ABCMdb

ABCC7 p.Arg352Ala

ClinVar:	c.1054C>T , p.Arg352Trp D , Likely pathogenic c.1055G>A , p.Arg352Gln D , Pathogenic
CF databases:	c.1055G>A , p.Arg352Gln D , CF-causing ; CFTR1: This missense mutation, at nucleotide position 1187 (G to A) in exon 7, has been detected in an Italian CF patient through DGGE and direct sequencing. The mutation generates an Arg to Gln substitution (R352Q) and creates a novel DdeI restriction site in the mutated allele. This mutation has been detected in a PS patient (paternal chromosome), associated with the haplotype A; the maternal chromosome carries a still uncharacterized mutation. It was found in one of 60 non-[delta] Italian CF chromosomes. c.1054C>G , p.Arg352Gly (CFTR1) ? , c.1054C>T , p.Arg352Trp (CFTR1) ? , The mutation was detected by SSCP/heteroduplex analysis and identified by direct DNA sequencing. The mutation was seen in a boy referred by West Midlands Regional Genetics Service, and whose other CF mutation was [delta]F508. We have seen it only once in over 150 samples tested.
Predicted by SNAP2:	A: D (91%), C: D (95%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (91%), K: D (85%), L: D (91%), M: D (95%), N: D (95%), P: D (95%), Q: D (59%), S: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
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,

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Publications
[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.

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101 Wild-type CFTR and all mutants except R352A showed an I- influx significantly higher (P < 0.01) than mock-transfected cells. Login to comment
112 More marked was the effect of R352A, which showed undetectable levels of CFTR activity. Login to comment
114 The absence of function did not allow us to measure the potency of CFTRinh-172 for the R352A mutant. Login to comment

[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.

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9 Finally, R352A-CFTR was sensitive to modification by externally applied MTSEA+ , while wild-type and R352E/D993R-CFTR were not. Login to comment
73 Channels formed by R352A, Q and E mutants and some double mutants exhibited multiple conductance levels, with s1 representing subconductance level 1; s2, subconductance level 2; f, full conductance level; and c, closed conductance level, as previously described (Zhang et al. 2005a, b). Login to comment
75 To determine how mutations at R352 affected the stability of the open state, single-channel records from WT-CFTR and R352A-CFTR were analyzed using Clampfit and QuB (http://www.qub.buffalo.edu) (Qin et al. 2006). Login to comment
111 Both R352A- and R352Q-CFTR showed three distinct open conductance states: s1, s2 and f.In contrast to WT-CFTR, channels formed by R352A- and R352Q-CFTR occupied the s1 and s2 states in the vast majority of open bursts, while transitions to the f conductance state were rare events. Login to comment
113 The transitions between the three open states in R352A- and R352Q-CFTR were random, showing no regular pattern. Login to comment
122 0 0.0 -0.4 -0.8 #ofevents 4000 -1.2 0.0 -0.4 -0.8 6000 #ofevents 0 -1.2 0.0 -0.4 -0.8 3000 #ofevents 0 -1.2 2500 #ofevents 0.0 -0.4 -0.8 0 -1.2 Current (pA) fc s1 s2 s1 s2 B C D A 0.4 pA 2 s c s1 s2 f R352A 0.4 pA 2 s 0.4 pA 2 s c s1 s2 f c f 0.4 pA 2 s c f WT R352Q R352K 00 s1 s2 s1 s2 Fig. 1 Sample traces of WT-CFTR and indicated R352 mutants from excised inside-out membrane patches with symmetrical 150 mM Cl- solution (left) and their all-points amplitude histograms (right). Login to comment
124 In R352A- and R352Q-CFTR, there are four current levels indicating the c, s1, s2 and f states. Login to comment
126 Solid lines in histograms are fit results to the gaussian function in Clampfit 9.0 To quantify the effect of mutations at R352 on the stability of the open state, we analyzed intraburst kinetics by determining the fraction of time that each channel spent in the s1, s2, f and IC states for WT-CFTR and R352A-CFTR. Login to comment
127 As shown in Fig. 3, WT-CFTR channels spent 96.7 ± 1.3% of each open burst in the f state, while R352A-CFTR channels spent only 65.5 ± 17.5% of each burst in the f state (P \ 0.02, mean ± SD for n = 3-4 records each). Login to comment
128 Consistent with previous results, R352A-CFTR channels spent a significantly larger fractional duration of each open burst in the s1 and s2 states than did WT-CFTR (P \ 0.001). Login to comment
129 We point out that while the data shown in the histograms of Fig. 1 reflect only the records displayed there, with a low number of bursts selected to emphasize transitions to subconductance states, the intraburst kinetic analysis presented here represents 870 s of recording for WT-CFTR, including 510 bursts, and 356 s of recording for R352A-CFTR, including 150 bursts. Login to comment
131 Dwell-time analysis of the same records indicated that the mean duration for the f state decreased from 632 ± 264 ms in WT-CFTR to 123 ± 63 ms in R352A-CFTR (mean ± SD), representing an 80% reduction in the stability of the fully open state (n = 3-4, P = 0.02). Login to comment
133 Substate behavior was observed in R352A-CFTR at all voltages (Fig. 4A), including both negative and positive membrane potentials. Login to comment
134 Figure 4 shows the i-V relationship for the f conductance states of WT-, R352A-, R352Q- and R352K-CFTR (Fig. 4B) and for the subconductance states of R352A- and R352Q-CFTR (Fig. 4C), at potentials ranging between VM = -100 and +100 mV. Login to comment
135 The f state slope conductance of R352A-CFTR at negative membrane potentials was not different from that of WT-CFTR, suggesting that the positive charge at R352 does not determine channel conductance; at positive membrane potentials, the slope conductance of the f state was larger in R352A-CFTR than in WT-CFTR (Table 1). Login to comment
139 The single-channel conductance of the f state exhibited significant outward rectification in R352A-, R352Q- and R352E-CFTR (Table 1). Login to comment
140 In sum, these results are not consistent with a simple role of R352 in providing positive charge to the intracellular vestibule; if this scenario were true, loss of the positive charge in R352A would be expected to reduce single-channel conductance at both positive and negative potentials but more drastically at a c d b c da b 0.5 s 0.2 pA 2 s 0.2 pA c s2 f s1 Fig. 2 Instability of open channel current does not reflect summed activity of multiple lower-conductance openings. Login to comment
143 In the lower part of the figure, these four openings are displayed at higher temporal resolution; these openings exhibit conductance transitions between open levels that are not found as transitions from the closed current level s2 fs1 0.1 0.01 0.001 1 State ** ** * FractionofOpenBurstDuration ** ** * IC Fig. 3 Stability of the open state and intraburst closed state of WT-CFTR and R352A-CFTR. Login to comment
144 Mean fraction of open burst duration is plotted for each state of two CFTR constructs (black bars, WT-CFTR; gray bars, R352A-CFTR). Login to comment
146 Anion Selectivity of R352A-, R352E- and R352K-CFTR To determine whether mutations at R352 affected the ability of CFTR channels to select between ions of similar charge, we studied the anion selectivity patterns of R352A-, R352E- and R352K-CFTR using inside-out macropatches and compared them to WT-CFTR. Login to comment
152 For calculation of Gx/GCl, we compared R352A-, R352E- and R352K-CFTR with WT-CFTR as well as R352E- and R352K-CFTR with R352A-CFTR. Login to comment
156 The normalization of relative conductances between the different anions tested likely f 0.2 pA 2 s -100mV -80mV -60mV -40mV 0.2 pA 2 s -100mV -80mV - - pA - - s1 s2 c s1 s2 f c s1 s2 f c f cA B C WT -CFTR R352Q R352A R352K WT -CFTR R352Q R352A R352K mV -100 -50 50 100 -0.8 -0.4 0.4 0.8 pA mV -100 -50 50 100 0.4 0.2 -0.2 -0.4 pA0.6 -0.6 -100 -50 50 100 0.4 0.2 -0.2 -0.4 0.6 -0.6 -100 -50 50 100 0.4 0.2 -0.2 -0.4 0.6 -0.6 R352Q s1 R352Q s2 R352A s1 R352A s2 R352Q s1 R352Q s2 f 0.2 pA 2 s -100mV -80mV -60mV -40mV - - pA - - 0.2 pA 2 s -100mV -80mV - - Fig. 4 Sample traces of R352A-CFTR and i-V relationships of the conducting states of WT-CFTR and R352 mutants. Login to comment
157 (A) Four traces of R352A-CFTR from a single patch at tested voltages indicated at the right. Login to comment
159 (B) Single-channel i-V relationships for f conductance states of R352A-, R352Q- and R352K-CFTR, with WT-CFTR for comparison. Login to comment
161 (C) The i-V relationship of the s1 and s2 subconductance states of R352A- and R352Q-CFTR. Login to comment
162 Slope conductances are summarized in Table 1 Table 1 Slope conductancea (in pS) of the f state of WT-CFTR and multiple single and double mutants CFTR n Negative VM Positive VM WT 7 6.82 ± 0.03 6.97 ± 0.06 R352A 6 6.80 ± 0.06 7.85 ± 0.07*, ** R352Q 6 5.29 ± 0.02* 6.28 ± 0.05*, ** R352K 5 6.87 ± 0.03 6.86 ± 0.01 R352E 5 3.78 ± 0.01* 6.03 ± 0.01*, ** R352E/E873R 6 3.84 ± 0.01* 5.64 ± 0.01*, ** R352E/ E1104R 6 4.36 ± 0.01* 5.86 ± 0.02*, ** R352E/D993R 5 5.90 ± 0.02* 6.44 ± 0.01*, ** D993R 7 8.27 ± 0.05* 7.13 ± 0.07** a Slope conductance indicates single-channel conductance calculated from 0 to +100 mV (positive VM) or to -100 mV (negative VM) by linear regression * P B 0.001 compared to the equivalent slope conductance in WT-CFTR, ** P B 0.001 compared to the slope conductance in the same mutant at negative VM reflects the loss of anion binding properties within the core of the permeation pathway, which contributes to the tight binding of SCN (Smith et al. 1999). Login to comment
163 It is interesting that the charge-destroying mutation, R352A, had minimal effects on relative permeabilities but very significant effects on relative conductances. Login to comment
166 Our present results suggest -300 -50 300 50 Br -100 100 NO3 Cl SCN pA mVBr NO3 SCN Cl -300 -50 300 50 Br -100 100 NO3 Cl SCNC pA mVBr NO3 SCN Cl R352E -4000 -50 4000 50 -100 100 -800 -50 800 50 -100 100 -6000 -50 6000 50 -100 100 A pA -50 800 50 -100 100 A SCN Br Cl NO3 NO3 Br Cl SCN Br NO3 SCN Cl Br NO3 SCN Cl -800 mV pA mV pA mV pA mV NO3 Br Cl SCN Br NO3 SCN Cl Br NO3 SCN Cl Br NO3 SCN Cl -4000 -50 4000 50 -100 100 D -800 -50 800 50 -100 100 E D993R -6000 -50 6000 50 -100 100 WT pA -50 800 50 -100 100 B SCN Br Cl NO3 NO3 Br Cl SCN Br NO3 SCN Cl Br NO3 SCN Cl -800 mV pA mV pA mV pA mV NO3 Br Cl SCN Br NO3 SCN Cl Br NO3 SCN Cl Br NO3 SCN Cl R352A R352K R352E/ Fig. 5 Mutations at R352 alter anion selectivity. Login to comment
167 Representative inside-out macropatches, recorded in the presence of cytoplasmic Cl- or Cl- plus substitute anions, with voltage ramps between -100 and +100 mV, are shown for (A) WT-CFTR, (B) R352A-CFTR, (C) R352E-CFTR, (D) R352K-CFTR and (E) the double mutant R352E/D993R-CFTR. Login to comment
171 Solutions were at pH 7.45 and are labeled as follows: 150 mM Cl- (black), 130 mM Cl- plus 20 mM NO3 - (purple), 130 mM Cl- plus 20 mM Br- (green) and 130 mM Cl- plus 20 mM SCN- (red) Table 2 Relative permeabilities of some anions in WT-CFTR and R352-CFTR mutants * Significant difference compared with WT-CFTR, P \ 0.05; ** Significant difference compared with R352A, P \ 0.05 CFTR n SCN Br NO3 WT 6 4.11 ± 0.17 1.45 ± 0.04 1.51 ± 0.02 R352A 10 4.18 ± 0.65 1.35 ± 0.21 1.70 ± 0.29 R352E 6 5.18 ± 0.32* 1.47 ± 0.08 1.64 ± 0.43 R352K 7 4.05 ± 0.12 1.52 ± 0.01 1.59 ± 0.03** R352E/D993R 6 3.62 ± 0.06* 1.48 ± 0.04 1.59 ± 0.02** Table 3 Relative conductances of some anions in WT-CFTR and R352-CFTR mutants CFTR n SCN Br NO3 WT 6 0.16 ± 0.02 0.67 ± 0.04 0.84 ± 0.04 R352A 10 1.59 ± 0.12* 1.31 ± 0.08* 1.59 ± 0.14* R352E 6 2.73 ± 0.31*, ** 1.49 ± 0.22* 1.54 ± 0.12* R352K 7 1.12 ± 0.08*, ** 0.99 ± 0.02*, ** 1.73 ± 0.26* R352E/ D993R 7 0.61 ± 0.05*, ** 0.98 ± 0.03*, ** 1.26 ± 0.13* Relative conductance was measured at VM = Vrev -25 mV * Significant difference compared with WT-CFTR, P\0.05; ** Significant difference compared with R352A, P\0.05 that loss of positive charge at position 352 destroyed the overall pore architecture, which subsequently changed the anion selectivity characteristics as seen in R352A- and R352E-CFTR. Login to comment
173 Furthermore, the finding that relative permeability values are nearly identical in R352A-, R352E- and R352K-CFTR suggests that the role of this site in determining anion selectivity is only indirect. Login to 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). Login to comment
178 Both R347A- and R352A-CFTR showed significantly weakened block by 200 lM glipizide, largely due to loss of the time-dependent component (Fig. 6). Login to comment
179 The average fractional block of WT-CFTR by 200 lM glipizide at VM = -120 mV (0.48 ± 0.02, n = 6) was significantly different from the block of R352A-CFTR (0.33 ± 0.03, n = 5, P = 0.004). Login to comment
181 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. Login to comment
183 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. Login to comment
184 However, the voltage dependence of block was clearly altered in R352A- and R347A-CFTR. Login to comment
185 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). Login to comment
186 In summary, mutations at R352 that destroyed the positive charge (R352A, R352E and R352Q) altered the pore architecture of CFTR and caused instability of the open state, changing anion selectivity and pore block by glipizide. Login to comment
189 100 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. Login to comment
190 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). Login to comment
192 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). Login to comment
207 As discussed above, block of R352A-CFTR by glipizide was different from that of WT-CFTR in that the time-dependent component of block was lost (Fig. 6). Login to comment
220 R352E/D993R-CFTR exhibited relative conductances to SCN- and Br- intermediate between that of WT-CFTR and R352A-CFTR (Table 3). Login to comment
221 These results also suggested that R352A-CFTR and R352E/D993R-CFTR have different pore architecture and that the selectivity properties of the pore of the double mutant might be slightly different from that of WT-CFTR. Login to comment
225 D993R-CFTR exhibited instability of the open state, with frequent transitions between all three open conductance levels (Fig. 9A, B); these three open states were even less stable than those of R352A-CFTR. Login to comment
242 MTSEA+ led to a transient increase in WT-CFTR current but a sustained increase in R352A-CFTR current (Fig. 10). Login to comment
243 After 5 min of incubation, WT-CFTR exhibited a 1.11 ± 0.05-fold increase, while R352A-CFTR exhibited a 1.30 ± 0.07-fold increase (n = 3 each, P = 0.021). Login to comment
246 First, channels bearing charge-destroying mutations at this site, including R352Q, R352E and R352A, exhibited instability of the open state compared to WT-CFTR, as indicated by frequent transitions between all three open conductance states (s1, s2, f). Login to comment
249 R352A-CFTR exhibited outward rectification in conditions of symmetrical [Cl- ], similar to that found in R347A-CFTR (Fig. 6). Login to comment
253 In c, points show mean ± SEM for n = 7 observations, and error bars are smaller than the symbols; lines are from linear regression WT 1 A 200 s Isoproterenol 0.4 A 100 s 0.4 A 100 s R352A R352E/D993R MTSEA MTSEA MTSEA WT 1 A 200 s Isoproterenol 0.4 A 100 s 0.4 A 100 s R352A R352E/D993R MTSEA MTSEA MTSEA Fig. 10 Mutation R352A results in appearance of sensitivity to a cysteine-modifying reagent. Login to comment
254 Oocytes expressing WT-CFTR (top trace), R352A-CFTR (middle trace) or R352E/D993R-CFTR (bottom trace), along with the b2-adrenergic receptor, were studied by two-electrode voltage clamp. Login to comment
300 We also note that while the relative conductance values for SCN- , Brand NO3 - are shifted in the same direction in R352K-CFTR as they are in R352A- or R352E-CFTR, the shifts for SCN- and Br- are smaller in R352K-CFTR than in the charge-destroying mutants. Login to comment

[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.

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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). Login to comment
140 Mutations R347A and R352A also represent a separate category from the rest. Login to comment
145 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. Login to comment
151 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. Login to comment
158 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. Login to comment
166 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). Login to comment
173 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). Login to comment
193 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). Login to comment
222 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]. Login to comment

[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.

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21 However, subconductance states are dominant events with short burst durations in CFTR channels bearing known salt bridge mutations, such as R352A, R347H, D993R, and D924R (13, 14). Login to comment
109 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. Login to comment
129 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. Login to comment
131 In R352A-CFTR, Arg347 can still interact with Asp924 and Asp993 . Login to comment
146 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). Login to comment
162 Recovery of Charge at R352C and D993C Rescued Channel Stability in the Full Open State-R352C-CFTR exhibited single channel behavior similar to that previously reported for R352A-, R352Q-, and R352E-CFTR (13). Login to comment
172 As a control, we show that R352A-CFTR was not sensitive to modification by MTS reagents (supplemental Fig. 2B). Login to comment
213 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). Login to comment

[hide] Three charged amino acids in extracellular loop 1 ... J Gen Physiol. 2014 Aug;144(2):159-79. doi: 10.1085/jgp.201311122. Epub 2014 Jul 14. Cui G, Rahman KS, Infield DT, Kuang C, Prince CZ, McCarty NA
Three charged amino acids in extracellular loop 1 are involved in maintaining the outer pore architecture of CFTR.
J Gen Physiol. 2014 Aug;144(2):159-79. doi: 10.1085/jgp.201311122. Epub 2014 Jul 14., [PMID:25024266]

Abstract [show]
The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) bears six extracellular loops (ECL1-6); ECL1 is the site of several mutations associated with CF. Mutation R117H has been reported to reduce current amplitude, whereas D110H, E116K, and R117C/L/P may impair channel stability. We hypothesized that these amino acids might not be directly involved in ion conduction and permeation but may contribute to stabilizing the outer vestibule architecture in CFTR. We used cRNA injected oocytes combined with electrophysiological techniques to test this hypothesis. Mutants bearing cysteine at these sites were not functionally modified by extracellular MTS reagents and were blocked by GlyH-101 similarly to WT-CFTR. These results suggest that these three residues do not contribute directly to permeation in CFTR. In contrast, mutants D110R-, E116R-, and R117A-CFTR exhibited instability of the open state and significantly shortened burst duration compared with WT-CFTR and failed to be locked into the open state by AMP-PNP (adenosine 5'-(beta,gamma-imido) triphosphate); charge-retaining mutants showed mainly the full open state with comparably longer open burst duration. These interactions suggest that these ECL1 residues might be involved in maintaining the outer pore architecture of CFTR. A CFTR homology model suggested that E116 interacts with R104 in both the closed and open states, D110 interacts with K892 in the fully closed state, and R117 interacts with E1126 in the open state. These interactions were confirmed experimentally. The results suggest that D110, E116, and R117 may contribute to stabilizing the architecture of the outer pore of CFTR by interactions with other charged residues.

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130 This is similar to our previous findings for TM6 mutants R334C-, R352A-, R347C/H-CFTR (Cotten and Welsh, 1999; Zhang et al., 2005b; Cui et al., 2008). Login to comment

[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.

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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). Login to comment
113 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). Login to comment
131 WT 0.0 0.1 0.2 0.3 Fractional inhibition by 2.5 &#b5;M GlyH-101 # R334C R334A T338A R352A # # # 0.4 0.5 0.4 &#b5;A 50 s ND96 ISO ISO+ GlyH ND96 ISO R334C- hCFTR A B C D ND96 ISO ISO+ GlyH ND96 ISO T338A-hCFTR 1 &#b5;A 50 s 1.0 &#b5;A 50 s ND96 ISO ISO+ GlyH ND96 ISO WT-hCFTR Fig. 5. Login to comment
167 We first investigated the effect of 2.5 òe;M GlyH-101 on hCFTR with mutations at selected amino acids that are also conserved in mCFTR, selected as follows: 1) R334 sits in the outer mouth of the CFTR pore, attracts Clafa; into the pore, and directly affects ion conduction, which might affect GlyH-101 binding in the pore; 2) T338 is located in the narrow part of the hCFTR pore and has been suggested as a possible binding site for GlyH-101 (21); 3) R352 forms a salt bridge with D993 and D924 and plays a key role in maintaining the open pore architecture of hCFTR (3, 37); consequently, mutation R352A disrupts the R352-D993-D924 salt bridge and may affect the GlyH-101 binding site by altering the hCFTR open pore A B D 800 pA 100 s hCFTR 1 mM ATP+ 127.6 U PKA ATP+PKA+ GlyH-101 control Fractional increase of mCFTR by GlyH-101 1.2 0 20 40 60 80 Kd = 0.60 nM Concentration (nM) 100 0.4 0.8 E 0.4 pA 2 s c f -GlyH-101 2000 # of events 0.0 0.2 -0.2 -0.4 -0.6 -0.8 -1.0 Current (pA) 4000 0.4 pA 2 s c f +GlyH-101 s1 2000 # of events 0.0 0.2 -0.2 -0.4 -0.6 -0.8 -1.0 Current (pA) 4000 6000 NPo 1.0 0 -GlyH +GlyH 2.0 * F 1 nA 100 s mCFTR 1mM ATP+ 127.6 U PKA ATP+PKA+ GlyH-101 control 200 pA 100 s mCFTR 1 mM ATP+ 638 U PKA ATP+PKA+ GlyH-101 control Fig. 7. Login to comment
181 In contrast, 2.5 òe;M GlyH-101 exhibited strengthened block of both T338A- and R352A-hCFTR (Fig. 5, C and D). Login to comment