ABCMdb

ABCC7 p.Lys892Gln

Predicted by SNAP2:	A: N (72%), C: D (53%), D: N (61%), E: N (82%), F: D (63%), G: N (66%), H: N (78%), I: N (66%), L: N (72%), M: N (72%), N: N (78%), P: N (53%), Q: N (82%), R: N (82%), S: N (82%), T: N (82%), V: N (66%), W: D (66%), Y: D (59%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N,

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[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.

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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
111 Block of K892Q was significantly strengthened by Co(CN)6 3- (Figure 5D) but was unaffected by Co(NO2)6 3- (Figure 5E). 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
212 Neutralization of another nearby positive charge, in K892Q, had a slightly different effect: block was slightly weakened relative to wild type, was unaffected by Co(NO2)6 3- and was actually significantly strengthened in the presence of external Co(CN)6 3- (Figures 5, 7), again consistent with altered interactions between external anions and cytoplasmic blocking substances 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

[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.

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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
69 Furthermore, in K892Q, the effect of external Pt(NO2)4 2-was reversed: in this mutant, block by internal Pt(NO2)4 2- ions was significantly strengthened by the presence of Pt(NO2)4 2- in the extracellular solution (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
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

[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.

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No. Sentence 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