 10385235:92:94
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93 The Kd for NPPB block of the current was increased from 166 mM for the wild-type to 371 and 1573 mM for the K335E and R347E mutants, respectively (Figures 5 and Figure 5 E€ect of NPPB on the K335E CFTR channel. Left panel: I/V relationship for the CFTR current measured in the presence and absence of 100 mM NPPB.
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ABCC7 p.Arg347Glu 10385235:93:118
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100 Figure 6 E€ect of NPPB on the R347E CFTR channel. Left panel: I/V relationship for the CFTR current measured in the presence and absence of 100 mM NPPB.
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ABCC7 p.Arg347Glu 10385235:100:35
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102 Right panel: concentration versus response curve for inhibition of the wild-type and R347E channels by NPPB.
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ABCC7 p.Arg347Glu 10385235:102:85
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105 For the R347E data, each point represents the mean+s.e.mean of three to ®ve experiments.
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ABCC7 p.Arg347Glu 10385235:105:8
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106 The theoretical curve for the R347E data provided a Kd of 1573 mM.
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ABCC7 p.Arg347Glu 10385235:106:30
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108 Although the sensitivity of the mutant channels to NPPB was reduced, both the K335E and R347E mutants displayed a voltage-dependence to NPPB block that was similar to the wild-type channel (Figure 7).
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ABCC7 p.Arg347Glu 10385235:108:88
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109 The slopes of the lines obtained from the relationship between the fractional block (Id/Io) and voltage had values of 0.23 (wild-type), 0.24 (K335E) and 0.30 (R347E).
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ABCC7 p.Arg347Glu 10385235:109:159
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116 Relationship between the fractional drug block (Id/I0) and the membrane potential determined for the wild-type (100 mM NPPB), K335E (400 mM NPPB) and R347E (1 mM NPPB) CFTR channels.
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ABCC7 p.Arg347Glu 10385235:116:150
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117 The slopes of the straight lines had values of 0.23 (wild-type), 0.24 (K335E) and 0.30 (R347E).
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ABCC7 p.Arg347Glu 10385235:117:88
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152 This insensitivity is most striking for the R347E mutant, suggesting that this positively charged residue serves as an important determinant in the binding of the negatively charged drug molecules.
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ABCC7 p.Arg347Glu 10385235:152:44
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153 While the concentration versus response curves for NPPB block of mutants K335E and R347E were shifted to higher concentrations, NPPB produced a voltage-dependent block in the mutants that was almost identical to that of the wild-type channel.
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ABCC7 p.Arg347Glu 10385235:153:83
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159 Although the anity of the R347E mutant for DPC has not been determined, it is likely that mutations at several sites in the M6 region will e€ect arylaminobenzoate block.
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ABCC7 p.Arg347Glu 10385235:159:32
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PMID: 10811966 [PubMed] Zhang ZR et al: "Direct comparison of NPPB and DPC as probes of CFTR expressed in Xenopus oocytes."
No. Sentence Comment
436 Two mutants were studied by Walsh: K335E, predicted to be at the extracellular end of TM6, and R347E, predicted to be at the cytoplasmic end of TM6.
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ABCC7 p.Arg347Glu 10811966:436:95
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439 Block of R347E-CFTR was also reduced (KD ‫ס‬ 1573 ␮M) and the voltage-dependence was increased significantly.
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ABCC7 p.Arg347Glu 10811966:439:9
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443 The results of the R347E mutation studied by Walsh [56] are difficult to interpret because this mutation causes disruption of channel structure due to loss of a salt bridge with an aspartic acid in TM8 [5].
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ABCC7 p.Arg347Glu 10811966:443:19
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PMID: 15857825 [PubMed] Wang W et al: "Activating cystic fibrosis transmembrane conductance regulator channels with pore blocker analogs."
No. Sentence Comment
68 To determine whether NPPB activates CFTR by binding to the same (or different) site that causes a pore block, we tested its effects on a CFTR pore mutant (R347E) that is resistant to block by NPPB (19).
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ABCC7 p.Arg347Glu 15857825:68:155
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69 Fig. 1 (E and F) shows that NPPB stimulated the currents mediated by R347E-CFTR at positive potentials to a greater extent compared with wild-type CFTR at moderate levels of phosphorylation (high PKA concentration (110 units/ml), followed by PKI).
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ABCC7 p.Arg347Glu 15857825:69:69
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70 Unlike the wild-type channel, the R347E-CFTR currents were stimulated by NPPB even at negative potentials.
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ABCC7 p.Arg347Glu 15857825:70:34
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71 Thus, NPPB behaves more as a pure agonist for the R347E pore mutant, which implies that this compound stimulates channel opening by binding to a site that is distinct from the pore-blocking site.
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ABCC7 p.Arg347Glu 15857825:71:50
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138 F, at low doses, NPPB stimulates currents in both directions for moderately phosphorylated R347E-CFTR channels in an HEK-293T patch.
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ABCC7 p.Arg347Glu 15857825:138:91
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PMID: 9518736 [PubMed] Briel M et al: "Cl- transport by cystic fibrosis transmembrane conductance regulator (CFTR) contributes to the inhibition of epithelial Na+ channels (ENaCs) in Xenopus oocytes co-expressing CFTR and ENaC."
No. Sentence Comment
35 The following CFTR mutations were generated: CF-associated mutations such as ÄF508, G551D and R117H as well as artificial mutations within MSD1 such as R347E and K335E (Hipper et al. 1995).
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ABCC7 p.Arg347Glu 9518736:35:157
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105 Finally, two artificial mutations (R347E and K335E) in the 6th transmembrane spanning domain were initially created in order to examine properties of the putative pore of CFTR (Anderson et al. 1991).
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ABCC7 p.Arg347Glu 9518736:105:35
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110 In contrast, other mutations, which still activated whole-cell Cl¦ conductance (R117H, R347E, K335E) downregulated ENaC currents.
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ABCC7 p.Arg347Glu 9518736:110:92
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PMID: 9813087 [PubMed] Jiang Q et al: "Cystic fibrosis transmembrane conductance regulator-associated ATP release is controlled by a chloride sensor."
No. Sentence Comment
6 Despite the lack of a need for Cl-conductance through CFTR to modulate ATP release, alterations in channel pore residues R347 and R334 caused changes in the relative ability of different halides to activate ATP efflux (wtCFTR, Cl ϾϾ Br; R347P, Cl ϾϾ Br; R347E, Br ϾϾ Cl; R334W, Cl ϭ Br).
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ABCC7 p.Arg347Glu 9813087:6:278
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80 The cAMP-stimulated Cl- and Br- conductances of the R347E mutant (5.3 Ϯ 2.1 ␮s and 12.15 Ϯ 3.3 ␮s, respectively) were much lower than that of the wild type.
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ABCC7 p.Arg347Glu 9813087:80:52
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89 For mutant R347, a cDNA segment was cut out from the PTM1-R347E CFTR construct (kindly provided by Dr. M. Welsh, University of Iowa) by restriction enzymes Erev,Br Erev,Cl RT F ------- PBr Br- [ ] PCl Cl- [ ] --------------------------       ln=- MroI and Bst1107I, and was subcloned into the pBQ-CFTR plasmid between the same restriction sites.
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ABCC7 p.Arg347Glu 9813087:89:58
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206 Mutations in the CFTR Channel Pore Alter the Halide Dependence of ATP Release To explore the mechanisms by which changes in the extracellular Cl-concentration affect activation of ATP release, we examined the CFTR mutants R334W, R347P, and R347E.
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ABCC7 p.Arg347Glu 9813087:206:240
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208 Immunoprecipitation studies using a rabbit antisera raised against a synthetic peptide corresponding to residues 45-65 in the CFTR NH2 terminus demonstrated similar levels of protein expression in wtCFTR, R334W, R347P, and R347E cRNA-injected oocytes (Fig. 6 A).
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ABCC7 p.Arg347Glu 9813087:208:223
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216 In contrast, the stimulated Cl-conductances were 4and 20-fold lower in the R334W and R347E mutants, respectively (Fig. 6 and Table II).
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ABCC7 p.Arg347Glu 9813087:216:85
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242 The halide dependence of CFTR-modulated ATP release was similarly analyzed in R334W, R347P, and R347E cRNA-injected oocytes.
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ABCC7 p.Arg347Glu 9813087:242:96
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247 Most importantly, the halide dependence in the R347E mutant was reversed to Br- ϾϾ Cl- ; JATP in the presence of Cl- (1.5 Ϯ 0.49 pmoles/min) was 7.3-fold lower than that in the presence of Br- (11 Ϯ 4.8 pmoles/min; Fig. 7 and Table II).
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ABCC7 p.Arg347Glu 9813087:247:47
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248 Interestingly, exposure of R347E-expressing oocytes to extracellular Br- had a lasting affect on JATP, even after extracellular Br- was replaced with Cl- .
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ABCC7 p.Arg347Glu 9813087:248:27
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279 Furthermore, the R347E had a greater than 20-fold reduced GBr as compared with wtCFTR, yet the magnitude of JATP in the presence of extracellular Br- was sixfold higher for R347E when compared with wtCFTR cRNA-injected oocytes.
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ABCC7 p.Arg347Glu 9813087:279:17
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ABCC7 p.Arg347Glu 9813087:279:173
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295 Mutants of CFTR that alter charged arginine residues within the channel pore including R334W, R347E, and R347P were used.
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ABCC7 p.Arg347Glu 9813087:295:94
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298 The Wc I/V relationships for wtCFTR, R347P, R347E, R334W cRNA, and water-injected oocytes are given in B.
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ABCC7 p.Arg347Glu 9813087:298:44
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303 These results depict average I/V relationships from N experiments for wtCFTR (N ϭ 5), R347E (N ϭ 5), R347P (N ϭ 5), R334W (N ϭ 8), and water (N ϭ 7)-injected oocytes from at least two independent batches of oocytes.
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ABCC7 p.Arg347Glu 9813087:303:92
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307 We therefore characterized the effects of arginine mutations R334W, R347P, and R347E on CFTR-modulated ATP release.
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ABCC7 p.Arg347Glu 9813087:307:79
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314 In contrast, the R347E mutation caused pronounced alterations of both electrophysiologic as well as ATP release properties of CFTR.
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ABCC7 p.Arg347Glu 9813087:314:17
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338 Summary of Electrophysiologic Measurements on CFTR Mutants Wild type R347P R347E R334W Mock ⌬GCl (cAMP) (␮s) 121 Ϯ 35 125 Ϯ 28 5.3 Ϯ 2.1 32 Ϯ 20 -0.61 Ϯ 0.62 (N ϭ 5) (N ϭ 5) (N ϭ 5) (N ϭ 8) (N ϭ 7) GBr /GCl 0.93 Ϯ 0.01 1.12 Ϯ 0.01 2.36 Ϯ 0.23 1.12 Ϯ 0.03 (N ϭ 5) (N ϭ 5) (N ϭ 5) (N ϭ 8) - PBr /PCl 1.13 Ϯ 0.02 1.02 Ϯ 0.01 1.00 Ϯ 0.04 1.22 Ϯ 0.02 (N ϭ 5) (N ϭ 5) (N ϭ 5) (N ϭ 8) - JATP (Cl) 8.7 Ϯ 3.4 2.0 Ϯ 0.98 1.5 Ϯ 0.49 3.4 Ϯ 1.3 0.021 Ϯ 0.001 (N ϭ 15) (N ϭ 9) (N ϭ 11) (N ϭ 7) (N ϭ 15) JATP (Br) 1.9 Ϯ 0.51 0.26 Ϯ 0.17 11.0 Ϯ 4.8 3.7 Ϯ 1.5 0.013 Ϯ 0.006 (N ϭ 15) (N ϭ 9) (N ϭ 11) (N ϭ 7) (N ϭ 15) JATP (Br)/JATP (Cl) 0.22 0.13 7.3 1.1 - CFTR cRNAs were injected into Xenopus oocytes and evaluated for both electrophysiologic and ATP release characteristics.
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ABCC7 p.Arg347Glu 9813087:338:75
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354 We thank Dr. Welsh for his mutant CFTR cDNAs R334W, R347E, and R347P, and his thoughtful review of this manuscript.
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ABCC7 p.Arg347Glu 9813087:354:52
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PMID: 9922375 [PubMed] Sheppard DN et al: "Structure and function of the CFTR chloride channel."
No. Sentence Comment
104 Thus, depending on the experimental tants, R347E and R1030E, did not alter the anion perme- conditions and whether block of the channel by I0 is con- ability sequence, although PI/PCl values were increased.
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ABCC7 p.Arg347Glu 9922375:104:43
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PMID: 9922376 [PubMed] Dawson DC et al: "CFTR: mechanism of anion conduction."
No. Sentence Comment
432 This behavior is consistent with the notion that iodide can reside in the channel with-(TM1), K335E (TM6), R347E (TM6), and R1030E (TM10).
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ABCC7 p.Arg347Glu 9922376:432:107
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437 The R347E reducing agent like thiosulfate ion.
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ABCC7 p.Arg347Glu 9922376:437:4
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557 Ineffect of mutations on anion binding suggests that permeant anions can interact with the channel sufficiently confirmation of these results, Smith and Dawson (unpublished data) found that blockade of CFTR by externalstrongly to impede conduction rates but that the ''tight binding`` is dependent on some element of pore conforma- SCN was abolished in R347E and also in R347Q CFTR, confirming the importance of the positive charge at thistion that is not critical to promote the entry of anions into the pore.
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ABCC7 p.Arg347Glu 9922376:557:353
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PMID: 7947814 [PubMed] Loo TW et al: "Mutations to amino acids located in predicted transmembrane segment 6 (TM6) modulate the activity and substrate specificity of human P-glycoprotein."
No. Sentence Comment
279 Mutation of Lys335 or Arg347 to Glu in TM6 altered the permeability and/or conductance ratios for halide ions (Anderson et al., 1991).
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ABCC7 p.Arg347Glu 7947814:279:22
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PMID: 9674722 [PubMed] Schwiebert EM et al: "Cystic fibrosis: a multiple exocrinopathy caused by dysfunctions in a multifunctional transport protein."
No. Sentence Comment
243 Other mutations in TMD1 which affect function and cause disease include other arginines in predicted ␣-helix 6, R334W, R347P, and R347E (97,98) and a glycine, G314E, in ␣-helix 5 (99).
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ABCC7 p.Arg347Glu 9674722:243:138
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PMID: 9879057 [PubMed] Gallet X et al: "Topological model of membrane domain of the cystic fibrosis transmembrane conductance regulator."
No. Sentence Comment
232 Mutations associated with mild forms of cystic fibrosis (R117H, R334W, and R347P) implicate three of our inner pore residues in the chloride conductance.50 In other studies, basic amino acids of membrane helices were replaced by acidic residues (K95D, K335E, R347E, and R1030E).
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ABCC7 p.Arg347Glu 9879057:232:259
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236 Mutations associated with mild forms of cystic fibrosis (R117H, R334W, and R347P) implicate three of our inner pore residues in the chloride conductance.50 In other studies, basic amino acids of membrane helices were replaced by acidic residues (K95D, K335E, R347E, and R1030E).
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ABCC7 p.Arg347Glu 9879057:236:259
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PMID: 9512029 [PubMed] Mansoura MK et al: "Cystic fibrosis transmembrane conductance regulator (CFTR) anion binding as a probe of the pore."
No. Sentence Comment
229 Hipper et al. (1995) reported that the mutations R334E, R334H, K335E, K335H, R347E, and R347H did not alter CFTR conduction properties, but careful inspection of the data presented revealed that the level of CFTR expression was very low so that altered properties of mutant CFTRs might have been easily obscured.
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ABCC7 p.Arg347Glu 9512029:229:77
status: NEW
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PMID: 9463368 [PubMed] Sugita M et al: "CFTR Cl- channel and CFTR-associated ATP channel: distinct pores regulated by common gates."
No. Sentence Comment
115 We confirmed the reduced single channel conductance of R347E CFTR (1.6 Ϯ 0.1 pS; nϭ3, p Ͻ0.01) (Figure 7C).
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ABCC7 p.Arg347Glu 9463368:115:55
status: NEW
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117 Effects of DIDS and R347E mutation on CFTR Cl- channels and CFTR-associated ATP channels.
X
ABCC7 p.Arg347Glu 9463368:117:20
status: NEW
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121 (C) Current traces from a MDCK cell expressing R347E in an inside-out patch with 100 mM ATP in the pipette and 140 mM Clin the bath.
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ABCC7 p.Arg347Glu 9463368:121:47
status: NEW
X
ABCC7 p.Arg347Glu 9463368:121:55
status: NEW
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122 R347E mutation had little effect on the slope conductance of the CFTR-associated ATP channels (5.08 Ϯ 0.27 pS; nϭ3) (Figure 7C).
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ABCC7 p.Arg347Glu 9463368:122:0
status: NEW
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128 We first examined the deletion mutant CFTR"06;R-S660A, Fig. 8.
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ABCC7 p.Arg347Glu 9463368:128:47
status: NEW
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189 Third, mutation of a residue in the CFTR Cl- channel pore, R347E, had little effect on the conductance of CFTR-associated ATP channels, in contrast to its effect on the CFTR Cl- conductance.
X
ABCC7 p.Arg347Glu 9463368:189:59
status: NEW
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272 Acknowledgements We thank M.Welsh for providing the CFTR∆R-S660A and CFTR S-oct-D mutants, R.Kopito for providing the K1250A and K464A mutants, J.Engelhardt for providing the R347E mutant, U.Patel for her precious technical help and D.Mak for helpful discussions.
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ABCC7 p.Arg347Glu 9463368:272:182
status: NEW
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123 Effects of DIDS and R347E mutation on CFTR Cl-channels and CFTR-associated ATP channels.
X
ABCC7 p.Arg347Glu 9463368:123:20
status: NEW
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129 R347E mutation had little effect on the slope conductance of the CFTR-associated ATP channels (5.08 afe; 0.27 pS; nafd;3) (Figure 7C).
X
ABCC7 p.Arg347Glu 9463368:129:0
status: NEW
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199 Third, mutation of a residue in the CFTR Cl-channel pore, R347E, had little effect on the conductance of CFTR-associated ATP channels, in contrast to its effect on the CFTR Cl-conductance.
X
ABCC7 p.Arg347Glu 9463368:199:58
status: NEW
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282 Acknowledgements We thank M.Welsh for providing the CFTRƊR-S660A and CFTR S-oct-D mutants, R.Kopito for providing the K1250A and K464A mutants, J.Engelhardt for providing the R347E mutant, U.Patel for her precious technical help and D.Mak for helpful discussions.
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ABCC7 p.Arg347Glu 9463368:282:180
status: NEW
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PMID: 9379167 [PubMed] Tabcharani JA et al: "Halide permeation in wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels."
No. Sentence Comment
214 In this regard, the mutant R347D had normal anion:cation permeability ratios (Linsdell and Hanrahan, unpublished observations) as did R347E (Anderson et al., 1991), but arg352, which has also been proposed to form part of the anion selectivity filter, remains a candidate.
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ABCC7 p.Arg347Glu 9379167:214:134
status: NEW
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264 Arg347 May Contribute to a Weak Field Strength Site for Iodide High macroscopic PI/PCl ratios have been reported previously for CFTR channels in which positively charged residues in the membrane spanning regions were mutated to negatively charged residues (K95E, 1.43; K335E, 1.37; R347E, 0.9; R1030E, 0.81; Anderson et al., 1991).
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ABCC7 p.Arg347Glu 9379167:264:282
status: NEW
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265 The PI/PCl ratio obtained for R347D under biionic conditions is intermediate between Iunbl and Ibl in the wild-type channel, and is consistent with the value of 0.9 reported previously for R347E (Anderson et al., 1991).
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ABCC7 p.Arg347Glu 9379167:265:189
status: NEW
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231 In this regard, the mutant R347D had normal anion:cation permeability ratios (Linsdell and Hanrahan, unpublished observations) as did R347E (Anderson et al., 1991), but arg352, which has also been proposed to form part of the anion selectivity filter, remains a candidate.
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ABCC7 p.Arg347Glu 9379167:231:134
status: NEW
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289 Arg347 May Contribute to a Weak Field Strength Site for Iodide High macroscopic PI/PCl ratios have been reported previously for CFTR channels in which positively charged residues in the membrane spanning regions were mutated to negatively charged residues (K95E, 1.43; K335E, 1.37; R347E, 0.9; R1030E, 0.81; Anderson et al., 1991).
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ABCC7 p.Arg347Glu 9379167:289:282
status: NEW
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290 The PI/PCl ratio obtained for R347D under biionic conditions is intermediate between Iunbl and Ibl in the wild-type channel, and is consistent with the value of 0.9 reported previously for R347E (Anderson et al., 1991).
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ABCC7 p.Arg347Glu 9379167:290:189
status: NEW
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PMID: 9089437 [PubMed] Cheung M et al: "Locating the anion-selectivity filter of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel."
No. Sentence Comment
20 The mutation R347E, however, had little or no effect on the halide permeability or conductance sequences (Anderson et al., 1991b).
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ABCC7 p.Arg347Glu 9089437:20:13
status: NEW
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205 Curiously, neither of these mutations nor the mutations R347E and R1030E were reported to alter the Cl- to Naϩ permeability ratio (PCl/PNa), and the latter two mutations had minimal effects on halide permeability or conductance ratios (Anderson et al., 1991b).
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ABCC7 p.Arg347Glu 9089437:205:56
status: NEW
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PMID: 8744306 [PubMed] Cheung M et al: "Identification of cystic fibrosis transmembrane conductance regulator channel-lining residues in and flanking the M6 membrane-spanning segment."
No. Sentence Comment
190 The multiple ion occupancy effects were eliminated by mutation of Arg347 to Asp or His, and the single-channel conductance was reduced (Tabcharani et al., 1993).
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ABCC7 p.Arg347Glu 8744306:190:24
status: NEW
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192 Curiously, the mutation R347E was reported to have little effect on the relative halide permeability or conductance sequences of the channel (Anderson et al., 1991b).
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ABCC7 p.Arg347Glu 8744306:192:24
status: NEW
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PMID: 7589561 [PubMed] Hipper A et al: "Mutations in the putative pore-forming domain of CFTR do not change anion selectivity of the cAMP activated Cl- conductance."
No. Sentence Comment
6 The following mutations were examined: K335E, R347E, R334E, K335H, R347H, R334H.
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ABCC7 p.Arg347Glu 7589561:6:46
status: NEW
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8 Moreover, anomalous mole fraction behavior for the cAMP activated current could not be detected: neither in wt-CFTR nor in R347E-CFTR.
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ABCC7 p.Arg347Glu 7589561:8:123
status: NEW
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20 (v) Mutations in the apparent 6th a-helical transmembrane domain of CFTR (K335E, R347E) resulted in changes in the halide selectivity of CFTR [1].
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ABCC7 p.Arg347Glu 7589561:20:81
status: NEW
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32 Synthesis of mutated CFTR-cDNA was induced by annealing of ampicillin repair oligonucleotide and oligonucleotide primers carrying the respective mutation changing positively charged to negatively charged amino acids (R334E, R347E, K335E) or replacing R and K at these positions by histidines (R334H, R347H, K335H).
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ABCC7 p.Arg347Glu 7589561:32:224
status: NEW
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80 Next, positively charged amino acids R334, R347, K335 located in the putative 6th pore forming transmembrane a-helical domain of CFTR, were exchanged by histidines (R334H, R347H, K335H) or by the negatively charged glutamate (R334E, R347E, K335E).
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ABCC7 p.Arg347Glu 7589561:80:233
status: NEW
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81 Wc conductances were activated significantly by IBMX in all 6 mutants but to variable degrees (AG in/.tS): 3.2 + 0.6 (R334E, n = 20), 2.7 + 0.6 (R334H, n = 13), 7.1 + 0.9 (K335E, n-- 20), 2.8 + 0.7 (K335H, n = 10), 3.2 + 0.04 (R347E, n = 32) and 1.8 + 0.3 (R347H, n = 10).
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ABCC7 p.Arg347Glu 7589561:81:227
status: NEW
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86 SCN conductance in wt and R347E CFTR and pH &sensitivity of histidine mutants Extracellular C1- (101 mmol/1) was partially (7 mmol/1) or almost completely (96 mmol/l) replaced by equal concentrations of SCN- and we currents were measured in IBMX stimulated oocytes.
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ABCC7 p.Arg347Glu 7589561:86:26
status: NEW
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87 For both wt CFTR and R347E-CFTR we found reduced wc conductance when C1- was replaced by SCN-.
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ABCC7 p.Arg347Glu 7589561:87:21
status: NEW
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88 Anomalous mole fraction behavior could neither be detected for wt-CFTR nor for R347E mutants (Fig. 4).
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ABCC7 p.Arg347Glu 7589561:88:79
status: NEW
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92 However, unlike in the previous study in R347H [7] no significant changes of G could be detected when extracellular pH was G wtCFTR R347E 10 - (n=17) 0.8- _T_ T 0~- ~ ~, ~, 0.4- ~ ~ o.2- ~ 0.0-I # 10 0.8 0.60.40.20.0- (n=14) T .__T_ i:I I L J Fig. 4. Summary of the conductance ratios obtained in wt and R347E-CFTR transfected oocytes stimulated by IBMX when 101 mmol/1 extracellular CI- was replaced by (mmol/1) 94 C1- and 7 SCN- (94/7) and 5 C1- and 96 SCN- (5/96), respectively.
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ABCC7 p.Arg347Glu 7589561:92:132
status: NEW
X
ABCC7 p.Arg347Glu 7589561:92:304
status: NEW
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105 In fact, in a previous study lysine and arginine were replaced by negatively charged amino acids in the first and sixth transmembrane domain (K95D, K335E, R347E), respectively [1].
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ABCC7 p.Arg347Glu 7589561:105:155
status: NEW
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108 In the present study we repeated some of the published (K335E, R347E, R347H) and performed additional mutations (R334E, R334H, K335H) which are all located in the putative sixth transmembrane domain and overexpressed the respective CFTRs in oocytes.
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ABCC7 p.Arg347Glu 7589561:108:63
status: NEW
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112 Comparable wc measurements were performed in the present study (K335E, R347E compared to R347D in [7]) with SCN- and C1- present in the extracellular bath solution at different concentration ratios.
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ABCC7 p.Arg347Glu 7589561:112:71
status: NEW
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114 Even more important, SCN- con- ductance was not different for the R347E mutant.
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ABCC7 p.Arg347Glu 7589561:114:66
status: NEW
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PMID: 7535742 [PubMed] Bonizzato A et al: "Analysis of the complete coding region of the CFTR gene in a cohort of CF patients from north-eastern Italy: identification of 90% of the mutations."
No. Sentence Comment
51 Genotypes were identified in six of them and were AF508/1898+3AG, R 1162X/ R347E AF508/2789 + 5G-~A, AFS08/R709X, 1717-1G--~ A/3849 + 10KbC-~T and R553X/2789 + 5G-~A.
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ABCC7 p.Arg347Glu 7535742:51:75
status: NEW
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PMID: 7515047 [PubMed] Akabas MH et al: "Amino acid residues lining the chloride channel of the cystic fibrosis transmembrane conductance regulator."
No. Sentence Comment
15 Mutation of Lys-95 to Asp, in M1, and Lys-335 and Arg-347 to Glu, in M6, altered the permeability andlor conductance ratios for halides (6).
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ABCC7 p.Arg347Glu 7515047:15:50
status: NEW
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PMID: 1322048 [PubMed] Anderson MP et al: "Chloride channels in the apical membrane of normal and cystic fibrosis airway and intestinal epithelia."
No. Sentence Comment
69 Relative anion permeability of CAMP-regulated channels in apical membrane and in cells expressing wild-type and mutant CFTR PXlPCl- Gx/Gcl- Br- ClI- Br ClI- CAMP 3T3 fibroblasts CFTR 1.11 1.00 0.59 1.26 1.00 0.29 HeLa cells CFTR 1.24 1.00 0.57 1.02 1.00 0.39 K95D 1.25 1.00 1.43 1.39 1.00 0.75 K335E 1.06 1.00 1.37 1.71 1.00 1.43 R347E 1.24 1.00 0.90 1.46 1.00 0.47 Rl030E 1.46 1.00 0.81 1.50 1.00 0.28 Human airway epithelia Apical ND 1.00 0.41 ND 1.00 0.35 T84 epithelia Apical 1.21 1.00 0.56 0.92 1.00 0.47 over cations.
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ABCC7 p.Arg347Glu 1322048:69:330
status: NEW
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PMID: 1375035 [PubMed] Welsh MJ et al: "Cystic fibrosis transmembrane conductance regulator: a chloride channel with novel regulation."
No. Sentence Comment
92 Mutation of 2 other basic residues, R347E and R1030E, did not change the selectivity sequence.
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ABCC7 p.Arg347Glu 1375035:92:36
status: NEW
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PMID: 23784545 [PubMed] Cai Z et al: "Acute inhibition of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel by thyroid hormones involves multiple mechanisms."
No. Sentence Comment
251 The occurrence of subconductance states is more frequent in some site-directed mutations in the MSDs [e.g., R334C-CFTR (70), R347E-CFTR (11), and R352E-CFTR (13)], while wild-type murine CFTR resides for prolonged periods in a minuscule subconductance state and only transitions infrequently to the full open-state (37).
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ABCC7 p.Arg347Glu 23784545:251:125
status: NEW
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PMID: 25236767 [PubMed] Lodwick D et al: "Sulfonylurea receptors regulate the channel pore in ATP-sensitive potassium channels via an intersubunit salt bridge."
No. Sentence Comment
192 Similar to Kir6.2-Lys338 , single charge reversal, Kir6.1-R347E, expressed with WT SUR2A, resulted in channels with decreased EC50 for pinacidil (0.71 + - 1.2 bc;M compared with WT, 43.9 + - 1.3 bc;M, Figures 9a and 9b).
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ABCC7 p.Arg347Glu 25236767:192:58
status: NEW
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193 Reinstatement of the salt bridge by charge reversals in both subunits (Kir6.1-R347E-SUR2A-E1318R) moved the EC50 back towards WT (23.5 + - 1.3 bc;M, Figure 9b).
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ABCC7 p.Arg347Glu 25236767:193:78
status: NEW
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194 Similarly, when glibenclamide sensitivity was considered, like Kir6.2-Lys338 , single charge reversal of Kir6.1-R347E resulted in an increase in IC50 (241.6 + - 1.1 nM compared with WT, 6.2 + - 1.1 nM), which was restored towards that of WT channels when single charge reversals in both subunits of residues involved in this salt bridge (Kir6.1-R347E-SUR2A-E1318R) were made (13.8 + - 1.1 nM) (Figure 9c).
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ABCC7 p.Arg347Glu 25236767:194:112
status: NEW
X
ABCC7 p.Arg347Glu 25236767:194:345
status: NEW
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197 Disruption of native Figure 9 Kir6.1-R347E-SUR2A-E1318R charge swap also restores WT channel pharmacology (a) Example recording of WT Kir6.1-SUR2A channel activation by 1, 10 and 100 bc;M pinacidil and inhibition with 10 bc;M glibenclamide.
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ABCC7 p.Arg347Glu 25236767:197:39
status: NEW
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199 Kir6.1-R347E and SUR2A WT formed channels which had a marked increase in sensitivity to pinacidil which was reversed by co-expression with the putative charge-swap partner SUR2A-E1318R (EC50 = 0.7 + - 0.1 bc;M compared with 44.9 + - 8.1 bc;M for WT channels, P <0.001, and 23.5 + - 13.4 bc;M with Kir6.1-R347E and SUR2A-E1318R, P > 0.05, ANOVA with Bonferroni`s post-hoc test, n6 cells for each data point).
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ABCC7 p.Arg347Glu 25236767:199:7
status: NEW
X
ABCC7 p.Arg347Glu 25236767:199:313
status: NEW
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201 Kir6.1-R347E co-expression with SUR2A WT showed a leftward shift in concentration-inhibition curve with an IC50 changing from 5.7 + - 0.1 nM to 480 + - 27 nM (P <0.0001).
X
ABCC7 p.Arg347Glu 25236767:201:7
status: NEW
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