ABCC7 p.Arg899Gln
Predicted by SNAP2: | A: N (87%), C: N (72%), D: N (61%), E: N (82%), F: N (66%), G: N (72%), H: N (93%), I: N (87%), K: N (97%), L: N (82%), M: N (82%), N: N (82%), P: N (78%), Q: N (87%), S: N (93%), T: N (93%), V: N (87%), W: D (63%), Y: N (72%), |
Predicted by PROVEAN: | A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, S: N, T: N, V: N, W: N, Y: N, |
[switch to compact view]
Comments [show]
None has been submitted yet.
[hide] Identification of positive charges situated at the... Pflugers Arch. 2008 Nov;457(2):351-60. Epub 2008 May 1. Zhou JJ, Fatehi M, Linsdell P
Identification of positive charges situated at the outer mouth of the CFTR chloride channel pore.
Pflugers Arch. 2008 Nov;457(2):351-60. Epub 2008 May 1., [PMID:18449561]
Abstract [show]
We have used site-directed mutagenesis and functional analysis to identify positively charged amino acid residues in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel that interact with extracellular anions. Mutation of two positively charged arginine residues in the first extracellular loop (ECL) of CFTR, R104, and R117, as well as lysine residue K335 in the sixth transmembrane region, leads to inward rectification of the current-voltage relationship and decreased single channel conductance. These effects are dependent on the charge of the substituted side chain and on the Cl(-) concentration, suggesting that these positive charges normally act to concentrate extracellular Cl(-) ions near the outer mouth of the pore. Side chain charge-dependent effects are mimicked by manipulating charge in situ by mutating these amino acids to cysteine followed by covalent modification with charged cysteine-reactive reagents, confirming the location of these side chains within the pore outer vestibule. State-independent modification of R104C and R117C suggests that these residues are located at the outermost part of the pore. We suggest that ECL1 contributes to the CFTR pore external vestibule and that positively charged amino acid side chains in this region act to attract Cl(-) ions into the pore. In contrast, we find no evidence that fixed positive charges in other ECLs contribute to the permeation properties of the pore.
Comments [show]
None has been submitted yet.
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).
X
ABCC7 p.Arg899Gln 18449561:122:267
status: NEW[hide] Pseudohalide anions reveal a novel extracellular s... Br J Pharmacol. 2012 Nov;167(5):1062-75. doi: 10.1111/j.1476-5381.2012.02041.x. Li MS, Cowley EA, Linsdell P
Pseudohalide anions reveal a novel extracellular site for potentiators to increase CFTR function.
Br J Pharmacol. 2012 Nov;167(5):1062-75. doi: 10.1111/j.1476-5381.2012.02041.x., [PMID:22612315]
Abstract [show]
BACKGROUND AND PURPOSE There is great interest in the development of potentiator drugs to increase the activity of the cystic fibrosis transmembrane conductance regulator (CFTR) in cystic fibrosis. We tested the ability of several anions to potentiate CFTR activity by a novel mechanism. EXPERIMENTAL APPROACH Patch clamp recordings were used to investigate the ability of extracellular pseudohalide anions (Co(CN)(6) (3-) , Co(NO(2) )(6) (3-) , Fe(CN)(6) (3-) , IrCl(6) (3-) , Fe(CN)(6) (4-) ) to increase the macroscopic conductance of mutant CFTR in intact cells via interactions with cytoplasmic blocking anions. Mutagenesis of CFTR was used to identify a possible molecular mechanism of action. Transepithelial short-circuit current recordings from human airway epithelial cells were used to determine effects on net anion secretion. KEY RESULTS Extracellular pseudohalide anions were able to increase CFTR conductance in intact cells, as well as increase anion secretion in airway epithelial cells. This effect appears to reflect the interaction of these substances with a site on the extracellular face of the CFTR protein. CONCLUSIONS AND IMPLICATIONS Our results identify pseudohalide anions as increasing CFTR function by a previously undescribed molecular mechanism that involves an interaction with an extracellular site on the CFTR protein. Future drugs could utilize this mechanism to increase CFTR activity in cystic fibrosis, possibly in conjunction with known intracellularly-active potentiators.
Comments [show]
None has been submitted yet.
No. Sentence Comment
37 In some experiments, additional mutations (R334Q, K892Q, R899Q) were introduced into this background using the QuikChange site-directed mutagenesis system.
X
ABCC7 p.Arg899Gln 22612315:37:57
status: NEW104 Examples of the macroscopic I-V relationships for R334Q, K892Q, R899Q and K892Q/R899Q (all in an E1371Q background) are shown in Figure 5A.
X
ABCC7 p.Arg899Gln 22612315:104:64
status: NEWX
ABCC7 p.Arg899Gln 22612315:104:80
status: NEW106 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.
X
ABCC7 p.Arg899Gln 22612315:106:9
status: NEW112 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).
X
ABCC7 p.Arg899Gln 22612315:112:77
status: NEWX
ABCC7 p.Arg899Gln 22612315:112:159
status: NEW113 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).
X
ABCC7 p.Arg899Gln 22612315:113:37
status: NEWX
ABCC7 p.Arg899Gln 22612315:113:50
status: NEW211 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.
X
ABCC7 p.Arg899Gln 22612315:211:10
status: NEW214 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.
X
ABCC7 p.Arg899Gln 22612315:214:36
status: NEW223 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.
X
ABCC7 p.Arg899Gln 22612315:223:29
status: NEW[hide] Evidence that extracellular anions interact with a... Can J Physiol Pharmacol. 2009 May;87(5):387-95. doi: 10.1139/y09-023. Zhou JJ, Linsdell P
Evidence that extracellular anions interact with a site outside the CFTR chloride channel pore to modify channel properties.
Can J Physiol Pharmacol. 2009 May;87(5):387-95. doi: 10.1139/y09-023., [PMID:19448737]
Abstract [show]
Extracellular anions enter into the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, interacting with binding sites on the pore walls and with other anions inside the pore. There is increasing evidence that extracellular anions may also interact with sites away from the channel pore to influence channel properties. We have used site-directed mutagenesis and patch-clamp recording to identify residues that influence interactions with external anions. Anion interactions were assessed by the ability of extracellular Pt(NO2)42- ions to weaken the pore-blocking effect of intracellular Pt(NO2)42- ions, a long-range ion-ion interaction that does not appear to reflect ion interactions inside the pore. 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)42- ions. These mutations do not affect unitary Cl- conductance or block of single-channel currents by extracellular Pt(NO2)42- ions, however, suggesting that the mutated residues are not in the channel pore region. These results suggest that extracellular anions can regulate CFTR pore properties by binding to a site outside the pore region, probably by a long-range conformational change. Our findings also point to a novel function of the long 4th extracellular loop of the CFTR protein in sensing and (or) responding to anions in the extracellular solution.
Comments [show]
None has been submitted yet.
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.
X
ABCC7 p.Arg899Gln 19448737:4:102
status: NEW13 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.
X
ABCC7 p.Arg899Gln 19448737:13:134
status: NEW68 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).
X
ABCC7 p.Arg899Gln 19448737:68:143
status: NEW75 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).
X
ABCC7 p.Arg899Gln 19448737:75:30
status: NEW77 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).
X
ABCC7 p.Arg899Gln 19448737:77:188
status: NEW80 Nevertheless, the apparent Cl- dependence of block was significantly reduced in both K335A and R899Q, and significantly enhanced in R117Q (Fig. 4D).
X
ABCC7 p.Arg899Gln 19448737:80:95
status: NEW97 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).
X
ABCC7 p.Arg899Gln 19448737:97:168
status: NEW100 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.
X
ABCC7 p.Arg899Gln 19448737:100:46
status: NEW104 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.
X
ABCC7 p.Arg899Gln 19448737:104:467
status: NEW110 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.
X
ABCC7 p.Arg899Gln 19448737:110:141
status: NEW111 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.
X
ABCC7 p.Arg899Gln 19448737:111:36
status: NEW117 (B) Control single-channel i-V relationship for wild type (*), K892Q (&), and R899Q (!).
X
ABCC7 p.Arg899Gln 19448737:117:78
status: NEW121 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.
X
ABCC7 p.Arg899Gln 19448737:121:133
status: NEWX
ABCC7 p.Arg899Gln 19448737:121:205
status: NEW127 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.
X
ABCC7 p.Arg899Gln 19448737:127:88
status: NEW136 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.
X
ABCC7 p.Arg899Gln 19448737:136:311
status: NEW143 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.
X
ABCC7 p.Arg899Gln 19448737:143:17
status: NEW[hide] The cystic fibrosis transmembrane conductance regu... Pflugers Arch. 2015 Aug;467(8):1783-94. doi: 10.1007/s00424-014-1618-8. Epub 2014 Oct 4. Broadbent SD, Ramjeesingh M, Bear CE, Argent BE, Linsdell P, Gray MA
The cystic fibrosis transmembrane conductance regulator is an extracellular chloride sensor.
Pflugers Arch. 2015 Aug;467(8):1783-94. doi: 10.1007/s00424-014-1618-8. Epub 2014 Oct 4., [PMID:25277268]
Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-) channel that governs the quantity and composition of epithelial secretions. CFTR function is normally tightly controlled as dysregulation can lead to life-threatening diseases such as secretory diarrhoea and cystic fibrosis. CFTR activity is regulated by phosphorylation of its cytosolic regulatory (R) domain, and ATP binding and hydrolysis at two nucleotide-binding domains (NBDs). Here, we report that CFTR activity is also controlled by extracellular Cl(-) concentration ([Cl(-)]o). Patch clamp current recordings show that a rise in [Cl(-)]o stimulates CFTR channel activity, an effect conferred by a single arginine residue, R899, in extracellular loop 4 of the protein. Using NBD mutants and ATP dose response studies in WT channels, we determined that [Cl(-)]o sensing was linked to changes in ATP binding energy at NBD1, which likely impacts NBD dimer stability. Biochemical measurements showed that increasing [Cl(-)]o decreased the intrinsic ATPase activity of CFTR mainly through a reduction in maximal ATP turnover. Our studies indicate that sensing [Cl(-)]o is a novel mechanism for regulating CFTR activity and suggest that the luminal ionic environment is an important physiological arbiter of CFTR function, which has significant implications for salt and fluid homeostasis in epithelial tissues.
Comments [show]
None has been submitted yet.
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).
X
ABCC7 p.Arg899Gln 25277268:81:70
status: NEW83 Typical I-V plots obtained in this series of experiments for WT, R899Q and the vector control are shown in Fig. 2b.
X
ABCC7 p.Arg899Gln 25277268:83:65
status: NEW102 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.
X
ABCC7 p.Arg899Gln 25277268:102:119
status: NEWX
ABCC7 p.Arg899Gln 25277268:102:172
status: NEW103 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).
X
ABCC7 p.Arg899Gln 25277268:103:141
status: NEWX
ABCC7 p.Arg899Gln 25277268:103:525
status: NEW110 **p<0.01 compared to WT CFTR interaction of Cl-with the extracellular domain of CFTR, we also studied the double-mutant R899Q-E1371Q.
X
ABCC7 p.Arg899Gln 25277268:110:121
status: NEW112 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.
X
ABCC7 p.Arg899Gln 25277268:112:1567
status: NEWX
ABCC7 p.Arg899Gln 25277268:112:1604
status: NEWX
ABCC7 p.Arg899Gln 25277268:112:1653
status: NEWX
ABCC7 p.Arg899Gln 25277268:112:1692
status: NEW126 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).
X
ABCC7 p.Arg899Gln 25277268:126:179
status: NEWX
ABCC7 p.Arg899Gln 25277268:126:247
status: NEW163 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.
X
ABCC7 p.Arg899Gln 25277268:163:27
status: NEW185 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.
X
ABCC7 p.Arg899Gln 25277268:185:37
status: NEW[hide] Interactions between permeant and blocking anions ... Biochim Biophys Acta. 2015 Jul;1848(7):1573-90. doi: 10.1016/j.bbamem.2015.04.004. Epub 2015 Apr 17. Linsdell P
Interactions between permeant and blocking anions inside the CFTR chloride channel pore.
Biochim Biophys Acta. 2015 Jul;1848(7):1573-90. doi: 10.1016/j.bbamem.2015.04.004. Epub 2015 Apr 17., [PMID:25892339]
Abstract [show]
Binding of cytoplasmic anionic open channel blockers within the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is antagonized by extracellular Cl(-). In the present work, patch clamp recording was used to investigate the interaction between extracellular Cl(-) (and other anions) and cytoplasmic Pt(NO2)4(2-) ions inside the CFTR channel pore. In constitutively open (E1371Q-CFTR) channels, these different anions bind to two separate sites, located in the outer and inner vestibules of the pore respectively, in a mutually antagonistic fashion. A mutation in the inner vestibule (I344K) that greatly increased Pt(NO2)4(2-) binding affinity also greatly strengthened antagonistic Cl(-):blocker interactions as well as the voltage-dependence of block. Quantitative analysis of ion binding affinity suggested that the I344K mutation strengthened interactions not only with intracellular Pt(NO2)4(2-) ions but also with extracellular Cl(-), and that altered blocker Cl(-)- and voltage-dependence were due to the introduction of a novel type of antagonistic ion:ion interaction inside the pore that was independent of Cl(-) binding in the outer vestibule. It is proposed that this mutation alters the arrangement of anion binding sites inside the pore, allowing both Cl(-) and Pt(NO2)4(2-) to bind concurrently within the inner vestibule in a strongly mutually antagonistic fashion. However, the I344K mutation does not increase single channel conductance following disruption of Cl(-) binding in the outer vestibule in R334Q channels. Implications for the arrangement of ion binding sites in the pore, and their functional consequences for blocker binding and for rapid Cl(-) permeation, are discussed.
Comments [show]
None has been submitted yet.
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.
X
ABCC7 p.Arg899Gln 25892339:65:72
status: NEW66 (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.
X
ABCC7 p.Arg899Gln 25892339:66:162
status: NEW67 (D) Mean Pt KD values obtained from such fits at different membrane potentials for E1371Q; results for R899Q/E1371Q were indistinguishable (not shown).
X
ABCC7 p.Arg899Gln 25892339:67:103
status: NEW69 (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- ].
X
ABCC7 p.Arg899Gln 25892339:69:153
status: NEW71 There was no significant difference between E1371Q and R899Q/E1371Q (P N 0.85).
X
ABCC7 p.Arg899Gln 25892339:71:55
status: NEW83 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.
X
ABCC7 p.Arg899Gln 25892339:83:35
status: NEW128 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.
X
ABCC7 p.Arg899Gln 25892339:128:201
status: NEW198 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).
X
ABCC7 p.Arg899Gln 25892339:198:113
status: NEW199 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].
X
ABCC7 p.Arg899Gln 25892339:199:5
status: NEW