ABCMdb

PMID: 11179391

Linsdell P
Relationship between anion binding and anion permeability revealed by mutagenesis within the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Physiol. 2001 Feb 15;531(Pt 1):51-66., 2001-02-15 [PubMed]

Sentences

No. Mutations Sentence Comment

19 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser Two mutations in CFTR which alter the anion permeability sequence, F337S and T338A, also altered the anion conductance sequence. Login to comment 34 ABCC7 p.Gly314Glu ABCC7 p.Arg347Asp ABCC7 p.Lys335Glu The hypothesis that anion permeability and anion binding are separable facets of the permeation process in the CFTR Cl¦ channel is supported by the fact that several mutations within the pore have been shown to alter anion binding without strongly affecting anion permeability (e.g. K335E, Anderson et al. 1991; R347D, Tabcharani et al. 1993; G314E, Mansoura et al. 1998). Login to comment 37 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala 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 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser 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 42 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ala The CFTR mutants F337A, L, S, W, Y and T338A were constructed and transfected into CHO and BHK cells by Alexandra Evagelidis and Shu-Xian Zheng in the laboratory of Dr John Hanrahan (McGill University, Montreal, Quebec, Canada), as described previously (Linsdell et al. 1998, 2000). Login to comment 43 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser In the present study, the permeation properties of two mutants, F337S and T338A, have been examined in detail. Login to comment 44 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala 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 ABCC7 p.Phe337Ser 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be expressed in CHO cells and studied using single channel recording (P. Linsdell, A. Evagelidis & J. W. Hanrahan, unpublished observations).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 ABCC7 p.Phe337Ser 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 48 ABCC7 p.Thr338Ala In contrast to mutations at F337, all mutations previously examined at T338 (T338A, N, S, V) significantly altered anion selectivity and Cl¦ conductance (Linsdell et al. 1998). Login to comment 49 ABCC7 p.Thr338Ala ABCC7 p.Thr338Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser 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 ABCC7 p.Thr338Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser ABCC7 p.Phe337Ser 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 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser 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 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser 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 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser 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 ABCC7 p.Thr338Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser ABCC7 p.Phe337Ser 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 ABCC7 p.Phe337Ser 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 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala 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 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser 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 ABCC7 p.Thr338Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser ABCC7 p.Phe337Ser 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 122 ABCC7 p.Phe337Tyr ------------------------------------------------------------------------------------------------------------------------ F337Y, a mutation which results in only a small change in amino acid side chain size. Login to comment 124 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser 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 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser 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 ABCC7 p.Phe337Ser 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 129 ABCC7 p.Thr338Ala Although smaller differences are apparent for T338A, in no case were these significantly different from wild-type (Table 1). Login to comment 130 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser The conductance sequences for wild-type, F337S and T338A are summarised in Table 2. Login to comment 132 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala 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 ABCC7 p.Phe337Ser ABCC7 p.Phe337Tyr 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 ABCC7 p.Phe337Ser 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 ABCC7 p.Phe337Ser 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 ABCC7 p.Phe337Ser ABCC7 p.Phe337Tyr 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 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser 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 144 ABCC7 p.Phe337Tyr ------------------------------------------------------------------------------------------------------------------------ (compared with wild-type) in gBrÏgCl and the 710% increase in gFÏgCl, were not reproduced in F337Y (Fig. 8). Login to comment 145 ABCC7 p.Phe337Ser 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 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser 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 ABCC7 p.Phe337Ser 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 ABCC7 p.Phe337Ser 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 162 ABCC7 p.Thr338Ala T338A Cl¦ currents were only weakly blocked by SCN¦ and ClOÚ¦, and not significantly blocked by other permeant anions including I¦ (Fig. 11C). Login to comment 164 ABCC7 p.Thr338Ala T338A Cl¦ currents were significantly increased in the presence of 25 mÒ intracellular NO×¦, suggesting that the high permeability and conductance of NO×¦ in this mutant (Table 1) allowed some degree of summation of Cl¦ and NO×¦ currents through the channel. Login to comment 165 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser 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 171 ABCC7 p.Thr338Ala Anion permeability in wild-type and T338A CFTR follows an approximate lyotropic sequence, with relative anion permeability being correlated with energy of hydration (Linsdell & Hanrahan, 1998; Linsdell et al. 1998; Fig. 12A). Login to comment 172 ABCC7 p.Thr338Ala In contrast, there is no obvious relationship between relative anion conductance and energy of hydration in either wild-type or T338A (Fig. 12B). Login to comment 175 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser 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 ABCC7 p.Thr338Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser ABCC7 p.Phe337Ser 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 ABCC7 p.Phe337Ser 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 184 ABCC7 p.Thr338Ala Interestingly, Cl¦ remains the anion with the highest conductance in this mutant, and the difference in conductance between Cl¦ and anions with similar energies of hydration (Br¦ and NO×¦) is greater than for wild-type or T338A (Fig. 12B). Login to comment 186 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser 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 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser 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 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser 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 200 ABCC7 p.Gly314Glu ABCC7 p.Lys335Glu Several mutations within the pore of CFTR alter permeant anion binding without strongly affecting anion selectivity (in terms of permeability ratios) (e.g. K335E, Anderson et al. 1991; G314E, Mansoura et al. 1998), suggesting that these mutations affect anion binding sites not intimately involved in the anion selectivity process (Smith et al. 1999; Linsdell et al. 2000). Login to comment 218 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser 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 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser In the present study, the anion binding properties of two mutants, F337S and T338A, were examined in detail. Login to comment 243 ABCC7 p.Phe337Ser 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 245 ABCC7 p.Thr338Ala In contrast, T338A did not significantly affect the relative conductance of different anions (Table 1), although some small changes in the conductance sequence were apparent (Table 2). Login to comment 246 ABCC7 p.Phe337Ser ABCC7 p.Phe337Tyr 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 ABCC7 p.Phe337Ser 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 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser ABCC7 p.Phe337Ser 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 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser 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 254 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Ala Interestingly, S1118 in TM11 has been suggested to occupy a position similar to that of T338 in TM6, and the mutations S1118A and S1118F cause small alterations in anion permeability (Zhang et al. 2000). Login to comment 256 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser 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 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser 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 264 ABCC7 p.Thr338Ala ABCC7 p.Thr338Ser Optimization of Cl¦ conductance, rather than Cl¦ permeability, is likely to be of greater importance to the physiological function of CFTR and other Cl¦ channels, although in CFTR the mutations T338A and T338S increase Cl¦ conductance, apparently without adversely affecting other channel properties (Linsdell et al. 1998). Login to comment 269 ABCC7 p.Phe337Ser 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 ABCC7 p.Phe337Ser 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