ABCMdb

PMID: 12679372

Gong X, Linsdell P
Molecular determinants and role of an anion binding site in the external mouth of the CFTR chloride channel pore.
J Physiol. 2003 Jun 1;549(Pt 2):387-97. Epub 2003 Apr 4., 2003-06-01 [PubMed]

Sentences

No. Mutations Sentence Comment

11 ABCC7 p.Arg334His Fixed positive charge at this site appears to play a role in Au(CN)2 _ binding, as judged by multiple substitutions of differently charged amino acid side chains and also by the pH dependence of block conferred by the R334H mutant. Login to comment 40 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser ABCC7 p.Lys335Ala 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 43 ABCC7 p.Arg334Cys In contrast, an additional mutation near the external end of TM6, R334C, not only caused a more dramatic weakening of Au(CN)2 _ block than previously studied mutants (Fig. 1), but also abolished or even reversed the Cl_ dependence of both apparent affinity (as judged by the Kd(0); Fig. 2A) and voltage dependence (as judgedbythefractionalelectricaldistance,d;Fig.2B). Login to comment 46 ABCC7 p.Arg334Cys Whereas in wild-type, Kd(0) increases dramatically as the extracellular anion is made more lyotropic (gluconate å Cl_ å SCN_ ), this trend is apparently abolished in R334C (Fig. 3C). Login to comment 49 ABCC7 p.Arg334Cys CharacterizationofR334asananionbindingsite The effects of the mutation R334C outlined above suggest that the positively charged arginine at this position normally contributes both to a lyotropic binding site and to a site which is crucial for electrostatic interactions between extracellular permeant ions and intracellular blocking ions. Login to comment 51 ABCC7 p.Arg334Cys ABCC7 p.Arg334Ala Previously we reported that the mutant R334A could not be expressed in BHK cells (Gong et al. 2002a); in the course of the present study, we confirmed this previous finding, but did find that, in addition to R334C, five other mutants could be studied (Fig. 4). Login to comment 53 ABCC7 p.Arg334Leu ABCC7 p.Arg334Gln ABCC7 p.Arg334Cys ABCC7 p.Arg334His ABCC7 p.Arg334Glu ABCC7 p.Arg334Lys Block of wild-type, R334C-, R334E-, R334H-, R334K-, R334L- and R334Q-CFTR by 100 mM and 1 mM intracellular Au(CN)2 _ are compared in Fig. 4B. Login to comment 54 ABCC7 p.Arg334Cys ABCC7 p.Arg334Glu ABCC7 p.Arg334Lys Block was affected in all mutants, depending on the ionic conditions used, but was particularly weakened in R334C, R334E and R334K (Fig. 5A-C). Login to comment 60 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser ABCC7 p.Phe337Ser ABCC7 p.Lys335Ala 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 61 ABCC7 p.Arg334Cys In contrast, in R334C extracellular Cl_ significantly weakens block and reduces its voltage dependence. Login to comment 65 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys Au(CN)2 _ block of R334C is relatively independent of the extracellular anion A, example R334C-CFTR I-V relationships recorded before (control) and after (+Au(CN)2) addition of 1 mM Au(CN)2 _ to the intracellular solution, with 150 mM NaCl, sodium gluconate or NaSCN present in the extracellular solution. Login to comment 67 ABCC7 p.Arg334Cys C, Kd(0) is strongly affected by the extracellular anion in wild-type (black bars) but not in R334C (grey bars). Login to comment 68 ABCC7 p.Arg334Cys D, the effect of changing the extracellular anion (from Cl_ to gluconate or from Cl_ to SCN_ ) on the strength of Au(CN)2 _ block, as quantified by the ratio of Kd(0) values estimated under different ionic conditions as described in Methods, is significantly reduced in R334C (grey bars) relative to wild-type (black bars) (**P < 0.001). Login to comment 79 ABCC7 p.Arg334His Adirectinvestigationoftheroleofpositivecharge A previous mutagenic investigation of arginine 334 emphasized the role played by the fixed positive charge at this position, and elegantly demonstrated this effect by titrating the side chain charge in R334H by changing the external pH (Smith et al. 2001). Login to comment 80 ABCC7 p.Arg334His Although we have studied mutants with neutral, positively charged and negatively charged side chains, the pH dependence of R334H provides a unique opportunity to examine the effect of side chain charge independently of side chain shape. Login to comment 81 ABCC7 p.Arg334His We therefore examined the effect of changing extracellular pH on Au(CN)2 _ block of wild-type and R334H-CFTR. Login to comment 82 ABCC7 p.Arg334His As shown in Fig. 7, Au(CN)2 _ blocked R334H more strongly at pH 5.5 than at pH 9.0, whereas block of wild-type was X. Gong and P. Linsdell392 J Physiol549.2 Figure 5. Login to comment 88 ABCC7 p.Arg334His However, the effect of changing the extracellular anion on the apparent affinity of Au(CN)2 _ block was not strongly pH dependent in R334H (Fig. 8), and at all pHs studied the effect of changing from extracellular gluconate to Cl_ , or from Cl_ to SCN_ , was small (as judged by the Kd(0) ratio) compared to the effect seen in wild-type at pH 7.4. Login to comment 89 ABCC7 p.Arg334His These experiments on R334H at different pHs strongly suggest that Au(CN)2 _ blocking affinity and the interaction between intracellular Au(CN)2 _ ions and extracellular anions show different dependencies on side chain charge at position 334. Login to comment 93 ABCC7 p.Arg334Glu ABCC7 p.Arg334Lys As noted by Smith et al. (2001), this effect was clearly charge dependent, being strongest in R334E and weak (but still significant) in R334K. Login to comment 94 ABCC7 p.Arg334His Consistent with this notion, and again as noted by Smith et al. (2001), rectification was pH dependent in R334H but not in wild-type (Fig. 9A). Login to comment 101 ABCC7 p.Arg334His ABCC7 p.Arg334His Extracellular pH modifies Au(CN)2 _ block of R334H- but not wild-type CFTR A, example I-V relationships for wild-type and R334H at extracellular pHs of 5.5 and 9.0, before (control) and after (+Au(CN)2) addition of 100 mM Au(CN)2 _ to the intracellular solution. Login to comment 103 ABCC7 p.Arg334His Mean of data from 3-5 patches, fitted as in Fig. 1B. C, effect of extracellular pH on Kd(0) in wild-type (0) and R334H (1). Login to comment 106 ABCC7 p.Arg334Leu However, SCN_ permeability was significantly increased in one mutant, R334L (Fig. 9B). Login to comment 111 ABCC7 p.Arg334Cys ABCC7 p.Arg334Glu Each of the mutations R334C, R334E and X. Gong and P. Linsdell394 J Physiol549.2 Figure 8. Login to comment 112 ABCC7 p.Arg334His Extracellular pH does not affect extracellular anion dependence of Au(CN)2 _ block in R334H-CFTR The effect of changing the extracellular anion (from Cl_ to gluconate or from Cl_ to SCN_ ), quantified by the Kd(0) ratio as in Fig. 3D, appears independent of pH in R334 and is significantly reduced relative to wild-type at pH 7.4. Login to comment 116 ABCC7 p.Arg334His Furthermore, this rectification acquires pH dependence in R334H not seen in wild-type. Login to comment 117 ABCC7 p.Arg334Leu Mean of data from 3-5 patches. B, relative SCN_ permeability, estimated from the reversal potential with 150 mM NaSCN in the extracellular solution, was unaltered in most mutants but significantly increased (**P < 0.001) in R334L. Login to comment 119 ABCC7 p.Arg334Lys R334K greatly decreased Au(CN)2 _ affinity (Figs B and 5), increasing Kd(0) 4-7-fold under conditions of high extracellular Cl_ (Fig. 5A), and 14-24-fold with the impermeant gluconate ion in the extracellular solution (Fig. 5B), conditions under which Au(CN)2 _ binding is studied in relative isolation from interactions with other anions. Login to comment 122 ABCC7 p.Arg334Cys Previously we showed that the R334C mutation significantly weakened block by intracellular lonidamine (Gong et al. 2002b), consistent with the idea that permeant and blocking ions may share common binding sites within the pore. Login to comment 131 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser ABCC7 p.Lys335Ala 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 141 ABCC7 p.Arg334Cys ABCC7 p.Arg334Glu ABCC7 p.Arg334Lys With either Cl_ or gluconate in the extracellular solution, Au(CN)2 _ block was most dramatically weakened in the mutants R334C, R334E and R334K, which involve replacement of the positively charged arginine side chain with one neutral side chain (cysteine), one negatively charged side chain (glutamate) and one positively charged side chain Anion binding site in the CFTR pore outer mouthJ Physiol 549.2 395 (lysine). Login to comment 143 ABCC7 p.Arg334His In R334H, positive charge does appear to enhance Au(CN)2 _ binding, since low pH, which is expected to favour protonation of this side chain (see also Smith et al. 2001), increases the apparent affinity of block (Fig. 7). Login to comment 147 ABCC7 p.Arg334His These different effects of side chain charge are clearly demonstrated in R334H, which shows pH-dependent rectification (Fig. 9A) and Au(CN)2 _ affinity (Fig. 7) but pH-independent interactions between intracellular Au(CN)2 _ and extracellular anions (Fig. 8). Login to comment 148 ABCC7 p.Arg334Leu ABCC7 p.Arg334His The fact that tight Au(CN)2 _ binding and strong Au(CN)2 _ -anion interactions are separable by mutagenesis is also evident in R334H and R334L (Fig. 5). Login to comment