ABCMdb

PMID: 11927667

Gong X, Burbridge SM, Cowley EA, Linsdell P
Molecular determinants of Au(CN)(2)(-) binding and permeability within the cystic fibrosis transmembrane conductance regulator Cl(-) channel pore.
J Physiol. 2002 Apr 1;540(Pt 1):39-47., 2002-04-01 [PubMed]

Sentences

No. Mutations Sentence Comment

12 ABCC7 p.Arg352Gln ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser ABCC7 p.Lys335Ala ABCC7 p.Ile344Ala ABCC7 p.Lys329Ala 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 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser 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 ABCC7 p.Arg352Gln ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser 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 70 ABCC7 p.Arg352Gln Block of PPi-stimulated wild-type and mutant CFTR Cl_ currents by Au(CN)2 _ A, example CFTR I-V relationships recorded from inside-out membrane patches from BHK cells transfected with wild-type (left) or R352Q-CFTR (right). Login to comment 72 ABCC7 p.Arg352Gln The PPi-induced increase in current amplitude was significantly different from wild-type only in R352Q (* P < 0.001, Student`s two-tailed t test). Login to comment 78 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Lys335Ala ABCC7 p.Ile344Ala ABCC7 p.Lys329Ala Currents carried by the CFTR mutants K329A, K335A, T338A, S341A and I344A were also stimulated an average of 2_3-fold by PPi (Fig. 2B). Login to comment 79 ABCC7 p.Phe337Ser 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 80 ABCC7 p.Arg352Gln In contrast, while only very small R352Q currents were recorded in the presence of PKA and ATP, currents were massively stimulated on addition of PPi (by an average of 26.3 ± 5.7-fold; Fig. 2A, B). Login to comment 81 ABCC7 p.Arg352Gln Although single channel recordings were not carried out, this implies that the channel open probability for R352Q in the absence of PPi is very low (< 0.04). Login to comment 82 ABCC7 p.Arg334Ala ABCC7 p.Thr351Ala Two other mutants were also transfected into BHK cells; however, even in the presence of PPi, currents carried by R334A and T351A were too small for proper analysis (not shown). Login to comment 86 ABCC7 p.Lys329Ala However, for all mutants studied except K329A, the blocking effects of 100 mM Au(CN)2 _ at _100 mV were significantly altered relative to wild-type (P < 0.05 in each case, Student`s two-tailed t test). Login to comment 87 ABCC7 p.Arg352Gln ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser ABCC7 p.Lys335Ala ABCC7 p.Ile344Ala ABCC7 p.Lys329Ala 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 88 ABCC7 p.Arg352Gln ABCC7 p.Ser341Ala At depolarised voltages, where the blocking effects of 100 mM Au(CN)2 _ are weak, block of most mutants was not significantly different from wild-type; the only differences in Au(CN)2 _ sensitivity at +60 mV were R352Q > S341A > wild-type. Login to comment 89 ABCC7 p.Arg352Gln Although block of PPi-stimulated channels by 100 mM Au(CN)2 _ was strongly voltage dependent in wild-type and most mutants, consistent with the 'conduction effect` of this anion (Linsdell & Gong, 2002), the strong inhibitory effects on R352Q were practically voltage independent. Login to comment 91 ABCC7 p.Arg352Gln ABCC7 p.Arg352Gln Together with the extreme stimulation of R352Q current by PPi (Fig. 2A and B), this suggests that we are unable to separate permeation from gating effects in this mutant and, as such, the potential role of R352Q in contributing to intrapore anion binding cannot be addressed in the present study. Login to comment 95 ABCC7 p.Lys329Ala Results for K329A were indistinguishable from wild-type (not shown). Login to comment 100 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser 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 101 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala Interestingly, S341A also significantly increased PAu(CN)2/PCl (Fig. 4), although to a lesser extent than T338A. Login to comment 116 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser 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 ABCC7 p.Arg352Gln ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser ABCC7 p.Lys335Ala ABCC7 p.Lys329Ala 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 ABCC7 p.Arg352Gln ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser ABCC7 p.Lys335Ala ABCC7 p.Ile344Ala ABCC7 p.Lys329Ala 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 126 ABCC7 p.Ser341Ala Interestingly, in spite of its previous association with disrupted anion binding within the pore (McDonough et al. 1994; Zhang et al. 2000), S341A showed significantly strengthened Au(CN)2 _ block at all potentials (Fig. 3), suggesting that the polar hydroxyl side chain of S341 does not contribute to lyotropic anion binding. Login to comment 128 ABCC7 p.Arg352Gln As discussed in the text, the effects of R352Q cannot be confidently ascribed to open channel block and, as such, we are unable to determine the role of R352 in anion binding in the pore. Login to comment 139 ABCC7 p.Thr338Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser ABCC7 p.Phe337Ser 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 140 ABCC7 p.Ser341Ala However, the dramatic increase in PAu(CN)2/PCl observed in S341A (Fig. 4) suggests that the region of the pore which predominantly controls selectivity between different anions may extend further towards the intracellular end of TM6 than previously appreciated. Login to comment 142 ABCC7 p.Thr338Ala Multiple TM6 residues contribute to anion binding, as determined by Au(CN)2 _ block of Cl_ permeation; the dramatic weakening of Au(CN)2 _ block in T338A suggests a particularly strong role of this residue in binding. Login to comment 147 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser 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 154 ABCC7 p.Arg352Gln ABCC7 p.Thr351Ala Unfortunately, we were unable to test the role of TM6 residues more intracellular than I344 on anion binding; R347 was not studied because of its association with pore stability (Cotten & Welsh, 1999), T351A was not well expressed in BHK cells and the blocking effects of Au(CN)2 _ on R352Q most likely do not reflect open channel block. Login to comment 157 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala Nevertheless, there does not seem to be a strong correlation between these two aspects of pore function, such that they may be controlled independently by the same structural featuresofthepore.Thus,F337Sisassociatedwithweakened Au(CN)2 _ binding and decreased Au(CN)2 _ permeability, T338A with weakened Au(CN)2 _ binding and increased Au(CN)2 _ permeability and S341A with strengthened Au(CN)2 _ bindingandincreasedpermeability(Figs3and4). Login to comment