ABCMdb

PMID: 18421494

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., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC7 p.Arg352Lys In contrast, R352K-CFTR was similar to wild-type. Login to comment 6 ABCC7 p.Arg352Glu 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. Login to comment 8 ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu 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. Login to comment 9 ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu ABCC7 p.Arg352Ala Finally, R352A-CFTR was sensitive to modification by externally applied MTSEA+ , while wild-type and R352E/D993R-CFTR were not. Login to comment 19 ABCC7 p.Arg347Asp Tabcharani et al. (1993) showed that anomalous mole-fraction behavior in mixtures of SCN- and Cl- was lost in R347D-CFTR and that this mutation also reduced single-channel conductance. Login to comment 21 ABCC7 p.Arg347Pro ABCC7 p.Arg347His Alternatively, Cotten and Welsh (1999) reported that R347 plays an important role in regulating the overall structure of the CFTR pore; R347P-CFTR exhibited significantly decreased single-channel conductance and unstable channel openings, while R347H-CFTR displayed a pH-dependent conductance and anomalous mole-fraction behavior. Login to comment 22 ABCC7 p.Arg347Lys Incontrast, R347K-CFTR exhibited permeation properties similar to those of wild-type (WT)-CFTR. Login to comment 24 ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg The D924R mutation in TM8 complemented the R347D mutation, reverting the channel to WT behavior, allowingtheauthorstoconcludethatR347functionsatleastin part by forming a salt bridge with D924 (Cotten and Welsh 1999). Login to comment 30 ABCC7 p.Arg352Gln ABCC7 p.Arg352Trp ABCC7 p.Arg352Gly Three cystic fibrosis (CF)-associated mutations have been identified at this position (R352G, R352W and R352Q), but the mechanisms by which these mutations cause disease are not clear (Cremonesi et al. 1992; Audre´zet et al. 1993; Brancolini et al. 1995). Login to comment 73 ABCC7 p.Arg352Ala 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 ABCC7 p.Arg352Ala 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 98 ABCC7 p.Arg347Cys ABCC7 p.Arg352Cys Previous studies showed that both R347C and R352C either were not accessible to membrane-impermeant MTS reagents (methanethiosulfonate ethyltrimethylammonium [MTSET+ ] or methanethiosulfonate ethylsulfonate [MTSES- ]) applied to the extracellular solution or lacked significant functional consequences when modified; this suggested that both sites either were at the predicted cytoplasmic end of the pore, and therefore cytoplasmic to the narrow region, or were not pore-facing residues (Smith et al. 2001). Login to comment 99 ABCC7 p.Arg352Cys R352C was, however, sensitive to membrane-permeant MTSEA+ . Login to comment 100 ABCC7 p.Arg352Gln Surprisingly, mutation R352Q also resulted in sensitivity to MTSEA+ , suggesting that loss of positive charge at position 352 caused an endogenous cysteine to become available for modification, perhaps reflecting loss of gross pore structure. Login to comment 105 ABCC7 p.Thr338Ala ABCC7 p.Arg334Cys Transitions to these subconductance levels occur rarely in WT-CFTR but more frequently in some pore-domain mutants, such as R334C and T338A, although the relative conductances between levels s1, s2, and f are maintained (Zhang et al. 2005a). Login to comment 111 ABCC7 p.Arg352Gln ABCC7 p.Arg352Gln ABCC7 p.Arg352Ala ABCC7 p.Arg352Ala 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 ABCC7 p.Arg352Gln ABCC7 p.Arg352Ala The transitions between the three open states in R352A- and R352Q-CFTR were random, showing no regular pattern. Login to comment 114 ABCC7 p.Arg334Cys In contrast, our previous experiments (Zhang et al. 2005a) with R334C-CFTR showed a more regular transition pattern: Within nearly every burst there were transitions to all three conductance states, with most bursts ending in the f state. Login to comment 120 ABCC7 p.Arg352Lys R352K-CFTR showed single-channel properties very similar to those of WT-CFTR: Transitions to the s1 and s2 conductance states were rare events in this mutant (Fig. 1D). Login to comment 122 ABCC7 p.Arg352Gln ABCC7 p.Arg352Lys ABCC7 p.Arg352Ala 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 ABCC7 p.Arg352Gln ABCC7 p.Arg352Ala In R352A- and R352Q-CFTR, there are four current levels indicating the c, s1, s2 and f states. Login to comment 126 ABCC7 p.Arg352Ala 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 ABCC7 p.Arg352Ala 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 ABCC7 p.Arg352Ala 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 ABCC7 p.Arg352Ala 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 ABCC7 p.Arg352Ala 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 ABCC7 p.Arg352Ala Substate behavior was observed in R352A-CFTR at all voltages (Fig. 4A), including both negative and positive membrane potentials. Login to comment 134 ABCC7 p.Arg352Gln ABCC7 p.Arg352Gln ABCC7 p.Arg352Lys ABCC7 p.Arg352Ala ABCC7 p.Arg352Ala 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 ABCC7 p.Arg352Ala ABCC7 p.Arg352Ala 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 136 ABCC7 p.Arg352Gln The f state slope conductance in R352Q-CFTR was slightly reduced at both negative and positive membrane potentials. Login to comment 137 ABCC7 p.Arg352Glu Both f state slope conductances were significantly reduced in R352E-CFTR (Table 1). Login to comment 138 ABCC7 p.Arg352Lys In contrast, the slope conductances of R352K-CFTR were very similar to those of WT-CFTR (Table 1). Login to comment 139 ABCC7 p.Arg352Gln ABCC7 p.Arg352Glu ABCC7 p.Arg352Ala The single-channel conductance of the f state exhibited significant outward rectification in R352A-, R352Q- and R352E-CFTR (Table 1). Login to comment 140 ABCC7 p.Arg352Ala 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 141 ABCC7 p.Arg352Gln Shown in the top trace is a single record for R352Q-CFTR in a patch with low open probability, VM = -80 mV. Login to comment 143 ABCC7 p.Arg352Ala 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 ABCC7 p.Arg352Ala 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 ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Lys ABCC7 p.Arg352Lys ABCC7 p.Arg352Ala ABCC7 p.Arg352Ala 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 ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Lys ABCC7 p.Arg352Lys ABCC7 p.Arg352Ala ABCC7 p.Arg352Ala 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 ABCC7 p.Arg352Gln ABCC7 p.Arg352Gln ABCC7 p.Arg352Gln ABCC7 p.Arg352Gln ABCC7 p.Arg352Gln ABCC7 p.Arg352Gln ABCC7 p.Arg352Lys ABCC7 p.Arg352Lys ABCC7 p.Arg352Ala ABCC7 p.Arg352Ala ABCC7 p.Arg352Ala ABCC7 p.Arg352Ala ABCC7 p.Arg352Ala 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 ABCC7 p.Arg352Ala (A) Four traces of R352A-CFTR from a single patch at tested voltages indicated at the right. Login to comment 159 ABCC7 p.Arg352Gln ABCC7 p.Arg352Lys ABCC7 p.Arg352Ala (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 ABCC7 p.Arg352Gln ABCC7 p.Arg352Ala (C) The i-V relationship of the s1 and s2 subconductance states of R352A- and R352Q-CFTR. Login to comment 162 ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg ABCC7 p.Glu1104Arg ABCC7 p.Arg352Gln ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Lys ABCC7 p.Arg352Ala ABCC7 p.Glu873Arg 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 ABCC7 p.Arg352Ala 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 ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Lys ABCC7 p.Arg352Ala 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 ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Lys ABCC7 p.Arg352Ala 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 ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Lys ABCC7 p.Arg352Lys ABCC7 p.Arg352Ala ABCC7 p.Arg352Ala ABCC7 p.Arg352Ala ABCC7 p.Arg352Ala ABCC7 p.Arg352Ala 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 ABCC7 p.Arg352Glu ABCC7 p.Arg352Lys ABCC7 p.Arg352Ala 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 ABCC7 p.Arg347Ala ABCC7 p.Arg352Ala 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 ABCC7 p.Arg347Ala ABCC7 p.Arg352Ala 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 ABCC7 p.Arg352Ala 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 180 ABCC7 p.Arg347Ala 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). Login to comment 181 ABCC7 p.Arg347Ala ABCC7 p.Arg352Ala 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 182 ABCC7 p.Arg352Lys In contrast, the average fractional block of R352K-CFTR by 200 lM glipizide was not significantly different from the block of WT-CFTR at this concentration (0.52 ± 0.02, n = 6, P = 0.119) (Fig. 6G). Login to comment 183 ABCC7 p.Arg352Lys ABCC7 p.Arg352Lys ABCC7 p.Arg352Lys ABCC7 p.Arg347Ala ABCC7 p.Arg352Ala 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 ABCC7 p.Arg347Ala ABCC7 p.Arg352Ala However, the voltage dependence of block was clearly altered in R352A- and R347A-CFTR. Login to comment 185 ABCC7 p.Arg352Lys ABCC7 p.Arg347Ala ABCC7 p.Arg352Ala 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 ABCC7 p.Arg352Gln ABCC7 p.Arg352Glu ABCC7 p.Arg352Ala 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 187 ABCC7 p.Arg352Lys R352K-CFTR, in contrast, maintained the positive charge and most characteristics of WT-CFTR. This strongly suggests that R352 may serve a critical role in preserving the gross structure of the channel pore, perhaps by contributing to an interfacial pair with a negatively charged amino acid at another position in CFTR. Login to comment 189 ABCC7 p.Arg352Lys ABCC7 p.Arg352Lys ABCC7 p.Arg347Ala ABCC7 p.Arg347Ala ABCC7 p.Arg352Ala ABCC7 p.Arg352Ala 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 ABCC7 p.Arg352Lys ABCC7 p.Arg347Ala ABCC7 p.Arg352Ala 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 ABCC7 p.Arg352Lys ABCC7 p.Arg347Ala ABCC7 p.Arg352Ala 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 198 ABCC7 p.Arg352Glu To identify the interaction partner for R352, we replaced R352 with an acidic residue (R352E) and introduced an arginine residue in the place of candidate interaction partners. Login to comment 199 ABCC7 p.Asp993Arg ABCC7 p.Glu1104Arg ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Glu873Arg We studied the conductance properties of CFTR channels bearing the following mutations: R352E, R352E/E873R, R352E/ D993R and R352E/E1104R. Login to comment 201 ABCC7 p.Glu1104Arg ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Glu873Arg Three of these mutants, R352E-, R352E/E873R- and R352E/E1104R-CFTR, exhibited instability of the open state, in which the amplitudes of the s1, s2 and f conductance states were very similar between the three mutants. Login to comment 202 ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu ABCC7 p.Arg352Lys R352E/D993R-CFTR, in contrast, exhibited stability of the full conductance state similar to that seen in WT-CFTR and R352K-CFTR (Fig. 1); transitions to the s1 and s2 states were rare events in this double mutant. Login to comment 204 ABCC7 p.Glu1104Arg ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Glu873Arg R352E-, R352E/ E873R- and R352E/E1104R-CFTR exhibited significant outward rectification, while WT-CFTR did not (Table 1). Login to comment 205 ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu The slope conductance of R352E/D993R-CFTR was slightly lower than that of WT-CFTR, although linearity of the i-V relation was mostly retained. Login to comment 206 ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu These data suggested that the D993R mutation at least partly compensated for the R352E mutation, although the double mutant R352E/ D993R-CFTR did not fully recapitulate the behavior of WT-CFTR. Login to comment 207 ABCC7 p.Arg352Ala 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 208 ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg ABCC7 p.Glu1104Arg ABCC7 p.Glu1104Arg ABCC7 p.Glu1104Arg ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Glu873Arg ABCC7 p.Glu873Arg ABCC7 p.Glu873Arg If D993 served as the interaction partner of R352, we would expect that block of R352E/D993R-CFTR would be similar to that 0.4 pA 2 s 0.4 pA 2 s 0.2 pA 2 s 0.2 pA 2 s c s1 s2 f c s1 s2 f c s1 s2 f c f R352E R352E/E873R R352E/E1104R R352E/D993R 0 4000 #ofevents 0.0 -0.4 -0.8 0.0 -0.4 -0.8 3000 #ofevents 0 #ofevents 0.0 -0.4 -0.8 3000 0 Currents (pA) 0.0 -0.4 0 2500#ofevents -0.8 fc s1 s2 s1 s2 s1 s2 0.4 pA 2 s 0.4 pA 2 s 0.2 pA 2 s 0.2 pA 2 s c s1 s2 f c s1 s2 f c s1 s2 f c f R352E R352E/E873R R352E/E1104R R352E/D993R 0.4 pA 2 s 0.4 pA 2 s 0.4 pA 2 s 0.4 pA 2 s 0.2 pA 2 s 0.2 pA 2 s 0.2 pA 2 s 0.2 pA 2 s c s1 s2 f c s1 s2 f c s1 s2 f c f R352E R352E/E873R R352E/E1104R R352E/D993R B C D A 0 4000 #ofevents 0.0 -0.4 -0.8 0 4000 #ofevents 0.0 -0.4 -0.8 0.0 -0.4 -0.8 3000 #ofevents 0 0.0 -0.4 -0.8 3000 #ofevents 0 #ofevents 0.0 -0.4 -0.8 3000 0 Currents (pA) #ofevents 0.0 -0.4 -0.8 3000 0 #ofevents 0.0 -0.4 -0.8 3000 0 Currents (pA) 0.0 -0.4 0 2500#ofevents -0.8 fc s1 s2 s1 s2 s1 s2 Fig. 7 Single-channel current tracings of R352E-CFTR and double mutants from excised inside-out patches (left) and resulting all-points amplitude histograms (right) under the same experimental conditions as in Fig. 1. Login to comment 210 ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu There are four current levels indicating the c, s1, s2 and f states in all but the revertant mutant R352E/ D993R-CFTR, which only exhibited the c and f states. Login to comment 213 ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu Figure 8B shows macropatch currents from R352E/D993R-CFTR in the presence and absence of 200 lM glipizide; time-dependent block was rescued in this double mutant. Login to comment 215 ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu This result suggested that the revertant double mutation, R352E/ D993R, recovered the time-dependent block by glipizide but did not completely recover the sensitivity to glipizide characteristic of WT-CFTR. This may reflect the difference in side chain volumes between aspartic and glutamic acids; the volume of a glutamic acid side chain is 20% larger than that of an aspartic acid side chain (Creighton 1993), which may result in a different pore structure. Login to comment 216 ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu To further explore the characteristics of R352E/D993R-CFTR, we studied anion selectivity between Cl- and four substitute monovalent anions. Login to comment 218 ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu Overall, both relative permeability and relative conductance values for WTand R352E/ D993R-CFTR were similar (Tables 2, 3). Login to comment 219 ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu R352E/D993R- CFTRcurrentsexhibitedthesameanionpermeabilitysequence as WT-CFTR: SCN- [NO3 - C Br- [Cl- (although PSCN/ PCl was clearly reduced in the double mutant). Login to comment 220 ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu ABCC7 p.Arg352Ala 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 ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu ABCC7 p.Arg352Ala 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 222 ABCC7 p.Asp993Arg The D993R Mutation Alone also Altered the Pore Architecture of CFTR D993 is localized to TM9 of CFTR, which has not been suggested to be a pore lining domain (McCarty 2000). Login to comment 223 ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu Because the data presented thus far suggested that D993 serves as the interacting partner of R352, we predicted that the D993R mutation alone would change channel activity in a manner similar to that of the R352E, -A or -Q mutation. Login to comment 224 ABCC7 p.Asp993Arg We studied D993R-CFTR with single-channel recording techniques using the same conditions as in Fig. 1. Login to comment 225 ABCC7 p.Asp993Arg ABCC7 p.Arg352Ala 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 226 ABCC7 p.Asp993Arg The slope conductance of the f state in D993R-CFTR was larger than that of WT-CFTR and indicated slight inward rectification (Fig. 9C, Table 1). Login to comment 227 ABCC7 p.Asp993Arg These results suggest that the D993R mutation alone also destroyed pore architecture in a manner similar to that of the charge-destroying mutations at R352. Login to comment 229 ABCC7 p.Arg352Cys We previously reported that a cysteine engineered at R352 either was not accessible to the membrane-impermeable reagents MTSES- and MTSET+ applied externally or lacked significant functional consequences when modified, although R352C-CFTR (but not WT-CFTR) did respond to prolonged exposure to the membrane-permeant MTSEA+ (Smith et al. 2001). Login to comment 230 ABCC7 p.Arg352Gln ABCC7 p.Arg352Cys Surprisingly, similar results were found in R352Q-CFTR, suggesting that the response to MTSEA+ in R352C-CFTR was nonspecific, not being due to modification of that engineered cysteine. Login to comment 232 ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg ABCC7 p.Glu1104Arg ABCC7 p.Glu1104Arg ABCC7 p.Glu1104Arg ABCC7 p.Glu1104Arg ABCC7 p.Glu1104Arg ABCC7 p.Glu1104Arg ABCC7 p.Glu1104Arg ABCC7 p.Glu1104Arg ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Glu873Arg ABCC7 p.Glu873Arg ABCC7 p.Glu873Arg ABCC7 p.Glu873Arg ABCC7 p.Glu873Arg ABCC7 p.Glu873Arg ABCC7 p.Glu873Arg ABCC7 p.Glu873Arg Hence, it is likely that MTSEA+ modified one (or more) of the endogenous cysteines, which B WT-CFTR R352E/D993R R352E R352E/E873R R352E/E1104R WT-CFTR R352E/D993R R352E R352E/E873R R352E/E1104R mV -100 -50 50 100 -0.8 -0.4 0.4 0.8 pA 100 ms 0.2 nA A WT-CFTR R352E/D993R R352E R352E/E873R R352E/E1104R WT-CFTR R352E/D993R R352E R352E/E873R R352E/E1104R mV -100 -50 50 100 -0.8 -0.4 0.4 0.8 pA WT-CFTR R352E/D993R R352E R352E/E873R R352E/E1104R WT-CFTR R352E/D993R R352E R352E/E873R R352E/E1104R WT-CFTR R352E/D993R R352E R352E/E873R R352E/E1104R WT-CFTR R352E/D993R R352E R352E/E873R R352E/E1104R mV -100 -50 50 100 -0.8 -0.4 0.4 0.8 pA 100 ms 0.2 nA Fig. 8 The double mutant R352E/D993R-CFTR recovers WT-like channel behavior. Login to comment 233 ABCC7 p.Asp993Arg ABCC7 p.Glu1104Arg ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Glu873Arg (A) Single channel i-V relationships are shown for full conductance states of WT-, R352E-, R352E/E873R-, R352E/ E1104R- and R352E/D993R-CFTR. Login to comment 235 ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu (B) Block of R352E/ D993R-CFTR macropatch currents by glipizide (200 lM) is time-dependent. Login to comment 239 ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu If this were true, we would expect that the revertant double mutant, R352E/D993R-CFTR, would not respond to MTSEA+ . Login to comment 242 ABCC7 p.Arg352Ala MTSEA+ led to a transient increase in WT-CFTR current but a sustained increase in R352A-CFTR current (Fig. 10). Login to comment 243 ABCC7 p.Arg352Ala 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 244 ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu In contrast, R352E/ D993R-CFTR was insensitive to exposure to MTSEA+ , which resulted in only a 1.08 ± 0.02-fold increase in current (n = 6), thus indicating that the double mutant exhibited WT-like behavior. Login to comment 246 ABCC7 p.Arg352Gln ABCC7 p.Arg352Glu ABCC7 p.Arg352Ala 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 248 ABCC7 p.Arg352Lys In contrast, channels bearing the charge-conserving mutation R352K showed characteristics similar to WT-CFTR. Login to comment 249 ABCC7 p.Arg347Ala ABCC7 p.Arg352Ala R352A-CFTR exhibited outward rectification in conditions of symmetrical [Cl- ], similar to that found in R347A-CFTR (Fig. 6). Login to comment 250 ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg These results strongly suggested that the loss of the positively charged side chain at position 352 shifted the pore architecture in such a way as to destabilize the full open state, 0.2 pA 2 s c s1 s2 f 0.2 pA 2 s c s1 s2 f Currents (pA) 0.0 -0.4 -0.8 2000 4000 6000 #ofevents mV -100 -50 50 100 -1.0 -0.5 0.5 1.0 WT-CFTR D993R pA 0.2 pA 2 s c s1 s2 f 0.2 pA 2 s c s1 s2 f A CB Currents (pA) 0.0 -0.4 -0.8 2000 4000 6000 #ofevents Currents (pA) 0.0 -0.4 -0.8 2000 4000 6000 #ofevents mV -100 -50 50 100 -1.0 -0.5 0.5 1.0 WT-CFTR D993R pA mV -100 -50 50 100 -1.0 -0.5 0.5 1.0 WT-CFTR D993R WT-CFTR D993R pA Fig. 9 Mutation D993R alone had effects similar to those of the charge-reversing mutations at R352. Login to comment 251 ABCC7 p.Asp993Arg Representative single-channel current tracing (A), all-points amplitude histogram (B) and i-V relationship for the f conductance state (C) are shown for the D993R mutant. Login to comment 253 ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Ala ABCC7 p.Arg352Ala ABCC7 p.Arg352Ala 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 ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu ABCC7 p.Arg352Ala 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 258 ABCC7 p.Asp993Arg ABCC7 p.Glu1104Arg ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Glu873Arg Second, we identified the interaction partner as D993 by use of double mutants; R352E/E873R-CFTR and R352E/E1104R-CFTR exhibited permeation properties similar to those of R352E-CFTR, while R352E/D993R-CFTR behaved more like WT-CFTR. Login to comment 261 ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu As predicted, D993R-CFTR exhibited instability of the open state similar to that seen in R352E-CFTR. Login to comment 263 ABCC7 p.Arg352Cys Designation of R352 as the anion selectivity filter was predominantly based upon differences in the rates of modification of engineered cysteines at this site (in R352C-CFTR) by positively and negatively charged sulfhydryl modifying reagents, MTSET+ and MTSES- (Cheung and Akabas 1997; Guinamard and Akabas 1999). Login to comment 265 ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg Also, near the predicted cytoplasmic end of the CFTR pore, substitutions of the arginine at position 347 by any residue other than lysine destabilized the pore structure, while the double mutation R347D/ D924R recovered open state stability, suggesting that R347 formed a salt bridge with D924 in the wild-type channel (Cotten and Welsh 1999). Login to comment 274 ABCC7 p.Asp836* However, other authors reported that the amino-terminal portion of CFTR, containing MSD1, NBD1 and the R domain (D836X-CFTR), formed regulated Cl-channels that differed somewhat from WT-CFTR (Sheppard et al. 1994). Login to comment 275 ABCC7 p.Asp836* ABCC7 p.Asp836* ABCC7 p.Asp836* Although the substate behavior of D836X-CFTR was not studied in detail, this mutant was reported to show instability of the open state compared to WT-CFTR; the authors suggested that residues in MSD2 might stabilize the channel complex, perhaps assisting in the arrangement of residues in MSD1 into a functional structure, based on the finding that the number of functional Cl-channels generated from the D836X construct was much lower than expected for WT-CFTR. Login to comment 281 ABCC7 p.Arg352Gln ABCC7 p.Asp993Tyr ABCC7 p.Arg352Trp ABCC7 p.Arg352Gly ABCC7 p.Asp993Gly Several R352 mutations are CF-associated mutations, including R352G, R352W and R352Q (Cremonesi et al. 1992; Audre´zet et al. 1993; Brancolini et al. 1995; Feldmann et al. 2003); similarly, D993Y and D993G are associated with disease (Tsui et al. 2007). Login to comment 295 ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu ABCC7 p.Arg352Lys Stability of the open state was retained in the case of a charge-conserving mutation, R352K, and in the double mutant R352E/D993R-CFTR. Login to comment 297 ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu Compared to WT-CFTR, R352E/D993R-CFTR channels exhibited lower slope conductance, weakened block by glipizide, and altered selectivity between Cl- and SCN- . Login to comment 298 ABCC7 p.Arg352Lys The fact that differences in anion selectivity remain between R352K- and WT-CFTR is consistent with the notion that interactions between lysine and the aspartic acid at D993 are not the same as the interactions between arginine and D993. Login to comment 300 ABCC7 p.Arg352Glu ABCC7 p.Arg352Lys ABCC7 p.Arg352Lys ABCC7 p.Arg352Ala 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 301 ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu We conclude that the double mutant R352E/D993R-CFTR retains the interaction between these residues but does not fully mimic the behavior of WT-CFTR, suggesting that permeation properties in the CFTR chloride channel are very sensitive to small changes in pore structure. Login to comment