ABCMdb

ABCC7 p.Phe337Ser

None has been submitted yet.

Publications

PMID: 11179391 [PubMed] Linsdell P et al: "Relationship between anion binding and anion permeability revealed by mutagenesis within the cystic fibrosis transmembrane conductance regulator chloride channel pore."

No. Sentence Comment

19 Two mutations in CFTR which alter the anion permeability sequence, F337S and T338A, also altered the anion conductance sequence. Login to comment
37 Most interestingly, two mutations which reduce amino acid side chain size at position 337, F337A and F337S, virtually abolished the normal lyotropic relationship between anion permeability and energy of hydration (Linsdell et al. 2000). Login to comment
38 The present study seeks to shed new light on the relationship between anion binding and anion permeability in CFTR channels by comparing the anion binding properties of wild-type CFTR with two mutants with altered anion selectivity, F337S and T338A. Login to comment
43 In the present study, the permeation properties of two mutants, F337S and T338A, have been examined in detail. Login to comment
44 Of those mutations introduced at F337, only F337A and F337S strongly altered anion selectivity, and the effects of these two mutations were very similar (Linsdell et al. 2000). Login to comment
46 However, of all the mutations introduced at F337, only F337S could be expressed in CHO cells and studied using single channel recording (P. Linsdell, A. Evagelidis & J. W. Hanrahan, unpublished observations). Login to comment
47 F337S was also more strongly expressed in BHK cells, allowing small macroscopic currents carried by anions with a very low conductance to be measured. Login to comment
49 For the present study, T338A was examined because: (1) it has a high single channel conductance, allowing single channel currents to be resolved, (2) its anion selectivity strongly follows the lyotropic sequence, in fact more strongly than that of wild-type CFTR, such that its effects on anion permeability might be considered 'opposite` to the effects of F337S, (3) replacing the threonine with a small, 'neutral` alanine is considered less likely to cause large changes in transmembrane helix structure, and (4) T338A is well expressed in both CHO and BHK cells (see Linsdell et al. 1998, for a full description of the permeation phenotype of T338A). Login to comment
83 Unitary properties of F337S and T338A CFTR A, single channel currents carried by wild-type, F337S and T338A, expressed in CHO cells, with symmetrical 150 mÒ NaCl, at a membrane potential of -50 mV. Login to comment
85 B, mean i-V relationships under these ionic conditions, wild-type 0, F337S 1 and T338A ±; mean of data from 3-9 patches. Login to comment
93 Estimation of unitary current amplitude from macroscopic current variance A, activation of macroscopic wild-type, F337S and T338A CFTR currents in BHK cell patches at +50 mV, in the symmetrical presence of 150 mÒ NaCl, by addition of PKA in the presence of ATP (see Methods). Login to comment
95 All three have been fitted by eqn (1) (see Methods), giving i = 0·118 pA and N = 374 for wild-type, i = 0·0387 pA and N = 1359 for F337S and i = 0·279 pA and N = 175 for T338A. Login to comment
105 Unitary Cl¦ currents carried by wild-type, F337S and T338A CFTR are compared in Fig. 3A. Mean slope conductance (Fig. 3B) was reduced in F337S (from 7·59 ± 0·10 pS (n = 12) to 1·76 ± 0·03 pS (n = 7)), and significantly increased in T338A (to 9·94 ± 0·14 pS, n = 6). Login to comment
106 Because of the low conductance of F337S, anion conductance was also estimated from the increase in current variance associated with activation of macroscopic CFTR currents at +50 mV in inside-out patches excised from BHK cells (Fig. 4), as described in Methods. Login to comment
110 Relative Cl¦ conductance appears to be weakly correlated with the size of the amino acid side chain present at position F337, with a large side chain favouring high conductance; this correlation results mainly from the low (and similar) conductances of the two mutants which strongly disrupted normal lyotropic anion selectivity, F337A and F337S. Login to comment
112 Macroscopic current variance analysis of relative anion conductance in wild-type, F337S and T338A Macroscopic currents were activated in BHK cell patches at +50 mV, in the symmetrical presence of the anion named on the far left, by addition of PKA in the presence of ATP (see Fig. 4). Login to comment
117 Relative anion permeabilities and conductances for wild-type and mutant CFTR ------------------------------------------------------------ PXÏPCl gXÏgCl ------------------ --------------------------- WT a F337S a T338A b WT c WT d F337S d T338A d ------------------------------------------------------------ Cl 1·00 ± 0·01 1·00 ± 0·08 1·00 ± 0·02 1·00 ± 0·01 1·00 ± 0·10 1·00 ± 0·16 1·00 ± 0·09 (10) (3) (11) (12) (9) (7) (4) Br 1·37 ± 0·07 0·50 ± 0·04 1·74 ± 0·04 0·48 ± 0·01 0·48 ± 0·13 0·16 ± 0·03* 0·44 ± 0·02 (8) (4) (3) (6) (4) (4) (3) I 0·83 ± 0·03 0·23 ± 0·02 2·09 ± 0·16 - - - - (6) (4) (5) F 0·10 ± 0·01 0·43 ± 0·02 0·12 ± 0·02 - 0·094 ± 0·017 0·76 ± 0·19* 0·054 ± 0·011 (9) (4) (4) (3) (4) (3) SCN 3·55 ± 0·26 0·93 ± 0·10 5·85 ± 0·27 - 0·060 ± 0·012 0·17 ± 0·04* 0·085 ± 0·007 (7) (5) (4) (5) (3) (4) NO× 1·58 ± 0·04 1·08 ± 0·02 2·04 ± 0·08 0·60 ± 0·02 0·73 ± 0·07 0·29 ± 0·08* 0·96 ± 0·05 (10) (4) (3) (5) (5) (3) (3) ClOÚ 0·25 ± 0·01 0·19 ± 0·00 1·35 ± 0·08 - 0·059 ± 0·014 0·041 ± 0·008 0·082 ± 0·011 (8) (3) (3) (6) (2) (4) Formate 0·24 ± 0·01 0·27 ± 0·02 0·45 ± 0·04 0·35 ± 0·01 0·49 ± 0·01 0·17 ± 0·02** 0·46 ± 0·07 (9) (3) (3) (6) (5) (3) (3) ------------------------------------------------------------a From Linsdell et al. (2000); b from Linsdell et al. (1998); c by single channel recording, d by current variance analysis. Login to comment
124 Macroscopic current variance analysis was also used to compare the relative conductances of different anions in wild-type, F337S and T338A (Fig. 6; Table 1). Login to comment
127 Relative conductances in wild-type estimated by these two methods (Fig. 7), as well as those in F337S and T338A estimated from macroscopic current variance analysis, are summarised in Table 1. Login to comment
128 For F337S, a number of significant differences from the wild-type pattern were observed; the relative conductance of NO×¦, Br¦ and formate were all significantly decreased, while the relative conductance of F¦ and SCN¦ increased. Login to comment
130 The conductance sequences for wild-type, F337S and T338A are summarised in Table 2. Login to comment
132 However, of those mutations involving substitution of F337 (Fig. 5), only F337A and F337S strongly disrupted selectivity (Linsdell et al. 2000). Login to comment
134 In order to determine whether the changes in relative anion conductance observed in F337S (Table 1) were associated with the change in selectivity or the change in conductance in this mutant, relative anion conductance was also examined in F337Y (Fig. 8). Login to comment
135 This mutant shows a reduction in Cl¦ conductance similar to that seen in F337S (Fig. 5), but its anion selectivity is identical to that of wild-type (Linsdell et al. 2000). Login to comment
136 Two of the most striking effects of F337S on relative anion conductance, the 67% reduction P. Linsdell J. Physiol. 531.158 Figure 7. Login to comment
140 Anion conductance changes observed in F337S are not seen in F337Y Relative Cl¦, Br¦ and F¦ conductances were estimated under symmetrical ionic conditions by macroscopic current variance analysis. Login to comment
143 Permeability and conductance sequences for wild-type and mutant CFTR ------------------------------------------------------------ Wild-Type F337S T338A ------------------------------------------------------------ Permeability sequence SCN > NO× > Br > Cl > NO× > Cl ü SCN > Br > SCN > I ü NO× > Br > I > ClOÚ formate > F F > formate > I > ClOÚ ClOÚ > Cl > formate > F Conductance sequence Cl > NO× > Br ü formate > Cl > F > NO× > SCN Cl ü NO× > formate Br > F > SCN ClOÚ formate Br > ClOÚ SCN ClOÚ > F ------------------------------------------------------------ Permeability and conductance sequences are derived from data given in Table 1. Login to comment
145 This strongly suggests that the alteration in anion relative conductance observed in F337S is related to the alteration in anion selectivity previously reported in this mutant (Linsdell et al. 2000). Login to comment
159 The ability of permeant anions to block Cl¦ currents was also examined in selectivity altering mutants, using macroscopic current variance experiments (for the low-conductance F337S) or single channel recording (for the high-conductance T338A), at -50 mV (Fig. 11). Login to comment
160 In F337S, block by SCN¦ and I¦ was somewhat weakened, and block by 10 mÒ ClOÚ¦ was abolished (although 25 mÒ ClOÚ¦ did significantly reduce Cl¦ current amplitude; data not shown). Login to comment
161 However, in contrast to wild-type, F337S Cl¦ currents were blocked by 25 mÒ Br¦ (Fig. 11B), although they were not significantly affected by 10 mÒ Br¦ (data not shown). Login to comment
165 The relationship between anion permeability and anion conductance The results summarised in Tables 1 and 2 indicate that the CFTR mutation F337S, which virtually abolishes the lyotropic pattern of anion permeability (Linsdell et al. 2000), also alters the relative conductance of different anions in the pore, whereas T338A, which strengthens the lyotropic nature of anion permeability (Linsdell et al. 1998), has no significant effect on relative conductance. Login to comment
175 The relationship between anion permeability and energy of hydration observed in wild-type and T338A is lost in F337S (Fig. 12A), which we previously suggested reflected a reduction in the relative importance of anion dehydration in determining anion P. Linsdell J. Physiol. 531.160 Figure 11. Login to comment
176 Block of wild-type, F337S and T338A CFTR by intracellular permeant anions Relative current amplitudes (at -50 mV) in the presence of different intracellular permeant anions were estimated from single channel recording (for wild-type, see Fig. 10, and also for T338A), or from macroscopic current variance analysis for F337S. Login to comment
183 However, anion conductance does appear weakly correlated with energy of hydration in F337S, although this is mainly due to the dramatic increase in relative conductance of the highly kosmotropic F¦ ion (Fig. 12B). Login to comment
186 This applies both to lyotropic anions with increased permeability in T338A (Br¦, NO×¦, ClOÚ¦) and the kosmotropic anion F¦, which shows increased permeability in F337S. Login to comment
189 The conductance ratio, gIÏgCl, was 0·20 ± 0·03 (n = 4) for wild-type, 0·14 ± 0·05 (n = 4) for F337S, and 0·59 ± 0·09 (n = 4) for T338A. Login to comment
197 Dependence of relative anion permeability (A) and relative anion conductance (B) on anion-free energy of hydration in wild-type, F337S and T338A CFTR Values of PXÏPCl and gXÏgCl in each case are as given in Table 1. Login to comment
218 Macroscopic I-V relationships under bi-ionic conditions (intracellular I¦, extracellular Cl¦), for wild-type, F337S and T338A CFTR expressed in BHK cells The different degrees of outward rectification suggest different relative I¦ conductances in these three CFTR variants. Login to comment
242 In the present study, the anion binding properties of two mutants, F337S and T338A, were examined in detail. Login to comment
243 F337S strongly altered the relative conductance of different anions, leading to significant decreases in the conductance of NO×¦, Br¦ and formate, and significant increases in the relative conductance of F¦ and SCN¦ (Table 1). Login to comment
246 The effects of the mutation F337S on relative anion conductance were not mimicked by another mutation, F337Y, which shows a similarly reduced Cl¦ conductance but unaltered anion selectivity (Fig. 8). Login to comment
247 This indicates that the mutation F337S causes a change in the architecture of the pore which alters anion permeability and anion conductance simultaneously, perhaps by modifying an anion binding site. Login to comment
249 Block by SCN¦, I¦ and ClOÚ¦ was significantly weakened in both F337S and T338A, and F337S showed significant block by high concentrations of Br¦ not evident in wild-type (Fig. 11). Login to comment
250 These results suggest that lyotropic anion binding is weakened in both F337S and T338A, in spite of the 'opposite` effect of these two mutations on anion selectivity. Login to comment
256 Implications for the mechanism of anion selectivity The mutations F337S and T338A caused co-ordinated changes in the relative permeability and relative conductance of Br¦, F¦, NO×¦, ClOÚ¦ and I¦ ions, with high permeability being associated with high conductance (Table 1; Fig. 13). Login to comment
261 Thus, not only in wild-type CFTR but also both F337S and T338A, Cl¦ is the anion with the highest conductance (Table 1), even though other anions may have higher permeabilities. Login to comment
269 In F337S, a reduction in the relative importance of anion dehydration allows kosmotropic anions such as F¦ to enter more easily, leading to a dramatic increase in both PFÏPCl and gFÏgCl in this mutant. Login to comment
270 In contrast, entry of Br¦, NO×¦ and I¦ is impeded in F337S, decreasing both the permeability and conductance of these relatively lyotropic anions. Login to comment

PMID: 11380256 [PubMed] Gupta J et al: "Asymmetric structure of the cystic fibrosis transmembrane conductance regulator chloride channel pore suggested by mutagenesis of the twelfth transmembrane region."

No. Sentence Comment

126 Although no information on the voltage dependence of SCN- block is obtained in this way, we have previously used this same protocol to show directly that intrapore anion binding is altered in the TM6 mutants F337S and T338A (21). Login to comment
177 Similar weakening of SCN- block was previously observed in the TM6 mutants F337S and T338A (21). Login to comment

PMID: 11478590 [PubMed] Linsdell P et al: "Thiocyanate as a probe of the cystic fibrosis transmembrane conductance regulator chloride channel pore."

No. Sentence Comment

131 In spite of this caveat, it is interesting to note that the effects of mutations within the pore that drastically alter SCN- permeability but have only small effects on SCN- binding (F337S, T338A; Linsdell 2001) may result in such a model from alterations in barrier height, implying that the effects of these mutations may result primarily from changes in anion access to the pore. Login to comment

PMID: 11927667 [PubMed] Gong X et al: "Molecular determinants of Au(CN)(2)(-) binding and permeability within the cystic fibrosis transmembrane conductance regulator Cl(-) channel pore."

No. Sentence Comment

12 Channel block by 100 mM Au(CN)2 _ , a measure of intrapore anion binding affinity, was significantly weakened in the CFTR mutants K335A, F337S, T338A and I344A, significantly strengthened in S341A and R352Q and unaltered in K329A. Login to comment
13 Relative Au(CN)2 _ permeability was significantly increased in T338A and S341A, significantly decreased in F337S and unaffected in all other mutants studied. Login to comment
42 Some of these have previously been associated with altered anion selectivity (F337S, T338A; Linsdelletal.1998,2000),alteredanion:cationselectivity(R352Q; Guinamard & Akabas, 1999), or disrupted open channel blocker binding (S341A; McDonough et al. 1994). Login to comment
79 The stimulation of F337S appeared somewhat less (1.46 ± 0.24-fold, n = 5), although this was not significantly different from wild-type (P > 0.1, Student`s two-tailed t test). Login to comment
87 Comparison between different channel variants at _100 mV reveals the sensitivity to this concentration of Au(CN)2 _ is R352Q > S341A > wild-type, K329A > I344A > K335A = F337S > T338A. Login to comment
100 Relative Au(CN)2 _ permeability was significantly decreased in F337S and significantly increased in T338A (Fig. 4), consistent with the previously described opposite effects of these mutants on CFTR lyotropic anion selectivity (Linsdell et al. 1998, 2000). Login to comment
116 Au(CN)2 _ permeability of different CFTR variants A, example CFTR I-V relationships recorded with 150 m KAu(CN)2 in the extracellular solution and 150 m KCl in the intracellular solution, for wild-type, F337S and T338A. Login to comment
123 At this voltage, block by 100 mM Au(CN)2 _ was significantly weakened in K335A, F337S, T338A and I334A, significantly strengthened in S341A and R352Q and unaffected in K329A (Fig. 3). Login to comment
124 The sequence of relative sensitivity to block by 100 mM Au(CN)2 _ at _100 mV (R352Q > S341A > wild-type, K329A > I344A > K335A = F337S > T338A) suggests that T338 normally makes the strongest contribution to Au(CN)2 _ binding within the pore, with nearby residues K335 and F337 also making large contributions. Login to comment
139 The effects of F337S and T338A on PAu(CN)2/PCl are consistent with the disruption (F337S; Linsdell et al. 2000) and strengthening (T338A; Linsdell et al. 1998) of lyotropic anion selectivity previously described in these two mutants. Login to comment
147 Only mutations in the central portion of TM6 (F337S, T338A, S341A) affected both Au(CN)2 _ binding and Au(CN)2 _ permeability (Figs 3_5). Login to comment

PMID: 12411425 [PubMed] Gong X et al: "Mechanism of lonidamine inhibition of the CFTR chloride channel."

No. Sentence Comment

7 5 Several point mutations within the sixth transmembrane region of CFTR (R334C, F337S, T338A and S341A) signi®cantly weakened block of macroscopic CFTR current, suggesting that lonidamine enters deeply into the channel pore from its intracellular end. Login to comment
116 As shown in Figure 7a, 55 mM lonidamine inhibited currents carried by R334C, K335A, F337S, T338A and S341A-CFTR. Login to comment
117 However, R334C, F337S and S341A were only weakly inhibited by this concentration relative to wild-type CFTR (see Figure 1). Login to comment
118 The eect of these mutations on block by lonidamine is more clearly seen in the dose-response curves shown in Figure 7b. Fits of these mean data by equation 1 suggests a Kd (at 7100 mV) of 58.5 mM for wild-type, 65.6 mM for K335A, 90.0 mM for T338A, 186 mM for F337S, 206 mM for S341A, and 338 mM for R334C. Login to comment
119 Similar analyses at other potentials showed a similar increase in Kd in R334C, F337S, S341A and (to a far lesser extent) T338A (Figure 7c). Login to comment
120 Fitting data from individual patches with equation 2 gave similar and, except in the case of K335A, signi®cant changes in Kd(-100): wild-type 60.6+5.2 mM (n=5), K335A 63.1+7.4 mM (n=5) (P40.05), T338A 93.4+4.1 mM (n=5) (P50.002), F337S 166+18 mM (n=5) (P50.0005), S341A 169+25 mM (n=5) (P50.005), R334C 260+19 mM (n=4) (P50.00001). Login to comment
121 These same ®ts also revealed changes in the voltage dependence of block, as judged by changes in d, although this was only statistically signi®cant in the case of R334C: wild-type 0.426+0.033 (n=5), K335A 0.484+0.024 (n=5) (P40.05), T338A 0.410+0.045 (n=5) (P40.05), F337S 0.365+0.015 (n=5) (P40.05), S341A 0.285+0.061 (n=5) (P40.05), R334C 0.233+0.066 (n=4) (P50.05). Login to comment
143 (a) Example I-V relationships for R334C, K335A, F337S, T338A and S341A-CFTR, before (solid lines) and following (dotted lines) addition of 55 mM lonidamine to the intracellular solution. Login to comment
145 (b) Concentration dependence of block at 7100 mV for wild-type, R334C, K335A, F337S, T338A and S341A. Login to comment
147 Each has been ®tted by equation 1, giving Kds of 58.5 mM (wild-type), 65.6 mM (K335A), 90.0 mM (T338A), 186 mM (F337S), 206 mM (S341A) and 338 mM (R334C). Login to comment
154 Lonidamine block was weakened in the TM6 mutants R334C, F337S and S341A (Figure 7), suggesting that these residues may normally contribute to lonidamine binding within the pore. Login to comment

PMID: 12679372 [PubMed] Gong X et al: "Molecular determinants and role of an anion binding site in the external mouth of the CFTR chloride channel pore."

No. Sentence Comment

40 conditions of high extracellular Cl_ concentration, Au(CN)2 _ block is weakened in the CFTR pore mutants K335A, F337S and T338A (Gong et al. 2002a), suggesting that these pore residues may contribute to lyotropic anion binding site(s) within the pore. Login to comment
60 In wild-type, K335A, F337S and T338A, high extracellular Cl_ significantly weakens Au(CN)2 _ block and (except in F337S) increases the fraction of the transmembrane electric field apparently experienced by the blocker. Login to comment
131 In contrast, mutation of other nearby TM6 residues associated with weakened Au(CN)2 _ binding (K335A, F337S, T338A) showed similar sensitivity to extracellular Cl_ concentration to that seen in wild-type (Figs 1 and 2). Login to comment

PMID: 14610019 [PubMed] Gong X et al: "Mutation-induced blocker permeability and multiion block of the CFTR chloride channel pore."

No. Sentence Comment

189 Previously, we showed that the mutations F337A and F337S, but not F337Y, disrupted the ability of the CFTR channel pore to select between permeant anions on the basis of free energy of hydration (Linsdell et al., 2000) and suggested that F337 contributes to a lyotropic anion "selectivity filter." Login to comment

PMID: 26606940 [PubMed] Wei S et al: "Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels."

No. Sentence Comment

70 Primer sequences for cloning and site-directed mutagenesis Ycf1p Forward cloning primer: CAACACAGGCATGTATATTA- AGAGC Reverse cloning primer: TTAAACTTATGGCGTCAGAG- TTGCC F565A: CATTGACTACTGACTTAGTTGCCCCTGCTTTG- ACTCTGTTC F565S: CATTGACTACTGACTTAGTTTCCCCTGCTTTGA- CTCTGTTC F565L: CATTGACTACTGACTTAGTTTTACCTGCTTTG- ACTCTGTTC G756D: AAGACAAACGAGCTTTTTGATCTCCAGATAAG- GAGATCCC D777N: ACAGCTGGCAAAGGATCATTAAGTAAATAAG- TGTCAGCTC Y1281G: GATCAAGCTCCGGCCTACCACGAGTGGAATA- ATTATTAAAC Yor1p Forward cloning primer: CTAATTGTACATCCGGTTTT- AACC Reverse cloning primer: TTGAGTCATTGCCCTTAA- AATGG F468S: AGGCAACCTGGTAATATTTCTGCCTCTTTATC- TTTATTTC F468A: AGGCAACCTGGTAATATTGCTGCCTCTTTATC- TTTATTTC F468L: AGGCAACCTGGTAATATTCTTGCCTCTTTATC- TTTATTTC G713D: GTGGTATTACTTTATCTGGTGATCAAAAGGCA- CGTATCAATTT Y1222G: ATAGGTAAACCAGGTCTACCGGCAAAATCAA- CATTTTCAA CFTR Forward cloning primer: GAAGAAGCAATGGAAAAA- ATGATTG Reverse cloning primer: TCGGTGAATGTTCTGACCT- TGG F337S: TCATCCTCCGGAAAATATCCACCACCATCTCA- TTCTGC F337A: TCATCCTCCGGAAAATAGCCACCACCATCTCA- TTCTGC F337L: TCATCCTCCGGAAAATATTAACCACCATCTCA- TTCTGC F337C: TCATCCTCCGGAAAATATGCACCACCATCTC- ATTCTGC Immunoblot analysis of CFTR protein expression Expression of the CFTR F337 mutants was verified by immunoblotting as described elsewhere (15). Login to comment
126 F337S-CFTR and F337A-CFTR exhibited robust GOF properties that included 1) substantial currents that remained following the removal of bath ATP with a scavenger (hexokinase/glucose) and subsequent perfusion with an ATP-free solution and 2) strong activation by the poorly hydrolyzable b,g-imidoadenosine 59-triphosphate (AMP-PNP), which is a weak agonist for WT CFTR (example record in Fig. 4A, data summaries in Fig. 4C, D) (4, 43). Login to comment
127 Because the F337S mutation exhibited strong GOF effects at the macroscopic current level, its Pos under various activation conditions (ATP, no nucleotide, AMP-PNP)wereestimatedforinside-outpatchesthatwere Figure 3. Login to comment
142 A) Macroscopic current record for excised patch containing hundreds of F337S-CFTR channels showing detectable current after ATP removal and strong AMP-PNP activation. Login to comment
152 Mean percent ATP-free currents 6 SEMs were as follows: WT (0.5 6 0.2%; n = 5); F337L (0.6 6 0.3%; n = 5); F337C (2.5 6 1.4%; n = 5), F337A (9.6 6 1.4%; n = 5), and F337S (15.8 6 4.5%; n = 10). Login to comment
153 The results for F337A and F337S were significantly different from WT by unpaired Student`s t test (P , 0.05). Login to comment
162 The results in Fig. 5 confirm that the extracellular F337S mutation increased Po especially following ATP removal and the subsequent addition of AMP-PNP. Login to comment
166 The extracellular F337S substitution enhances the ATP sensitivities of CFTR channels including the Y1219G ATP binding mutant GOF mutations that increase the ligand-free activities of allosteric proteins such as hormone receptors and neurotransmitter-gated channels also reciprocally enhance ligand sensitivity by biasing the equilibrium toward those conformations with the higher ligand affinities (i.e., the activated receptor or open channel) (14, 15, 44-46). Login to comment
167 The ATP titration data in Fig. 6 indicate that such reciprocity is also apparent for the F337S mutation. Login to comment
173 F337S-CFTR channels have increased Pos under all conditions examined. Login to comment
174 A) Unitary current recordings at a single holding potential (260 mV; record inverted for presentation) for an excised inside-out patch containing 6 F337S-CFTR channels. Login to comment
179 B) Corresponding unitary current recordings for a patch containing 5 WT-CFTR channels. Conditions and methods were identical to A. Note the different current scales indicating the lower unitary currents exhibited by the F337S mutant. Login to comment
180 C-E) Mean Pos for WT-CFTR and F337S-CFTR channels estimated for the indicated conditions. Login to comment
182 Numbers (n) are 4 and 5 for WT and F337S, respectively. Login to comment
185 The F337S substitution markedly increases the activities of CFTR constructs that cannot be activated by ATP We previously observed that cytosolic GOF mutations increased the channel activity of the most common CF regulation mutant G551D-CFTR (14, 15). Login to comment
187 G551D-CFTR channel activity in excised membrane patches is strongly increased by bath addition of the natural compound curcumin (14, 15, 48), which reveals the presence of gating-defective channels in the patch (Fig. 7A). Login to comment
188 Introducing the F337S substitution markedly increased the control currents mediated by G551D-CFTR channels and correspondingly reduced the relative stimulation by curcumin (Fig. 7B-D). Login to comment
190 Figure 8 shows that the F337S substitution also substantially increased the activity of a CFTR truncation mutant that lacks NBD2 (D1198-CFTR). Login to comment
191 This construct behaves similarly to the G551D CF mutant in that it exhibits very low, ATP-unresponsive currents in excised patches under control conditions but can be stimulated by curcumin (Fig. 8A) (14, 15, 48). Login to comment
192 As for G551D-CFTR, the F337S substitution substantially increased the control currents and correspondingly reduced the relative stimulation by curcumin when introduced into this NBD2 deletion construct (Fig. 8B-D). Login to comment
193 The currents mediated by F337S/D1198-CFTR were insensitive to addition of the ATP scavenger as expected for a construct lacking 1 of the 2 NBDs that dimerize to form the composite ATP binding sites (Fig. 8E). Login to comment
196 The results of Figs. 7 and 8 confirm that the F337S substitution is a bona fide GOF mutation that promotes the unliganded activities of CFTR constructs that cannot be stimulated by ATP. Login to comment
197 PKA sensitivity is also enhanced by the extracellular F337S mutation A GOF mutation that increases unliganded channel opening might also affect the PKA sensitivity of channel activationgiventhatRdomainphosphorylationisrequired to open the channel even in the absence of ATP binding (Fig. 8) (14). Login to comment
198 This prediction is analogous to the effect of a GOF mutation to increase ligand sensitivity, as was verified for the F337S mutant in the ATP titration experiments shown in Fig. 6. Login to comment
201 To test this idea, the extracellular F337S mutation was combined with the previously characterized K978C mutation, which locates tothe cytosolic side below TM9 (14, 15). Login to comment
202 The K978C substitution increases ATP-free channel activity and enhances the ATP and PKA sensitivities of CFTR activation similar to that shown here for the F337S mutation (14). Login to comment
204 Removing bath ATP in the standard excised macropatch protocol decreased the currents mediated by F337S/K978C-CFTR by less than 20% (Fig. 10A, B). Login to comment
207 More strikingly, F337S/K978C-CFTR appeared to be nearly maximally active in the absence of both PKA and ATP (Fig. 10C-F). Login to comment
208 Substantial F337S/K978C-CFTR-mediated currents could be detected for macropatches that were excised in the absence of both PKA and ATP in Figure 6. Login to comment
209 The extracellular F337S mutation increases the ATP sensitivity of CFTR activation either in the WT background or in the Y1219G ATP binding mutant. Login to comment
220 In support of this interpretation, the Po of F337S/K978C-CFTR estimatedfrom multichannel records in theabsence ofbothPKAandATP(examplerecordinFig.10D)ranged from 0.34 to 0.95 with a mean Po (6SEM) of 0.53 6 0.08 (n = 7 patches;estimated using the conventional Clampfit protocol; see Methods). Login to comment
228 The F337S mutation increases the activity of the common CF regulation mutant, G551D-CFTR. Login to comment
230 Macroscopic currents mediated by G551D-CFTR without or with the F337S substitution. Activation conditions were the same as for Fig.4. Login to comment
232 Note the much larger control current and lower relative activation by curcumin for F337S/G551D-CFTR. Login to comment
233 C) Scatter plot showing the generally larger macroscopic control currents at 180 mV for G551D-CFTR channels containing the F337S substitution. Login to comment
237 Mean control currents (6SEMs) were as follows: G551D (0.5 6 0.1 pA; n = 5) and F337S/ G551D (115.5 6 59.2; n = 5). Login to comment
239 The much lower relative activation of F337S/G551D-CFTR is due to its higher control or baseline currents. Login to comment
245 The F337S mutation enhances the ATP-independent activities of channels lacking NBD2. Login to comment
246 A, B) Macroscopic currents mediated by D1198-CFTR channels without or with the F337S substitution. Activation conditions and curcumin concentrations were identical to Fig. 7. Login to comment
247 B) The F337S/D1198-CFTR current was inhibited by adding a high concentration of the voltage-dependent CFTR pore blocker, glibenclamide (300 mM). Login to comment
248 C) Scatter plot showing the larger macroscopic control currents at 180 mV for D1198-CFTR channels with the F337S substitution. Login to comment
249 Mean control currents (6SEMs) were as follows: D1198 (2.7 6 2.3 pA; n = 6) and F337S/ D1198 (177.0 6 75.6; n = 7). Login to comment
251 *P , 0.05 by unpaired Student`s t test. E) Macroscopic current record showing that the control currents mediated by F337S/D1198-CFTR are not reduced by scavenging ATP with hexokinase/ glucose (see Fig. 4 legend for hexokinase/glucose concentrations). Login to comment
253 F) Macroscopic current record showing that F337S/D1198-CFTR channel activity is strongly dependent on PKA phosphorylation. Login to comment
268 The extracellular F337S mutation also increases CFTR sensitivity to cytosolic PKA. Login to comment
270 C) Mean PKA titration data for F337S-CFTR and WT-CFTR. Login to comment
276 The F337S/K978C double mutant is nearly fully active in the absence of both exogenous PKA and ATP. Login to comment
277 A) Representative macroscopic current record showing that ATP removal by scavenger addition followed by bath perfusion with an ATP-free solution only modestly decreases the activities of F337S/K978C-CFTR channels. Conditions were identical to Fig. 4. Login to comment
280 The F337S data set is from Figure 4C. Login to comment
282 C) Representative macroscopic current record showing that F337S/K978C-CFTR channels are nearly maximally active in excised patches in the absence of both ATP and exogenous PKA. Login to comment
284 D) Unitary current recording at a single holding potential for an excised patch containing 3 F337S/K978C-CFTR channels. Login to comment
288 E) Gap-free record at a single holding potential for an excised patch containing 80-100 F337S/K978C-CFTR channels showing substantial activity in the absence of bath PKA and ATP. Login to comment
289 F) Stationary noise plot for F337S/K978C-CFTR channels in the absence of PKA and ATP. Login to comment
300 The present results confirmed these earlier findings and also revealed that a subset of F337 substitutions are strong GOF mutants, notably, F337S and F337A. Login to comment
301 GOF effects on CFTR channel gating were operationally defined as 1) large fractional currents that persist following ATP removal, 2) robust activation by the normally weak agonist, AMP-PNP, and 3) substantial increases in channel activities when introduced into CFTR constructs that cannot be activated by ATP, namely, the most common CF regulation mutant (G551D-CFTR) or a truncation mutant lacking NBD2 (D1198-CFTR). Login to comment
302 The F337S substitution also increased the ATP sensitivity of CFTR channel activation when introduced either into the WT background or into the Y1219G-CFTR ATP binding mutant. Login to comment
313 The F337S GOF mutation also provides insights into how PKA phosphorylation regulates CFTR PKA-mediated phosphorylation is essential for WT CFTR channel activity (9). Login to comment
315 Two findings from our analysis of the F337S GOF mutant impact our understanding of how PKA regulates CFTR channel activity. Login to comment
316 First, the strong PKA dependence of the channel activity of the F337S/D1198-CFTR truncation construct (Fig. 8F) argues for a regulatory mechanism that is independent of any effect that phosphorylation might have on NBD dimerization (see also 14). Login to comment
319 The F337S/ D1198-CFTR construct lacks NBD2, cannot form an NBD1-NBD2 dimer and is unresponsive to ATP. Login to comment
324 The second finding that impacts our understanding of the PKA regulatory mechanism was the long-range allosteric effect of the extracellular F337S mutation on PKA sensitivity (Fig. 9). Login to comment
329 Engineering a superactive CFTR The F337S/K978C double mutant has the highest single-channel activity in the absence of exogenous PKA and ATP of any CFTR construct that we have characterized to date. Login to comment
332 We anticipated that the F337S and K978C mutations would have additive GOF effects on ATP-free CFTR activity because they locate to opposite sides of the pore where they presumably impact CFTR structure in different ways. Login to comment
69 Primer sequences for cloning and site-directed mutagenesis Ycf1p Forward cloning primer: CAACACAGGCATGTATATTA- AGAGC Reverse cloning primer: TTAAACTTATGGCGTCAGAG- TTGCC F565A: CATTGACTACTGACTTAGTTGCCCCTGCTTTG- ACTCTGTTC F565S: CATTGACTACTGACTTAGTTTCCCCTGCTTTGA- CTCTGTTC F565L: CATTGACTACTGACTTAGTTTTACCTGCTTTG- ACTCTGTTC G756D: AAGACAAACGAGCTTTTTGATCTCCAGATAAG- GAGATCCC D777N: ACAGCTGGCAAAGGATCATTAAGTAAATAAG- TGTCAGCTC Y1281G: GATCAAGCTCCGGCCTACCACGAGTGGAATA- ATTATTAAAC Yor1p Forward cloning primer: CTAATTGTACATCCGGTTTT- AACC Reverse cloning primer: TTGAGTCATTGCCCTTAA- AATGG F468S: AGGCAACCTGGTAATATTTCTGCCTCTTTATC- TTTATTTC F468A: AGGCAACCTGGTAATATTGCTGCCTCTTTATC- TTTATTTC F468L: AGGCAACCTGGTAATATTCTTGCCTCTTTATC- TTTATTTC G713D: GTGGTATTACTTTATCTGGTGATCAAAAGGCA- CGTATCAATTT Y1222G: ATAGGTAAACCAGGTCTACCGGCAAAATCAA- CATTTTCAA CFTR Forward cloning primer: GAAGAAGCAATGGAAAAA- ATGATTG Reverse cloning primer: TCGGTGAATGTTCTGACCT- TGG F337S: TCATCCTCCGGAAAATATCCACCACCATCTCA- TTCTGC F337A: TCATCCTCCGGAAAATAGCCACCACCATCTCA- TTCTGC F337L: TCATCCTCCGGAAAATATTAACCACCATCTCA- TTCTGC F337C: TCATCCTCCGGAAAATATGCACCACCATCTC- ATTCTGC Immunoblot analysis of CFTR protein expression Expression of the CFTR F337 mutants was verified by immunoblotting as described elsewhere (15). Login to comment
125 F337S-CFTR and F337A-CFTR exhibited robust GOF properties that included 1) substantial currents that remained following the removal of bath ATP with a scavenger (hexokinase/glucose) and subsequent perfusion with an ATP-free solution and 2) strong activation by the poorly hydrolyzable b,g-imidoadenosine 59-triphosphate (AMP-PNP), which is a weak agonist for WT CFTR (example record in Fig. 4A, data summaries in Fig. 4C, D) (4, 43). Login to comment
141 A) Macroscopic current record for excised patch containing hundreds of F337S-CFTR channels showing detectable current after ATP removal and strong AMP-PNP activation. Login to comment
151 Mean percent ATP-free currents 6 SEMs were as follows: WT (0.5 6 0.2%; n = 5); F337L (0.6 6 0.3%; n = 5); F337C (2.5 6 1.4%; n = 5), F337A (9.6 6 1.4%; n = 5), and F337S (15.8 6 4.5%; n = 10). Login to comment
161 The results in Fig. 5 confirm that the extracellular F337S mutation increased Po especially following ATP removal and the subsequent addition of AMP-PNP. Login to comment
165 The extracellular F337S substitution enhances the ATP sensitivities of CFTR channels including the Y1219G ATP binding mutant GOF mutations that increase the ligand-free activities of allosteric proteins such as hormone receptors and neurotransmitter-gated channels also reciprocally enhance ligand sensitivity by biasing the equilibrium toward those conformations with the higher ligand affinities (i.e., the activated receptor or open channel) (14, 15, 44-46). Login to comment
172 F337S-CFTR channels have increased Pos under all conditions examined. Login to comment
178 B) Corresponding unitary current recordings for a patch containing 5 WT-CFTR channels. Conditions and methods were identical to A. Note the different current scales indicating the lower unitary currents exhibited by the F337S mutant. Login to comment
181 Numbers (n) are 4 and 5 for WT and F337S, respectively. Login to comment
184 The F337S substitution markedly increases the activities of CFTR constructs that cannot be activated by ATP We previously observed that cytosolic GOF mutations increased the channel activity of the most common CF regulation mutant G551D-CFTR (14, 15). Login to comment
189 Figure 8 shows that the F337S substitution also substantially increased the activity of a CFTR truncation mutant that lacks NBD2 (D1198-CFTR). Login to comment
195 The results of Figs. 7 and 8 confirm that the F337S substitution is a bona fide GOF mutation that promotes the unliganded activities of CFTR constructs that cannot be stimulated by ATP. Login to comment
200 To test this idea, the extracellular F337S mutation was combined with the previously characterized K978C mutation, which locates tothe cytosolic side below TM9 (14, 15). Login to comment
203 Removing bath ATP in the standard excised macropatch protocol decreased the currents mediated by F337S/K978C-CFTR by less than 20% (Fig. 10A, B). Login to comment
206 More strikingly, F337S/K978C-CFTR appeared to be nearly maximally active in the absence of both PKA and ATP (Fig. 10C-F). Login to comment
219 In support of this interpretation, the Po of F337S/K978C-CFTR estimatedfrom multichannel records in theabsence ofbothPKAandATP(examplerecordinFig.10D)ranged from 0.34 to 0.95 with a mean Po (6SEM) of 0.53 6 0.08 (n = 7 patches;estimated using the conventional Clampfit protocol; see Methods). Login to comment
227 The F337S mutation increases the activity of the common CF regulation mutant, G551D-CFTR. Login to comment
229 Macroscopic currents mediated by G551D-CFTR without or with the F337S substitution. Activation conditions were the same as for Fig.4. Login to comment
231 Note the much larger control current and lower relative activation by curcumin for F337S/G551D-CFTR. Login to comment
236 Mean control currents (6SEMs) were as follows: G551D (0.5 6 0.1 pA; n = 5) and F337S/ G551D (115.5 6 59.2; n = 5). Login to comment
238 The much lower relative activation of F337S/G551D-CFTR is due to its higher control or baseline currents. Login to comment
244 The F337S mutation enhances the ATP-independent activities of channels lacking NBD2. Login to comment
250 *P , 0.05 by unpaired Student`s t test. E) Macroscopic current record showing that the control currents mediated by F337S/D1198-CFTR are not reduced by scavenging ATP with hexokinase/ glucose (see Fig. 4 legend for hexokinase/glucose concentrations). Login to comment
252 F) Macroscopic current record showing that F337S/D1198-CFTR channel activity is strongly dependent on PKA phosphorylation. Login to comment
267 The extracellular F337S mutation also increases CFTR sensitivity to cytosolic PKA. Login to comment
269 C) Mean PKA titration data for F337S-CFTR and WT-CFTR. Login to comment
275 The F337S/K978C double mutant is nearly fully active in the absence of both exogenous PKA and ATP. Login to comment
279 The F337S data set is from Figure 4C. Login to comment
281 C) Representative macroscopic current record showing that F337S/K978C-CFTR channels are nearly maximally active in excised patches in the absence of both ATP and exogenous PKA. Login to comment
283 D) Unitary current recording at a single holding potential for an excised patch containing 3 F337S/K978C-CFTR channels. Login to comment
287 E) Gap-free record at a single holding potential for an excised patch containing 80-100 F337S/K978C-CFTR channels showing substantial activity in the absence of bath PKA and ATP. Login to comment
299 The present results confirmed these earlier findings and also revealed that a subset of F337 substitutions are strong GOF mutants, notably, F337S and F337A. Login to comment
312 The F337S GOF mutation also provides insights into how PKA phosphorylation regulates CFTR PKA-mediated phosphorylation is essential for WT CFTR channel activity (9). Login to comment
314 Two findings from our analysis of the F337S GOF mutant impact our understanding of how PKA regulates CFTR channel activity. Login to comment
318 The F337S/ D1198-CFTR construct lacks NBD2, cannot form an NBD1-NBD2 dimer and is unresponsive to ATP. Login to comment
323 The second finding that impacts our understanding of the PKA regulatory mechanism was the long-range allosteric effect of the extracellular F337S mutation on PKA sensitivity (Fig. 9). Login to comment
328 Engineering a superactive CFTR The F337S/K978C double mutant has the highest single-channel activity in the absence of exogenous PKA and ATP of any CFTR construct that we have characterized to date. Login to comment
331 We anticipated that the F337S and K978C mutations would have additive GOF effects on ATP-free CFTR activity because they locate to opposite sides of the pore where they presumably impact CFTR structure in different ways. Login to comment

PMID: 10827976 [PubMed] Linsdell P et al: "Molecular determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator chloride channel pore."

No. Sentence Comment

71 In contrast, both F337A and F337S showed dramatically altered anion selectivity (Fig. 2 and Tables 1 and 2), characterized by large reductions in the relative permeability of lyotropic anions (Brafa; , Iafa; , SCNafa; , NO3 afa; ) and greatly increased permeability of the small, kosmotropic Fafa; anion. Login to comment
74 As described previously for wild-type CFTR (Linsdell and Hanrahan, 1998a), the mutants F337A, F337S, and F337Y all showed negligible Naaf9; permeability (Table 1). Login to comment
76 The altered anion selectivity of F337A and F337S led to a disruption of the relationship between anion permeability and hydration energy in these mutants (Fig. 3). Login to comment
78 In contrast, for both F337A and F337S, there was no obvious correlation between anion permeability and energy of hydration (Fig. 3), suggesting that lyotropic selectivity is greatly diminished in these mutants. Login to comment
82 In contrast, the two mutations that strongly affect selectivity, F337A and F337S, both involve a substantial reduction in amino acid side-chain volume. Login to comment
101 Note that the range of reversal potentials with different anions is greatly reduced in both F337A and F337S, indicating a reduced ability of the channel to discriminate between different anions. Login to comment
104 We used a similar approach to determine whether the altered anion selectivity of F337A and F337S was associated with any change in functional pore diameter (Table 3). Login to comment
106 The permeabilities of F337A and F337S to extracellular formate, acetate, and propanoate ions were not significantly different from those observed in wild-type CFTR, and both pyruvate and methane sulfonate were not measurably permeant in wild type, F337A, or F337S. Login to comment
111 However, the effects of the mutations F337A and F337S, which virtually abolish the normal lyotropic anion selectivity sequence (Tables 1 and 2 and Fig. 3) by decreasing the relative permeability of lyotropic anions and increasing that of kosmotropic anions (Fig. 4), support an alternative explanation, namely that selectivity is determined at a discrete region unaffected by previously studied mutations. Login to comment
116 Although we cannot rule out this possibility, we feel that the fact that mutations at two adjacent TM6 residues, F337 (this study) and T338 (Linsdell et al., 1998), significantly affect TABLE 1 Relative permeability of intracellular ions in wild-type and mutant CFTR Cld1a; channels Wild type F337A F337S F337L F337Y F337W I344A Cl 1.00 afe; 0.01 (10) 1.00 afe; 0.04 (6) 1.00 afe; 0.08 (3) 1.00 afe; 0.02 (5) 1.00 afe; 0.02 (6) 1.00 afe; 0.03 (5) 1.00 afe; 0.01 (9) Br 1.37 afe; 0.07 (8) 0.60 afe; 0.04 (4)** 0.50 afe; 0.04 (4)** 1.22 afe; 0.04 (5) 1.39 afe; 0.04 (3) 1.12 afe; 0.05 (4)* 1.74 afe; 0.01 (3)* I 0.83 afe; 0.03 (6) 0.23 afe; 0.04 (5)** 0.23 afe; 0.02 (4)** 0.39 afe; 0.01 (3)** 0.69 afe; 0.03 (7)* - 0.99 afe; 0.05 (4)* F 0.103 afe; 0.007 (9) 0.35 afe; 0.01 (4)** 0.43 afe; 0.02 (4)** 0.15 afe; 0.02 (3)* 0.095 afe; 0.009 (3) 0.081 afe; 0.009 (3) 0.075 afe; 0.012 (5)* SCN 3.55 afe; 0.26 (7) 0.97 afe; 0.05 (4)** 0.93 afe; 0.10 (5)** 2.85 afe; 0.20 (4) 3.05 afe; 0.29 (4) 4.42 afe; 0.56 (4) 3.27 afe; 0.30 (5) NO3 1.58 afe; 0.04 (10) 1.30 afe; 0.03 (3)* 1.08 afe; 0.02 (4)** 1.38 afe; 0.03 (4)* 1.43 afe; 0.04 (3) 1.62 afe; 0.03 (3) 1.71 afe; 0.06 (4) ClO4 0.25 afe; 0.01 (8) 0.19 afe; 0.00 (3)* 0.17 afe; 0.03 (4)* 0.23 afe; 0.04 (3) 0.15 afe; 0.01 (4)** - 0.24 afe; 0.02 (3) Formate 0.24 afe; 0.01 (9) 0.27 afe; 0.02 (3) 0.33 afe; 0.03 (4)* 0.35 afe; 0.02 (3)* 0.24 afe; 0.01 (3) - 0.28 afe; 0.01 (3) Acetate 0.091 afe; 0.003 (10) 0.073 afe; 0.004 (3)* 0.12 afe; 0.02 (5) - 0.092 afe; 0.014 (4) - 0.076 afe; 0.007 (3) Naaf9; 0.007 afe; 0.010 (24) 0.001 afe; 0.018 (3) 0.001 afe; 0.021 (5) - 0.002 afe; 0.004 (3) - - Relative permeabilities for different anions present in the intracellular solution under biionic conditions were calculated from macroscopic current reversal potentials (e.g., Fig. 2), according to Eq. 1 (see Materials and Methods). Login to comment
122 TABLE 2 Anion selectivity sequences for wild-type and mutant CFTR Cld1a; channels Wild-type SCNafa; b0e; NO3 afa; b0e; Brafa; b0e; Clafa; b0e; Iafa; b0e; ClO4 afa; b07; form b0e; Fafa; b0e; ace F337A NO3 afa; b0e; Clafa; c56; SCNafa; b0e; Brafa; b0e; Fafa; b0e; form c56; Iafa; b0e; ClO4 afa; b0e; ace F337S NO3 afa; b0e; Clafa; c56; SCNafa; b0e; Brafa; b0e; Fafa; b0e; form b0e; Iafa; b0e; ClO4 afa; b0e; ace F337L SCNafa; b0e; NO3 afa; b0e; Brafa; b0e; Clafa; b0e; Iafa; b0e; form b0e; ClO4 afa; b0e; Fafa; F337Y SCNafa; b0e; NO3 afa; c56; Brafa; b0e; Clafa; b0e; Iafa; b0e; form b0e; ClO4 afa; b0e; Fafa; b07; ace I344A SCNafa; b0e; Brafa; c56; NO3 afa; b0e; Clafa; b07; Iafa; b0e; form b0e; ClO4 afa; b0e; ace b07; Fafa; Sequences were derived from the relative anion permeabilities given in Table 1. form, formate; ace, acetate. Login to comment
125 Furthermore, the mutations F337A and F337S altered selectivity between different anions without disrupting the ability of the channel to select for Clafa; over Naaf9; (Table 1), supporting the hypothesis that the CFTR pore uses different mechanisms to determine lyotropic anion selectivity and anion:cation selectivity (Linsdell et al., 1998; Guinamard and Akabas, 1999). Login to comment
129 Nevertheless, it is clear that in CFTR, interactions between permeating anions and the pore do influence anion selectivity, because point mutations in the channel (F337A and F337S) disrupt the selectivity sequence. Login to comment
130 Both F337A and F337S compromise the relationship between anion permeability and hydration energy (Fig. 3), suggesting a reduction in the relative importance of anion FIGURE 3 Relationship between relative anion permeability and hydration energy for wild-type and F337-mutated CFTR. Login to comment
142 One possible explanation for the loss of the relationship between anion permeability and hydration energy in F337A and F337S is that anions are able to pass through the pores of these mutants with more of their associated waters of hydration intact than in wild type, so reducing the degree of anion dehydration required for permeation. Login to comment
151 In fact, for intracellular anions, the mutations F337A and F337S had a much stronger effect on the permeability of small anions (halides, SCNafa; , NO3 afa; ) than on larger anions (ClO4 afa; , formate, acetate), suggesting that removal of a steric barrier is not the primary effect of these mutations (Table 1). Login to comment
152 Furthermore, neither F337A nor F337S showed greatly altered permeability to extracellular organic anions (Table 3), the permeabilities of which do appear to be limited by unhydrated anion size (Linsdell et al., 1997, 1998; Linsdell and Hanrahan, 1998a). Login to comment
153 Although the relationship between the permeability of such organic anions, when present in the extracellular solution, and the actual physical dimensions of the pore, is unclear (Linsdell and Hanrahan, 1998a), the results summarized in Table 3 do not suggest a strong alteration in the functional dimensions of the pore in F337A or F337S. Login to comment
154 A reduction in the relative importance of anion dehydration in determining permeability, as is suggested in F337A and F337S, could result not only from a decrease in the degree of anion dehydration, but also from an increase in the strength of the interaction between permeating anions and the channel pore. Login to comment
158 While the mutants F337L, F337Y, and I344A maintain Eisenman sequence III, both F337A and F337S convert the channel to a relatively strong field strength sequence (Clafa; b0e; Brafa; b0e; Fafa; b0e; Iafa; ; Eisenman sequence V) (Table 2). Login to comment
159 This increase in field strength might imply that permeating anions interact more strongly with the pores of F337A and F337S than with wild-type CFTR. Login to comment
160 Consistent with this, CFTR single-channel conductance (measured with symmetrical 154 mM Clafa; ) is reduced from 7.6 afe; 0.1 pS (n afd; 12) for wild type to 1.8 afe; 0.0 pS (n afd; 7) for F337S (P. Linsdell, unpublished observations). Login to comment
165 However, SCNafa; permeability is reduced to a similar extent in F337A (hydrophobic) and F337S (polar), but is not altered in F337Y (polar) (Table 1), suggesting that SCNafa; permeability is not influenced by hydrophobic interactions with the large, hydrophobic side chain of F337. Login to comment
166 How, then, might we explain the effects of the mutations F337A and F337S on anion selectivity? Login to comment
169 Reduction of this steric effect in both F337A and F337S would allow the permeating anion TABLE 3 Relative permeability of extracellular organic anions in wild-type and mutant CFTR Cld1a; channels Wild type F337A F337S Formate 0.129 afe; 0.007 (4) 0.157 afe; 0.013 (3) 0.112 afe; 0.002 (3) Acetate 0.038 afe; 0.007 (4) 0.026 afe; 0.008 (4) 0.029 afe; 0.013 (3) Propanoate 0.022 afe; 0.003 (4) 0.024 afe; 0.001 (3) 0.024 afe; 0.002 (3) Pyruvate b0d;0.011 (4) b0d;0.011 (3) b0d;0.011 (3) Methane sulfonate b0d;0.011 (3) b0d;0.011 (3) b0d;0.011 (2) Relative permeabilities for different organic anions present in the extracellular solution under biionic conditions were calculated as described in the legend to Table 1. Login to comment

PMID: 25673337 [PubMed] Rubaiy HN et al: "Location of a permeant anion binding site in the cystic fibrosis transmembrane conductance regulator chloride channel pore."

No. Sentence Comment

32 Specifically, mutations that reduce side-chain volume (F337A, F337S) disrupt the relationship between the anion permeability and anion free energy of hydration [18]. Login to comment
97 This permeability sequence is disrupted by mutations at the putative narrow region of the pore, located more extracellularly in the pore than K95, in particular F337A and F337S (see ''Introduction``). Login to comment

PMID: 26229102 [PubMed] Corradi V et al: "Cystic Fibrosis Transmembrane Conductance Regulator (CFTR): CLOSED AND OPEN STATE CHANNEL MODELS."

No. Sentence Comment

138 Narrow Region and Inner Vestibule-Residues of TM6 have been shown to be critical for both anion permeation and channel gating (72), with residues Phe-337-Ser-341 forming the narrow portion of the pore (68, 72, 79-81), and a number of TM6 residues up to Gln-353 lining the permeation pathway further toward the cytosol (68, 72). Login to comment