ABCC7 p.Arg347Ala
| ClinVar: |
c.1039C>T
,
p.Arg347Cys
?
, not provided
c.1040G>A , p.Arg347His D , Pathogenic c.1040G>T , p.Arg347Leu D , Pathogenic c.1040G>C , p.Arg347Pro D , Pathogenic |
| CF databases: |
c.1040G>C
,
p.Arg347Pro
D
, CF-causing ; CFTR1: This mutation destroys a Hha I restriciton site and creates an NcoI site and occurred in a family diagnosed as PS. The mutation have been identified and analyzed using the SSCP technique.
c.1040G>A , p.Arg347His D , CF-causing ; CFTR1: The patient is of Italian origin and carries the [delta]F508 mutation on the other chromosome. Initially we thought this was the same mutation as R347 because it destroys the same hhai site; however, R347H does not create the NcoI site. c.1040G>T , p.Arg347Leu (CFTR1) D , A nucleotide change, G->T at position 1172, was detected leading to R347L. The other chromosome carries a [delta]F508. This mutation was found on one chromosome among 150 CF chromosomes screened. c.1039C>T , p.Arg347Cys (CFTR1) ? , This mutation was identified by DGGE and direct sequencing. |
| Predicted by SNAP2: | A: D (95%), C: D (95%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (71%), I: D (95%), K: D (95%), L: D (80%), M: D (95%), N: D (95%), P: D (75%), Q: D (95%), S: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%), |
| Predicted by PROVEAN: | A: N, C: D, D: D, E: N, F: D, G: D, H: N, I: D, K: N, L: N, M: N, N: N, P: N, Q: N, S: N, T: N, V: D, W: D, Y: D, |
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Comments [show]
None has been submitted yet.
[hide] Evidence for direct CFTR inhibition by CFTR(inh)-1... Biochem J. 2008 Jul 1;413(1):135-42. Caci E, Caputo A, Hinzpeter A, Arous N, Fanen P, Sonawane N, Verkman AS, Ravazzolo R, Zegarra-Moran O, Galietta LJ
Evidence for direct CFTR inhibition by CFTR(inh)-172 based on Arg347 mutagenesis.
Biochem J. 2008 Jul 1;413(1):135-42., 2008-07-01 [PMID:18366345]
Abstract [show]
CFTR (cystic fibrosis transmembrane conductance regulator) is an epithelial Cl- channel inhibited with high affinity and selectivity by the thiazolidinone compound CFTR(inh)-172. In the present study, we provide evidence that CFTR(inh)-172 acts directly on the CFTR. We introduced mutations in amino acid residues of the sixth transmembrane helix of the CFTR protein, a domain that has an important role in the formation of the channel pore. Basic and hydrophilic amino acids at positions 334-352 were replaced with alanine residues and the sensitivity to CFTR(inh)-172 was assessed using functional assays. We found that an arginine-to-alanine change at position 347 reduced the inhibitory potency of CFTR(inh)-172 by 20-30-fold. Mutagenesis of Arg347 to other amino acids also decreased the inhibitory potency, with aspartate producing near total loss of CFTR(inh)-172 activity. The results of the present study provide evidence that CFTR(inh)-172 interacts directly with CFTR, and that Arg347 is important for the interaction.
Comments [show]
None has been submitted yet.
No. Sentence Comment
5 We found that an arginine-to-alanine change at position 347 reduced the inhibitory potency of CFTRinh-172 by 20-30-fold.
X
ABCC7 p.Arg347Ala 18366345:5:17
status: NEW84 Experiments were carried out on FRT cells with stable expression of wild-type or R347A CFTR.
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ABCC7 p.Arg347Ala 18366345:84:81
status: NEW97 Figure 1 Mutants of the sixth TMD (A) Representative traces showing normalized cell fluorescence recordings and quenching upon I- addition in COS-7 cells transfected with wild-type (top panel) or R347A (bottom panel) CFTR.
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ABCC7 p.Arg347Ala 18366345:97:196
status: NEW110 T338A and R347A were similar to wild-type CFTR.
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ABCC7 p.Arg347Ala 18366345:110:10
status: NEW118 The exception was R347A.
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ABCC7 p.Arg347Ala 18366345:118:18
status: NEW124 **Indicate that the R347A Ki was significantly higher (P < 0.01) compared with wild-type CFTR.
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ABCC7 p.Arg347Ala 18366345:124:20
status: NEW125 For Arg347 mutants other than R347A, sensitivity to CFTRinh-172 was so low that the Ki could not be determined precisely (see Experimental section).
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ABCC7 p.Arg347Ala 18366345:125:30
status: NEW127 CFTR form CFTRinh-172 Ki (μM) Hill coefficient I- influx (mM/s) n Wild-type 1.32 + - 0.25 1.03 + - 0.07 0.1336 + - 0.0107 10 S341A 0.57 + - 0.17 1.21 + - 0.37 0.0297 + - 0.0064 4 T338A 3.20 + - 0.86 1.13 + - 0.20 0.1260 + - 0.0225 4 R347A 44.98 + - 4.71** 0.91 + - 0.04 0.1288 + - 0.0154 7 R334A 2.39 + - 0.74 0.93 + - 017 0.0313 + - 0.062 4 A349S 1.23 + - 0.41 1.11 + - 0.25 0.1500 + - 0.011 4 R347D >50 Not determined 0.1160 + - 0.0136 7 R347D/D924R >50 Not determined 0.1008 + - 0.0504 4 R347C >50 Not determined 0.1437 + - 0.0123 4 Mock 0.003 + - 0.001 10 introduced a mutation at position 349 (an alanine residue replaced by a serine residue).
X
ABCC7 p.Arg347Ala 18366345:127:239
status: NEW132 As found for R347A, the mutants R347C and R347D also showed a normal rate of anion transport but altered sensitivity to CFTRinh-172 (Figures 2A and 2B).
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ABCC7 p.Arg347Ala 18366345:132:13
status: NEW143 FRT cells were stably transfected with wild-type, R334A, R347A and R347D CFTR, and transepithelial Cl- currents were measured.
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ABCC7 p.Arg347Ala 18366345:143:57
status: NEW144 The R347A and R347D mutants showed Figure 2 Mutagenesis of Arg347 and Asp924 residues (A) Rate of I- transport measured in COS-7 cells transfected with the indicated constructs.
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ABCC7 p.Arg347Ala 18366345:144:4
status: NEW155 The calculated Ki for wild-type CFTR, R347A and R347D was 0.85 +- 0.13 μM (n = 8), 17.35 +- 3.90 μM (n = 13) and 53.10 +- 4.74 μM (n = 6) respectively (Figure 3E).
X
ABCC7 p.Arg347Ala 18366345:155:38
status: NEW158 Interestingly, the R347D mutant, although insensitive to CFTRinh-172, was fully inhibited by the open-channel blocker GlyH-101 Figure 3 CFTR Cl- current inhibition by CFTRinh-172 (A-D) Representative traces showing recordings of transepithelial Cl- currents measured in FRT cells with stable expression of wild-type (WT), R347A, R334A and R347D-CFTR. Cells were first stimulated with 20 μM forskolin to activate CFTR and then tested with increasing concentrations of CFTRinh-172.
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ABCC7 p.Arg347Ala 18366345:158:325
status: NEW169 In cells expressing the R347A mutant (n = 6), cAMP stimulation elicited currents with a moderate outward rectification of the current-voltage relationship (Figures 6C and 6D).
X
ABCC7 p.Arg347Ala 18366345:169:24
status: NEW173 of the R347A mutant were only partially inhibited (Figures 6C and 6D).
X
ABCC7 p.Arg347Ala 18366345:173:7
status: NEW175 50% reduction of R347A currents whereas the inhibition of wild-type currents at 5 μM was nearly total.
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ABCC7 p.Arg347Ala 18366345:175:17
status: NEW202 Interestingly, when we generated the double mutant R347D/D924R, in which the positions of positive and negative charges are inverted but the salt bridge is maintained [25], we Figure 6 Patch-clamp analysis of CFTR inhibition by CFTRinh-172 (A and C) Superimposed membrane currents recorded from cells expressing wild-type and the R347A mutant at membrane potentials between -100 and +100 mV.
X
ABCC7 p.Arg347Ala 18366345:202:330
status: NEW203 Currents were recorded in the absence and in the presence of CFTRinh-172 (5 μM for wild-type and 10 μM for R347A).
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ABCC7 p.Arg347Ala 18366345:203:119
status: NEW205 (E) Summary of block caused by CFTRinh-172 at all membrane potentials on wild-type (at 5 μM) and R347A (at 10 μM).
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ABCC7 p.Arg347Ala 18366345:205:103
status: NEW206 Values are means + - S.E.M. (n = 6 for both wild-type and R347A).
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ABCC7 p.Arg347Ala 18366345:206:58
status: NEW211 We hypothesized that the negatively charged carboxyl group in CFTRinh-172 interacts with the positive charge of Arg347 , such that the effects of Arg347 mutations could be explained by loss of electrostatic attraction (R347A) or generation of electrostatic repulsion (R347D).
X
ABCC7 p.Arg347Ala 18366345:211:219
status: NEW[hide] Mutations at arginine 352 alter the pore architect... J Membr Biol. 2008 Mar;222(2):91-106. Epub 2008 Apr 18. Cui G, Zhang ZR, O'Brien AR, Song B, McCarty NA
Mutations at arginine 352 alter the pore architecture of CFTR.
J Membr Biol. 2008 Mar;222(2):91-106. Epub 2008 Apr 18., [PMID:18421494]
Abstract [show]
Arginine 352 (R352) in the sixth transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) previously was reported to form an anion/cation selectivity filter and to provide positive charge in the intracellular vestibule. However, mutations at this site have nonspecific effects, such as inducing susceptibility of endogenous cysteines to chemical modification. We hypothesized that R352 stabilizes channel structure and that charge-destroying mutations at this site disrupt pore architecture, with multiple consequences. We tested the effects of mutations at R352 on conductance, anion selectivity and block by the sulfonylurea drug glipizide, using recordings of wild-type and mutant channels. Charge-altering mutations at R352 destabilized the open state and altered both selectivity and block. In contrast, R352K-CFTR was similar to wild-type. Full conductance state amplitude was similar to that of wild-type CFTR in all mutants except R352E, suggesting that R352 does not itself form an anion coordination site. In an attempt to identify an acidic residue that may interact with R352, we found that permeation properties were similarly affected by charge-reversing mutations at D993. Wild-type-like properties were rescued in R352E/D993R-CFTR, suggesting that R352 and D993 in the wild-type channel may interact to stabilize pore architecture. Finally, R352A-CFTR was sensitive to modification by externally applied MTSEA+, while wild-type and R352E/D993R-CFTR were not. These data suggest that R352 plays an important structural role in CFTR, perhaps reflecting its involvement in forming a salt bridge with residue D993.
Comments [show]
None has been submitted yet.
No. Sentence Comment
174 Mutations R352A and R347A Abolished Time-Dependent Block by Glipizide Glipizide is a CFTR pore blocker from the sulfonylurea family of compounds which includes glibenclamide (Sheppard and Welsh 1992; Schultz et al. 1996; Sheppard and Robinson 1997; Zhang et al. 2004a, b).
X
ABCC7 p.Arg347Ala 18421494:174:20
status: NEW178 Both R347A- and R352A-CFTR showed significantly weakened block by 200 lM glipizide, largely due to loss of the time-dependent component (Fig. 6).
X
ABCC7 p.Arg347Ala 18421494:178:5
status: NEW180 Similar results were found for block of R347A-CFTR (fractional block was 0.11 ± 0.02, n = 5, P \ 0.001 compared to WT-CFTR).
X
ABCC7 p.Arg347Ala 18421494:180:40
status: NEW181 The gross change in pore architecture induced by both the R347A and R352A mutations appeared to have altered the kinetics of interaction with the site underlying slow block by glipizide, resulting in the loss of time-dependent inhibition.
X
ABCC7 p.Arg347Ala 18421494:181:58
status: NEW183 Figure 6B, D, F, H shows the macroscopic i-V relationships for WT-, R352A-, R347A- and R352K-CFTR in representative experiments, indicating that glipizide blocked the currents primarily at negative membrane potentials in WTand R352K-CFTR.
X
ABCC7 p.Arg347Ala 18421494:183:76
status: NEW184 However, the voltage dependence of block was clearly altered in R352A- and R347A-CFTR.
X
ABCC7 p.Arg347Ala 18421494:184:75
status: NEW185 Finally, R352A- and R347A-CFTR, but not R352K-CFTR, exhibited outward rectification of macroscopic currents in the absence of blocker, consistent with the outward rectification of single-channel amplitudes (Fig. 4, Table 1) (Cotten and Welsh 1999).
X
ABCC7 p.Arg347Ala 18421494:185:20
status: NEW189 100 ms 200 pA 100 ms 2 nA 20 pA 100 ms -100 -600 -400 -200 200 400 600 ATP ATP + Glip 200 50 100 mV -50 pA -100 pA mV 50 100 -60 20 40 ATP + Glip 200 ATP-40 60 -20 -50 mV -100 -50 50 100 -4 -2 2 4 nA ATP ATP + Glip 200 mV -100 -50 50 100 -400 -200 200 400 ATP ATP + Glip 200 pA mV -100 -50 50 100 -400 -200 200 400 ATP ATP + Glip 200 pA 200 pA 100 ms 200 pA 100 ms 100 ms 200 pA 100 ms 200 pA 100 ms 2 nA 100 ms 2 nA 20 pA 100 ms 20 pA 100 ms -100 -600 -400 -200 200 400 600 ATP ATP + Glip 200 50 100 mV -50 pA -600 -400 -200 200 400 600 ATP ATP + Glip 200 50 100 mV -50 pA -100 pA mV 50 100 -60 20 40 ATP + Glip 200 ATP-40 60 -20 -50-100 pA mV 50 100 -60 20 40 ATP + Glip 200 ATP-40 60 -20 -50-100 pA mV 50 100 -60 20 40 ATP + Glip 200 ATP-40 60 -20 -50 mV -100 -50 50 100 -4 -2 2 4 nA ATP ATP + Glip 200 mV -100 -50 50 100 -4 -2 2 4 nA ATP ATP + Glip 200 mV -100 -50 50 100 -400 -200 200 400 ATP ATP + Glip 200 pA mV -100 -50 50 100 -400 -200 200 400 ATP ATP + Glip 200 pA 200 pA 100 ms 200 pA 100 ms R347A-CFTR WT-CFTR R352K-CFTR R352A-CFTR 100 ms 200 pA 100 ms 2 nA 20 pA 100 ms -100 -600 -400 -200 200 400 600 ATP ATP + Glip 200 50 100 mV -50 pA -100 pA mV 50 100 -60 20 40 ATP + Glip 200 ATP-40 60 -20 -50 mV -100 -50 50 100 -4 -2 2 4 nA ATP ATP + Glip 200 mV -100 -50 50 100 -400 -200 200 400 ATP ATP + Glip 200 pA mV -100 -50 50 100 -400 -200 200 400 ATP ATP + Glip 200 pA 200 pA 100 ms 200 pA 100 ms 100 ms 200 pA 100 ms 200 pA 100 ms 2 nA 100 ms 2 nA A B D E F 20 pA 100 ms 20 pA 100 ms -100 -600 -400 -200 200 400 600 ATP ATP + Glip 200 50 100 mV -50 pA -600 -400 -200 200 400 600 ATP ATP + Glip 200 50 100 mV -50 pA G H -100 pA mV 50 100 -60 20 40 ATP + Glip 200 ATP-40 60 -20 -50-100 pA mV 50 100 -60 20 40 ATP + Glip 200 ATP-40 60 -20 -50-100 pA mV 50 100 -60 20 40 ATP + Glip 200 ATP-40 60 -20 -50 mV -100 -50 50 100 -4 -2 2 4 nA ATP ATP + Glip 200 mV -100 -50 50 100 -4 -2 2 4 nA ATP ATP + Glip 200 C mV -100 -50 50 100 -400 -200 200 400 ATP ATP + Glip 200 pA mV -100 -50 50 100 -400 -200 200 400 ATP ATP + Glip 200 pA 200 pA 100 ms 200 pA 100 ms R347A-CFTR WT-CFTR R352K-CFTR R352A-CFTR Fig. 6 Mutations at R352 alter pore pharmacology.
X
ABCC7 p.Arg347Ala 18421494:189:1003
status: NEWX
ABCC7 p.Arg347Ala 18421494:189:2063
status: NEW190 Left Block of CFTR macropatch currents by glipizide (glip) was time-dependent in WT-CFTR (A) and R352K-CFTR (G) but not in R352A-CFTR (C) or R347A-CFTR (E).
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ABCC7 p.Arg347Ala 18421494:190:141
status: NEW192 Right i-V relationships for WT-CFTR (B), R352A-CFTR (D), R347A-CFTR (F) and R352K-CFTR (H) were constructed from voltage ramps performed in the absence (black) and in the presence of 200 lM glipizide (red).
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ABCC7 p.Arg347Ala 18421494:192:57
status: NEW249 R352A-CFTR exhibited outward rectification in conditions of symmetrical [Cl- ], similar to that found in R347A-CFTR (Fig. 6).
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ABCC7 p.Arg347Ala 18421494:249:105
status: NEW[hide] Differential contribution of TM6 and TM12 to the p... Pflugers Arch. 2012 Mar;463(3):405-18. Epub 2011 Dec 13. Cui G, Song B, Turki HW, McCarty NA
Differential contribution of TM6 and TM12 to the pore of CFTR identified by three sulfonylurea-based blockers.
Pflugers Arch. 2012 Mar;463(3):405-18. Epub 2011 Dec 13., [PMID:22160394]
Abstract [show]
Previous studies suggested that four transmembrane domains 5, 6, 11, 12 make the greatest contribution to forming the pore of the CFTR chloride channel. We used excised, inside-out patches from oocytes expressing CFTR with alanine-scanning mutagenesis in amino acids in TM6 and TM12 to probe CFTR pore structure with four blockers: glibenclamide (Glyb), glipizide (Glip), tolbutamide (Tolb), and Meglitinide. Glyb and Glip blocked wildtype (WT)-CFTR in a voltage-, time-, and concentration-dependent manner. At V (M) = -120 mV with symmetrical 150 mM Cl(-) solution, fractional block of WT-CFTR by 50 muM Glyb and 200 muM Glip was 0.64 +/- 0.03 (n = 7) and 0.48 +/- 0.02 (n = 7), respectively. The major effects on block by Glyb and Glip were found with mutations at F337, S341, I344, M348, and V350 of TM6. Under similar conditions, fractional block of WT-CFTR by 300 muM Tolb was 0.40 +/- 0.04. Unlike Glyb, Glip, and Meglitinide, block by Tolb lacked time-dependence (n = 7). We then tested the effects of alanine mutations in TM12 on block by Glyb and Glip; the major effects were found at N1138, T1142, V1147, N1148, S1149, S1150, I1151, and D1152. From these experiments, we infer that amino acids F337, S341, I344, M348, and V350 of TM6 face the pore when the channel is in the open state, while the amino acids of TM12 make less important contributions to pore function. These data also suggest that the region between F337 and S341 forms the narrow part of the CFTR pore.
Comments [show]
None has been submitted yet.
No. Sentence Comment
119 The major effects of increasing or decreasing sensitivity to Glyb were seen with mutations R334A, K335A, F337A, S341A, I344A, R347A, M348A, V350A, and R352A (Fig. 3 left).
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ABCC7 p.Arg347Ala 22160394:119:126
status: NEW140 Mutations R347A and R352A also represent a separate category from the rest.
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ABCC7 p.Arg347Ala 22160394:140:10
status: NEW145 The present data show that mutations R347A and R352A significantly reduced block by all three blockers; for Glyb and Glip, block became strictly time-independent, perhaps reflecting the gross loss of pore architecture leading to loss of the binding site underlying slow pore block.
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ABCC7 p.Arg347Ala 22160394:145:37
status: NEW151 The surprising finding that mutations at six adjacent positions Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT ** ** ** ** ** ** * * * 0.8 0.6 0.4 0.2 0 Fractional block by Glyb50 μM Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT ** ** ** ** ** ** ** ** * * * * * * ** ** Fractional block by Tolb300 μM 0.8 0.6 0.4 0.2 0 Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT * ** ** ** ** ** ** ** ** Fractional block by Glip200 μM 0.8 0.6 0.4 0.2 0 Fig. 3 Alanine-scanning in TM6 to identify the amino acids that interact with the three blockers.
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ABCC7 p.Arg347Ala 22160394:151:100
status: NEWX
ABCC7 p.Arg347Ala 22160394:151:301
status: NEWX
ABCC7 p.Arg347Ala 22160394:151:521
status: NEW158 Among the 20 single amino acid mutants of TM12 that we tested in this paper, none of them exhibited significant change in their single-channel conductance compared to WT-CFTR, while we know that mutations R334A, F337A, S341A, R347A, and R352A in TM6 all exhibited significant change in their single-channel conductance [11, 12, 29, and the present manuscript]; these data strongly suggest that TM6 and TM12 do not equally contribute to the pore of CFTR.
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ABCC7 p.Arg347Ala 22160394:158:226
status: NEW166 Double asterisks indicate significantly different compared to WT-CFTR (p<0.01) Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT 0.3 0.2 0.1 0 * * ** ** 0.4 Initial block by 50 μM Glyb Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT 0.4 0.3 0.2 0.1 0 ** ** * Initial block by 200 μM Glip Fig. 5 Initial block of WT-CFTR and selected TM6 mutants by 50 μM Glyb (left) and 200 μM Glip (right) in symmetrical 150 mM Cl- solution. Data are shown only for those mutants which exhibited significant changes in steady-state fractional block according to Fig. 3 (bars show mean±SEM, n=5-10).
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ABCC7 p.Arg347Ala 22160394:166:115
status: NEWX
ABCC7 p.Arg347Ala 22160394:166:300
status: NEW173 Mutation S341A caused the largest decrease in block by Glyb and Glip (aside from R347A and R352A, which have non-canonical effects as described above; Fig. 3).
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ABCC7 p.Arg347Ala 22160394:173:81
status: NEW193 Probable orientation of drugs in the pore Glyb and Glip are identical molecules along most of their lengths, differing only in the substituents on the ring at the Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT 0.8 0.6 0.2 0 ** ** ** ** Time-dependent block by 50 μμM Glyb Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT ** ** * ** * Time-dependent block by 200 μM Glip 0.4 0.8 0.6 0.2 00.4 Fig. 6 Time-dependent block of WT-CFTR and selected TM6 mutants by 50 μM Glyb (left) and 200 μM Glip (right) in symmetrical 150 mM Cl- solution. Data are shown only for those mutants which exhibited significant changes in fractional block according to Fig. 3 (bars show mean±SEM, n=5-10).
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ABCC7 p.Arg347Ala 22160394:193:199
status: NEWX
ABCC7 p.Arg347Ala 22160394:193:396
status: NEW222 Likewise, the effects of mutations R347A and R352A are also indirect, because charge-destroying substitutions at these sites alter the gross architecture of the pore, with pleiotropic effects [11, 12].
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ABCC7 p.Arg347Ala 22160394:222:35
status: NEW[hide] Two salt bridges differentially contribute to the ... J Biol Chem. 2013 Jul 12;288(28):20758-67. doi: 10.1074/jbc.M113.476226. Epub 2013 May 24. Cui G, Freeman CS, Knotts T, Prince CZ, Kuang C, McCarty NA
Two salt bridges differentially contribute to the maintenance of cystic fibrosis transmembrane conductance regulator (CFTR) channel function.
J Biol Chem. 2013 Jul 12;288(28):20758-67. doi: 10.1074/jbc.M113.476226. Epub 2013 May 24., [PMID:23709221]
Abstract [show]
Previous studies have identified two salt bridges in human CFTR chloride ion channels, Arg(352)-Asp(993) and Arg(347)-Asp(924), that are required for normal channel function. In the present study, we determined how the two salt bridges cooperate to maintain the open pore architecture of CFTR. Our data suggest that Arg(347) not only interacts with Asp(924) but also interacts with Asp(993). The tripartite interaction Arg(347)-Asp(924)-Asp(993) mainly contributes to maintaining a stable s2 open subconductance state. The Arg(352)-Asp(993) salt bridge, in contrast, is involved in stabilizing both the s2 and full (f) open conductance states, with the main contribution being to the f state. The s1 subconductance state does not require either salt bridge. In confirmation of the role of Arg(352) and Asp(993), channels bearing cysteines at these sites could be latched into a full open state using the bifunctional cross-linker 1,2-ethanediyl bismethanethiosulfonate, but only when applied in the open state. Channels remained latched open even after washout of ATP. The results suggest that these interacting residues contribute differently to stabilizing the open pore in different phases of the gating cycle.
Comments [show]
None has been submitted yet.
No. Sentence Comment
89 A, representative current samples of WT-, R347A-, R347D-, D924R-, R347K-, and R347D/D924R-CFTR were recorded from excised inside-out patch from Xenopus oocytes with 150 mM Clafa; symmetrical solution in the presence of 1 mM Mg-ATP and 50 nM PKA at VM afd; afa;100 mV (n afd; 4-6 for each mutant).
X
ABCC7 p.Arg347Ala 23709221:89:42
status: NEW94 R347A-CFTR showed a very long and stable s1 state with very brief openings to s2 or f states, whereas R347D-CFTR only exhibits a long stable s1 state and appears to never get out of s1 (at the resolution of our recording apparatus), as if introduction of negative charge at this position confers electrostatic repulsion with other negative charges in the native channel and thereby greatly interferes with the ability to go beyond the s1 state.
X
ABCC7 p.Arg347Ala 23709221:94:0
status: NEW96 D924R-CFTR exhibits all three open states in contrast to R347A- and R347D-CFTR, although the stability of the open state is compromised; indeed, the fractional occupancies of both s1 and s2 states are greatly increased in this mutant (Fig. 2B).
X
ABCC7 p.Arg347Ala 23709221:96:57
status: NEW101 In addition, breakingthissaltbridgedisruptedthestabilityofthes2andfstates but did not significantly affect s1; therefore, both R347A and R347D showed long stable s1 states, although R347A endeavored to reach the s2 and f states but failed to maintain them, whereas R347D completely lost the ability to open to s2 and f state.
X
ABCC7 p.Arg347Ala 23709221:101:127
status: NEWX
ABCC7 p.Arg347Ala 23709221:101:182
status: NEW109 We therefore hypothesized that Arg347 might also interact with Asp993 to rescue the CFTR channel pore to a stable f state and tested this hypothesis in three double mutants; TABLE 1 Summary of the effects of mutations studied Mutant Main features of open bursts Impact on f state R347A Emphasizes s1 state, brief transitions to s2 and f Can reach f but not stable R347D Emphasizes s1 state, no transitions to s2 and f Cannot reach f D924R Brief transitions to all conductance levels Can reach f but not stable R347K Wild type-like Wild type-like R347D/D924R Emphasizes s2 state, rare and brief transitions to f Can reach f but not stable R352E Opens to all 3 levels; s1 much more stable than in WT, s2 unstable, f unstable Can reach f but not stable D993R Opens to all 3 levels, but none are stable Can reach f but not stable R352E/D993R Wild type-like, with increased transitions to s1 and s2; slightly reduced single-channel conductance Wild type-like R352E/D924R Opens to all 3 levels, but none are stable Can reach f but not stable R347D/D993R Very stable s2; rare and brief transitions to both s1 and f Can reach f but not stable R347A/R352A Opens to all 3 levels; s1 much more stable than in WT, s2 unstable, f unstable Can reach f but not stable R347D/D924R/D993R Opens to all 3 levels; s1 much more stable than in WT, s2 relatively stabilized, f unstable Can reach f but not stable R347D/D924R/R352E/D993R Primarily flickers between s2 and f; s1 much more stable than in WT, slightly reduced single channel conductance Can reach f but not stable FIGURE 3.
X
ABCC7 p.Arg347Ala 23709221:109:280
status: NEWX
ABCC7 p.Arg347Ala 23709221:109:1135
status: NEW129 As we show in Fig. 4, R347A/ R352A-CFTR behaves just like R352A-CFTR, opening to all three conductance states with little stability of either state, as we reported before.
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ABCC7 p.Arg347Ala 23709221:129:22
status: NEW146 Representative current samples of R347A/R352A-, R347D/D924R/D993R-, and R347D/D924R/D993R/R352E-CFTR were recorded under the same conditions as in Fig. 3 (n afd; 5-6 for each mutant) (A).
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ABCC7 p.Arg347Ala 23709221:146:34
status: NEW213 We conclude that the subconductance states in CFTR probably also represent pore conformational change for the following reasons: 1) the CFTR channel pore forms from one polypeptide as a monomer and only bears one permeation pathway (12); 2) the s1 and s2 states occur as rare events in some point mutations, such as T338A/Cand K335A/C-CFTR, which do not appear to affect gross pore architecture, whereas they are frequent events in CFTR channels bearing salt bridge mutations, such as R352A- and R347A-CFTR, as discussed above; 3) mutations at sites involved in salt bridges (such as Arg347 , Arg352 , Asp924 , and Asp993 ) result in much more frequent occupancy of subconductance states; 4) mutations at sites involved in salt bridges (such as Arg347 and Arg352 ) lead to greatly altered sensitivity to pore blockers (7, 13); and 5) the subconductance behavior is not affected by different concentrations of Clafa; or by changes in membrane potential (12, 16).
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ABCC7 p.Arg347Ala 23709221:213:496
status: NEW226 R347A-CFTR single channel traces clearly show that the channel first opens from the c to s1 state and then attempts to further open to the s2 and f state; we never saw the channel directly open from c to s2 or f in these mutants.
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ABCC7 p.Arg347Ala 23709221:226:0
status: NEW[hide] Murine and human CFTR exhibit different sensitivit... Am J Physiol Lung Cell Mol Physiol. 2015 Oct 1;309(7):L687-99. doi: 10.1152/ajplung.00181.2015. Epub 2015 Jul 24. Cui G, McCarty NA
Murine and human CFTR exhibit different sensitivities to CFTR potentiators.
Am J Physiol Lung Cell Mol Physiol. 2015 Oct 1;309(7):L687-99. doi: 10.1152/ajplung.00181.2015. Epub 2015 Jul 24., [PMID:26209275]
Abstract [show]
Development of therapeutic molecules with clinical efficacy as modulators of defective CFTR includes efforts to identify potentiators that can overcome or repair the gating defect in mutant CFTR channels. This has taken a great leap forward with the identification of the potentiator VX-770, now available to patients as "Kalydeco." Other small molecules with different chemical structure also are capable of potentiating the activity of either wild-type or mutant CFTR, suggesting that there are features of the protein that may be targeted to achieve stimulation of channel activity by structurally diverse compounds. However, neither the mechanisms by which these compounds potentiate mutant CFTR nor the site(s) where these compounds bind have been identified. This knowledge gap partly reflects the lack of appropriate experimental models to provide clues toward the identification of binding sites. Here, we have compared the channel behavior and response to novel and known potentiators of human CFTR (hCFTR) and murine (mCFTR) expressed in Xenopus oocytes. Both hCFTR and mCFTR were blocked by GlyH-101 from the extracellular side, but mCFTR activity was increased with GlyH-101 applied directly to the cytoplasmic side. Similarly, glibenclamide only exhibited a blocking effect on hCFTR but both blocked and potentiated mCFTR in excised membrane patches and in intact oocytes. The clinically used CFTR potentiator VX-770 transiently increased hCFTR by approximately 13% but potentiated mCFTR significantly more strongly. Our results suggest that mCFTR pharmacological sensitivities differ from hCFTR, which will provide a useful tool for identifying the binding sites and mechanism for these potentiators.
Comments [show]
None has been submitted yet.
No. Sentence Comment
110 The amplitude of s1 was b03;25% and s2 was b03;65% of f, which is different from the ratios of s1 and s2 to f in WT-, R334C-, R352A-, and R347A-hCFTR (s1 is b03;40% and s2 is b03;70% of f) (21, 27, 28).
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ABCC7 p.Arg347Ala 26209275:110:144
status: NEW113 As we previously reported, R334C-, R347A-, and R352A-hCFTR generally open from the closed state (c), to s1, then opened to s2 and f states (6, 37, 40).
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ABCC7 p.Arg347Ala 26209275:113:35
status: NEW