ABCMdb

ABCC7 p.Arg899Gln

None has been submitted yet.

Publications

PMID: 18449561 [PubMed] Zhou JJ et al: "Identification of positive charges situated at the outer mouth of the CFTR chloride channel pore."

No. Sentence Comment

122 Considering the data at +80 mV, where the inhibitory effects of Pt(NO2)4 2- are strongest, suggests that Pt(NO2)4 2- inhibition is significantly weakened in R104Q, K335A, and (to a lesser extent) R1128Q but not significantly altered in K114C, R117Q, K329A, K829Q, or R899Q (Fig. 8c). Login to comment

PMID: 22612315 [PubMed] Li MS et al: "Pseudohalide anions reveal a novel extracellular site for potentiators to increase CFTR function."

No. Sentence Comment

37 In some experiments, additional mutations (R334Q, K892Q, R899Q) were introduced into this background using the QuikChange site-directed mutagenesis system. Login to comment
104 Examples of the macroscopic I-V relationships for R334Q, K892Q, R899Q and K892Q/R899Q (all in an E1371Q background) are shown in Figure 5A. Login to comment
106 Block of R899Q in intact cells was not significantly affected by extracellular Co(CN)6 3- Figure 2 External pseudohalide anions weaken the apparent blocking effect of cytosolic anions under low extracellular chloride concentration conditions. Login to comment
112 Neutralization of both of these extracellular positive charges, in the K892Q/R899Q/ E1371Q triple mutant, resulted in apparent blocker sensitivity that, as in R899Q/E1371Q, was not significantly affected by extracellular Co(CN)6 3- or Co(NO2)6 3- (Figure 5D,E). Login to comment
113 Each of these mutants (R334Q, K892Q, R899Q, K892Q/R899Q) also abolished the sensitivity of current inhibition in intact cells to extracellular Cl- ions (Figure 6). Login to comment
211 Thus, the R899Q mutation showed similar sensitivity to current inhibition in intact cells as wild type, but this inhibition was completely insensitive to extracellular Co(CN)6 3- and Co(NO2)6 3- (Figures 5, 7), suggesting a specific disruption of external anion effect in this mutant. Login to comment
214 Based on our results with K892Q and R899Q (summarized in Figure 7), we speculate that pseudohalide anions interact with a site away from the channel pore to weaken channel interactions with cytoplasmic blocking substances and so promote elevated overall channel conductance. Login to comment
223 The effects of the K892Q and R899Q mutations point to some involvement of the fourth extracellular loop of CFTR in the regulation of CFTR by extracellular anions. Login to comment

PMID: 19448737 [PubMed] Zhou JJ et al: "Evidence that extracellular anions interact with a site outside the CFTR chloride channel pore to modify channel properties."

No. Sentence Comment

4 We found that mutations that remove positive charges in the 4th extracellular loop of CFTR (K892Q and R899Q) significantly alter the interaction between extracellular and intracellular Pt(NO2)4 2- ions. Login to comment
13 Nous croyons que les mutations qui e &#b4;liminent les charges positives dans la quatrie `me boucle extracellulaire du CFTR (K892Q et R899Q) modifient significativement l`interaction entre les ions Pt(NO2)4 2- intracellulaires et extracellulaires. Login to comment
68 In contrast, external Pt(NO2)4 2- ions did not significantly affect the apparent affinity of block by internal Pt(NO2)4 2- in R104Q, R117Q, or R899Q (Fig. 2). Login to comment
75 To confirm that the K892Q and R899Q mutations did not directly alter pore properties, we further investigated interactions with external Pt(NO2)4 2- at the single-channel level (Fig. 3). Login to comment
77 Furthermore, under these ionic conditions, voltage-dependent inhibition by extracellular Pt(NO2)4 2- ions in the extracellular solution appeared indistinguishable in wild type, K892Q, and R899Q (Figs. 3A, 3D, and 3E). Login to comment
80 Nevertheless, the apparent Cl- dependence of block was significantly reduced in both K335A and R899Q, and significantly enhanced in R117Q (Fig. 4D). Login to comment
97 Our present results suggest that this interaction is dependent on the presence of positive charges in ECL4 (K892, R899), since neutralization of these charges removes (R899Q) or reverses (K892Q) the effect of external Pt(NO2)4 2- ions on the blocking effect of internal Pt(NO2)4 2- (Fig. 2). Login to comment
100 Thus, charge-neutralizing mutations K892Q and R899Q have no effect on unitary conductance (Fig. 3), as we have previously reported for the charge-re- Fig. 2. Login to comment
104 Curves were fitted to mean data by eq. 1, giving the following values: for wild type, (*) Kd(0) = 89.5 mmol/L and -zd = -0.270, (*) Kd(0) = 274.1 mmol/L and -zd = -0.262; for R104Q, (*) Kd(0) = 131.1 mmol/L and -zd = -0.261, (*) Kd(0) = 200.41 mmol/L and -zd = -0.326; for R117Q, (*) Kd(0) = 44.8 mmol/L and -zd = -0.222, (*) Kd(0) = 65.0 mmol/L and -zd = -0.304; for K892Q, (*) Kd(0) = 58.4 mmol/L and -zd = -0.275, (*) Kd(0) = 21.1 mmol/L and -zd = -0.250; and for R899Q, (*) Kd(0) = 102.8 mmol/L and -zd = -0.312, (*) Kd(0) = 92.8 mmol/L and -zd = -0.334. Login to comment
110 Furthermore, inhibition of Cl-current by extracellular Pt(NO2)4 2- ions-probably a pore-mediated effect (see above)-is unaltered in K892Q or R899Q (Fig. 3), suggesting these mutations do not directly affect extracellular Pt(NO2)4 2- interactions with the pore. Login to comment
111 We believe the effects of K892Q and R899Q on interactions between extracellular and intracellular Pt(NO2)4 2- ions shown in Fig. 2 therefore reflect interactions between extracellular Pt(NO2)4 2- ions and the extracellular-facing part of the CFTR protein at some distance from the pore. Login to comment
117 (B) Control single-channel i-V relationship for wild type (*), K892Q (&), and R899Q (!). Login to comment
121 In each case data were fitted by eq. 1, giving Kd (at 0 mV) of 5.98 mmol/L for wild type, 5.74 mmol/L for K892Q, and 5.33 mmol/L for R899Q, and zd of -0.382 for wild type, -0.390 for K892Q, and -0.434 for R899Q. Login to comment
127 The effects of external Pt(NO2)4 2are sensitive to mutations away from the pore (K892Q, R899Q) but not deep within the pore (K335A, R334Q), which would be expected to alter ion-ion interactions in the pore. Login to comment
136 For 154 mmol/L Cl- (*), fitted curves gave the following values: Kd(0) = 308.8 mmol/L and -zd = -0.441 for wild type; Kd(0) = 356.4 mmol/L and -zd = -0.378 for R104Q; Kd(0) = 234.9 mmol/L and -zd = -0.376 for R117Q; Kd(0) = 204.2 mmol/L and -zd = -0.395 for K892Q; and Kd(0) = 169.4 mmol/L and -zd = -0.462 for R899Q. Login to comment
143 Nevertheless, in R899Q there is a significant decrease in the ability of external Cl-ions to antagonize block by internal Pt(NO2)4 2- (Fig. 4), suggesting that at least some part of the effect of external Cl-ions is mediated by a non-pore-dependent mechanism involving ECL4. Login to comment

PMID: 25277268 [PubMed] Broadbent SD et al: "The cystic fibrosis transmembrane conductance regulator is an extracellular chloride sensor."

No. Sentence Comment

81 Figure 2b, c shows that removing the positive charge at position 899 (R899Q) completely abolished [Cl- ]o sensing by CFTR (high Cl- stimulation; 4.3&#b1; 6.6 %, n=7), whereas all the other ECL charge neutralising mutants had no significant effect on the response (Fig. 2c). Login to comment
83 Typical I-V plots obtained in this series of experiments for WT, R899Q and the vector control are shown in Fig. 2b. Login to comment
102 To check that the restoration of [Cl- ]o sensing by E1371Q CFTR after phosphorylation (Fig. 3d) was due to an C A B WT R899Q VEC WT (i) (ii) (iii) (iv) (i) (ii) (iii) (iv) R899Q Fig. 2 Arginine residue 889 in extracellular loop 4 of CFTR is essential for [Cl- ]o sensing. Login to comment
103 a Representative fWCR current recordings measured between &#b1;100 mV in 20 mV steps from HEK cells transfected with wild type (WT) CFTR and R899Q CFTR, as indicated. The current traces are from the top down: (i) unstimulated in 155.5 mM [Cl- ]o, (ii) forskolin (FSK)-stimulated in 155.5 mM [Cl- ]o, (iii) FSK-stimulated in 35.5 mM [Cl- ]o and (iv) FSK-stimulated in 155.5 mM [Cl- ]o. Dotted line to the right of the current traces indicates zero current level. b Representative IV plots for the Vector Control (VEC), WT and R899Q CFTR, for unstimulated in 155.5 mM [Cl- ]o (black squares); FSK-stimulated in 155.5 mM [Cl- ]o (black rhombus); FSK-stimulated in 35.5 mM [Cl- ]o (white circles) and FSK-stimulated in 155.5 mM [Cl- ]o washoff (white triangles). Login to comment
110 **p<0.01 compared to WT CFTR interaction of Cl-with the extracellular domain of CFTR, we also studied the double-mutant R899Q-E1371Q. Login to comment
112 To explore the role of phosphorylation further, we studied the effect of deleting the R domain from CFTR (residues 634-836) [12, 7], which removes all the major PKA/PKC Table 1 Summary of the FSK stimulation of whole cell currents and Erev shifts observed with the CFTR constructs used in this study CFTR Construct n FSK Stimulation (%&#b1;SEM) Erev shift (mV&#b1;SEM) WT (50 bc;M ATP) 5 180&#b1;96 15.0&#b1;3.6 WT (100 bc;M ATP) 6 12,000&#b1;6,000 15.2&#b1;3.0 WT (300 bc;M ATP) 8 1,200&#b1;600 17.0&#b1;3.0 WT (1 mM ATP) 24 13,000&#b1;6,000 23.7&#b1;1.8 WT (1.3 mM ATP) 9 1,400&#b1;900 16.7&#b1;2.6 WT (2 mM ATP) 24 6,100&#b1;5,300 16.7&#b1;1.6 WT (5 mM ATP) 7 1,600&#b1;1,000 20.1&#b1;4.4 WT (50 bc;M ATP + 50 bc;M P-ATP) 7 224&#b1;130 15.3&#b1;1.0 WT + Genistein 4 7,600&#b1;5,200 26.1&#b1;5.4 WT + AMP-PNP 5 2,800&#b1;2,500 21.8&#b1;5.5 WT (3 mM MgCl2) 7 28,000&#b1;17,000 18.3&#b1;3.1 R104Q 5 4,600&#b1;1,600 28.6&#b1;4.7 K114C 5 12,000&#b1;6,700 29.2&#b1;3.0 R117Q 4 33,000&#b1;20,000 30.1&#b1;3.4 K329A 5 13,000&#b1;10,000 33.7&#b1;2.1 R334Q 9 13,000&#b1;6,700 27.3&#b1;2.9 K335A 5 3,200&#b1;1,500 20.8&#b1;7.1 W401G 7 2,600&#b1;1,800 18.5&#b1;4.8 Delta-R (No Stim) 5 - 25.1&#b1;2.7 Delta-R (No FSK, Genistein) 5 140&#b1;13 22.7&#b1;3.0 Delta-R (FSK, No Genistein) 4 89&#b1;14 15.6&#b1;6.0 Delta-R (FSK + Genistein) 6 639&#b1;432 25.1&#b1;4.9 Delta-R-E1371S (No FSK) 9 - 21.4&#b1;4.8 Delta-R-E1371S (FSK) 4 2,600&#b1;1,400 15.3&#b1;4.7 K892Q 7 16,000&#b1;9,500 36.8&#b1;4.8 R899E 4 1,200&#b1;400 25.0&#b1;2.7 R899K 4 1,600&#b1;900 26.6&#b1;2.9 R899Q 7 5,400&#b1;2,800 30.0&#b1;1.3 R899Q + AMP-PNP 4 72,000&#b1;50,000 15.2&#b1;2.8 R899Q-E1371Q (No FSK) 4 - 18.4&#b1;5.9 R899Q-E1371Q (FSK) 6 107&#b1;48 15.6&#b1;3.0 R1128Q 6 14,000&#b1;6,100 41.1&#b1;4.2 Y1219G 6 3,200&#b1;2,500 19.2&#b1;3.3 E1371Q (No FSK) 6 - 25.5&#b1;3.5 E1371Q (FSK) 8 -28&#b1;9 22.3&#b1;4.0 E1371Q (FSK, No ATP, No GTP) 8 270&#b1;130 19.4&#b1;4.5 E1371Q + AMP-PNP (No FSK) 4 - 24.7&#b1;6.5 E1371Q + AMP-PNP (FSK) 8 180&#b1;170 17.4&#b1;4.0 Vector Control 4 15&#b1;38 - FSK stimulation was calculated as the percentage increase in current density at -60 mV from the Erev, after 5-min exposure to 10 bc;M FSK. Login to comment
126 c, e Representative I-V plots for the data presented in b and d. f Percentage stimulation of either basal or FSK-activated currents by [Cl- ]o for WT CFTR (n=24), the ECL4 mutant R899Q, the hydrolysis-deficient mutant E1371Q and the double-mutant R899Q-E1371Q (see Fig. 1) under different conditions as indicated (n=4-8). Login to comment
163 It is conceivable that the R899Q/E mutations cause abnormal folding of CFTR, which results in a change in the interaction between Cl- and some other residue in the protein. Login to comment
185 In addition, whether substitution of arginine for glutamine at position 899 in ECL4 leads to a change in ATPase activity of the CFTR is an important area for future research. Login to comment

PMID: 25892339 [PubMed] Linsdell P et al: "Interactions between permeant and blocking anions inside the CFTR chloride channel pore."

No. Sentence Comment

65 (A, B, E, F) Example macroscopic I-V relationships for E1371Q (A, B) or R899Q/E1371Q (E, F) under high (154 mM; A, E) or low (4 mM; B, F) extracellular [Cl- ] conditions. In each case currents were recorded before (control) and after the addition of 100 bc;M and 1 mM Pt(NO2)4 2-to the intracellular solution. Login to comment
66 (C, G) Mean fraction of control current remaining after addition of different concentrations of Pt(NO2)4 2- at a membrane potential of -100 mV for E1371Q (C) and R899Q/E1371Q (G) under high (filled circles) and low (open circles) extracellular [Cl- ] conditions. Data have been fitted using Eq. (1) as described in the Materials and methods. Login to comment
67 (D) Mean Pt KD values obtained from such fits at different membrane potentials for E1371Q; results for R899Q/E1371Q were indistinguishable (not shown). Login to comment
69 (H) Mean values of Pt KD at 0 mV membrane potential obtained from fits of Pt KD-V relationships such as those shown in D for E1371Q (filled squares) and R899Q/E1371Q (open squares) at different extracellular [Cl- ]. Login to comment
71 There was no significant difference between E1371Q and R899Q/E1371Q (P N 0.85). Login to comment
83 Identical results were obtained in R899Q/ E1371Q channels (Fig. 2; Table 1), both at high and low [Cl- ]o conditions, indicating that extracellular Cl- interactions with this externally-located arginine residue (Fig. 1) do not contribute to these antagonistic effects. Login to comment
128 Pt KD(0) (4 mM Cl- ) (bc;M) zb4; (4 mM Cl- ) Pt KD(0) (154 mM Cl- ) (bc;M) zb4; (154 mM Cl- ) E1371Q 183.7 &#b1; 33.2 (9) -0.397 &#b1; 0.030 (9) 441.0 &#b1; 28.6 (7) -0.503 &#b1; 0.026 (7) R899Q/E1371Q 189.9 &#b1; 55.0 (6) -0.362 &#b1; 0.063 (6) 434.0 &#b1; 32.2 (7) -0.458 &#b1; 0.048 (7) K95Q/E1371Q 1110 &#b1; 172 (6)** -0.244 &#b1; 0.022 (6)* 1422 &#b1; 218 (6)** -0.193 &#b1; 0.041 (6)** I344K/E1371Q 6.92 &#b1; 1.48 (6)** -1.589 &#b1; 0.125 (6)** 164.7 &#b1; 27.5 (7)** -1.604 &#b1; 0.080 (7)** R334Q/E1371Q 1081 &#b1; 220 (4)** -0.637 &#b1; 0.106 (4)* 1112 &#b1; 144 (4)** -0.621 &#b1; 0.051 (4)* R334Q/I344K/E1371Q 39.24 &#b1; 7.94 (4)* -1.093 &#b1; 0.037 (4)** 258.3 &#b1; 30.7 (5)* -1.075 &#b1; 0.033 (5)** Fig. 4. Effect of mutations that weaken or strengthen intracellular Pt(NO2)4 2- block. Login to comment
198 Thus, block by cytoplasmic Pt(NO2)4 2- ions - and its dependence on extracellular [Cl- ] - is independent of the R899Q mutation (in an E1371Q background) (Fig. 2; Table 1). Login to comment
199 This R899Q mutation has previously been shown to disrupt the antagonistic effects of Cl- and other extracellular anions on block by different cytoplasmic anions, including Pt(NO2)4 2- [45,46]. Login to comment