ABCMdb

PMID: 9512029

Mansoura MK, Smith SS, Choi AD, Richards NW, Strong TV, Drumm ML, Collins FS, Dawson DC
Cystic fibrosis transmembrane conductance regulator (CFTR) anion binding as a probe of the pore.
Biophys J. 1998 Mar;74(3):1320-32., [PubMed]

Sentences

No. Mutations Sentence Comment

11 ABCC7 p.Lys95Asp Substitution of an aspartic acid for K95 (K95D) in TM1 altered anion selectivity (Anderson et al., 1991), and three cysteine-substituted residues in this TM (G91, K95, and Q98) were identified as being accessible to water-soluble, charged, sulfhydryl-specific reagents (Akabas et al., 1994), but a CFTR construct in which the first 118 amino acids, including those that constitute TM1, were deleted produced channels with conduction properties similar to wild type (Carroll et al., 1995). Login to comment 27 ABCC7 p.Gly91Arg Glycine 91 was one of three residues in TM1 identified as being accessible to water-soluble thiol reagents, and this residue is also the site of a patient mutation, G91R, associated with a pancreatic sufficient phenotype (Guillermit et al., 1993). Login to comment 28 ABCC7 p.Gly314Glu ABCC7 p.Gly314Arg Another residue in TM1, K95, was implicated as being important for anion selectivity (Anderson et al., 1991), but mutants at this locus did not give rise to robust expression in Xenopus oocytes. TM5 has a relatively high frequency of patient mutations compared with the other 11 putative TMs (Cystic Fibrosis Genetic Analysis Consortium, unpublished data), and two missense patient mutations, G314E (Golla et al., 1994) and G314R (Nasr et al., 1996), have been identified at G314. Login to comment 62 ABCC7 p.Gly314Glu ABCC7 p.Lys335Glu ABCC7 p.Gly91Arg ABCC7 p.Lys335Ala ABCC7 p.Gly91Glu ABCC7 p.Gly314Ala ABCC7 p.Gly314Gln ABCC7 p.Lys335Asp ABCC7 p.Gly91Ala ABCC7 p.Gly314Asp ABCC7 p.Lys335Arg Expression levels Wild-type and 11 mutant CFTR constructs were used in this study: G91A, G91E, G91R, G314A, G314D, G314E, G314Q, K335R, K335A, K335D, and K335E. Login to comment 97 ABCC7 p.Lys1250Ala Finally, dose-dependent inhibition of CFTR conductance by [SCN]o (see below) was identical in wtCFTR and a CFTR mutant exhibiting highly altered gating, K1250A (Wilkinson et al., 1996). Login to comment 98 ABCC7 p.Lys1250Ala Finally, dose-dependent inhibition of CFTR conductance by [SCN]o (see below) was identical in wtCFTR and a CFTR mutant exhibiting highly altered gating, K1250A (Wilkinson et al., 1996). Login to comment 103 ABCC7 p.Lys335Glu Only one mutation, K335E, significantly altered the sequence of permeability ratios derived from shifts in reversal potential. Login to comment 104 ABCC7 p.Lys335Glu Only one mutation, K335E, significantly altered the sequence of permeability ratios derived from shifts in reversal potential. Login to comment 106 ABCC7 p.Lys335Asp The aspartic acid substitution (K335D) altered the permeability ratios for Br, HCOO, and I, but the average values did not differ markedly from those seen with wtCFTR, and the sequence was not changed. Login to comment 107 ABCC7 p.Lys335Ala ABCC7 p.Lys335Ala ABCC7 p.Lys335Asp ABCC7 p.Lys335Arg Neither the arginine (K335R) nor the alanine substitution (K335A) resulted in any substantial change in the permeability ratios (with the exception of SCN for K335A). Login to comment 108 ABCC7 p.Lys335Ala ABCC7 p.Lys335Ala ABCC7 p.Lys335Arg Neither the arginine (K335R) nor the alanine substitution (K335A) resulted in any substantial change in the permeability ratios (with the exception of SCN for K335A). Login to comment 112 ABCC7 p.Lys335Ala ABCC7 p.Lys335Arg The conductance ratios for anions judged by shifts in reversal potential to be most highly permeant (SCN, NO3, and Br) were all increased significantly by substituting a negatively charged residue (D or E) for K335, whereas mutants in which the charge was conserved (K335R) or removed (K335A) were identical to wtCFTR. Login to comment 113 ABCC7 p.Lys335Glu ABCC7 p.Lys335Glu ABCC7 p.Lys335Ala ABCC7 p.Lys335Asp ABCC7 p.Lys335Arg The K335D and K335E constructs also exhibited increased conductance ratios for I, a result previously reported by Anderson et al. (1991) for K335E CFTR. Login to comment 114 ABCC7 p.Lys335Glu ABCC7 p.Lys335Glu ABCC7 p.Lys335Asp The K335D and K335E constructs also exhibited increased conductance ratios for I, a result previously reported by Anderson et al. (1991) for K335E CFTR. Login to comment 115 ABCC7 p.Gly314Glu Most dramatic here was the substitution of glutamic acid (G314E), which raised conductance ratios for SCN, NO3, and Br. Login to comment 116 ABCC7 p.Gly314Glu ABCC7 p.Gly314Gln ABCC7 p.Gly314Asp It was of particular interest that the introduction of a glutamine (G314Q) also produced increased conductance ratios for the highly permeant anions and I, whereas the aspartic-acid-substituted construct (G314D) was not different from wtCFTR. Login to comment 117 ABCC7 p.Gly314Gln ABCC7 p.Gly314Asp It was of particular interest that the introduction of a glutamine (G314Q) also produced increased conductance ratios for the highly permeant anions and I, whereas the aspartic-acid-substituted construct (G314D) was not different from wtCFTR. Login to comment 120 ABCC7 p.Gly314Glu As an example, the I-V plots shown in Fig. 2, A and B, illustrate the effect of [SCN]o substitution on wild-type and G314E CFTR. Login to comment 121 ABCC7 p.Gly314Glu As an example, the I-V plots shown in Fig. 2, A and B, illustrate the effect of [SCN]o substitution on wild-type and G314E CFTR. Login to comment 122 ABCC7 p.Gly314Glu In the G314E mutant the attenuation of gCl by 2% [SCN]o was virtually abolished. Login to comment 123 ABCC7 p.Gly314Glu In the G314E mutant the attenuation of gCl by 2% [SCN]o was virtually abolished. Login to comment 131 ABCC7 p.Gly314Asp The behavior of G314D CFTR was virtually identical to that of wtCFTR. Login to comment 132 ABCC7 p.Gly314Asp The behavior of G314D CFTR was virtually identical to that of wtCFTR. Login to comment 135 ABCC7 p.Gly314Ala ABCC7 p.Gly314Asp There was no readily discernible voltage dependence to the dose-dependent attenuation of wtCFTR conductance by SCN; and similar results were obtained for G314D and G314A. Login to comment 136 ABCC7 p.Gly314Glu ABCC7 p.Gly314Ala ABCC7 p.Gly314Gln ABCC7 p.Gly314Asp In the case of the G314E and G314Q mutants, however, the SCN effect appeared to be moderately voltage dependent (Fig. 2 TABLE 2 Summary of permeability and conductance ratios from anion substitution experiments n SCN NO3 Br HCOO I A. Login to comment 137 ABCC7 p.Gly314Glu ABCC7 p.Gly314Glu ABCC7 p.Gly314Glu ABCC7 p.Lys335Glu ABCC7 p.Lys335Glu ABCC7 p.Gly91Arg ABCC7 p.Gly91Arg ABCC7 p.Lys335Ala ABCC7 p.Lys335Ala ABCC7 p.Gly91Glu ABCC7 p.Gly91Glu ABCC7 p.Gly314Ala ABCC7 p.Gly314Ala ABCC7 p.Gly314Gln ABCC7 p.Gly314Gln ABCC7 p.Gly314Gln ABCC7 p.Lys335Asp ABCC7 p.Lys335Asp ABCC7 p.Gly91Ala ABCC7 p.Gly91Ala ABCC7 p.Gly314Asp ABCC7 p.Gly314Asp ABCC7 p.Lys335Arg ABCC7 p.Lys335Arg Permeability Ratios Wild type 4-9 3.42 Ϯ 0.28 1.42 Ϯ 0.04 1.22 Ϯ 0.02 0.39 Ϯ 0.01 0.44 Ϯ 0.03 G91A 3-6 3.24 Ϯ 0.26 1.53 Ϯ 0.04 1.27 Ϯ 0.02 0.37 Ϯ 0.04 0.40 Ϯ 0.04 G91E 3-7 3.50 Ϯ 0.54 1.59 Ϯ 0.04 1.27 Ϯ 0.01 0.35 Ϯ 0.01 0.51 Ϯ 0.04 G91R 3-4 5.26 ؎ 0.46* 1.60 Ϯ 0.03 1.40 ؎ 0.01* 0.32 Ϯ 0.04 0.64 ؎ 0.04* G314A 3-4 2.87 Ϯ 0.17 1.45 Ϯ 0.03 1.19 Ϯ 0.02 0.31 Ϯ 0.03 0.33 Ϯ 0.03 G314D 4 3.42 Ϯ 0.34 1.44 Ϯ 0.05 1.25 Ϯ 0.04 0.33 Ϯ 0.03 0.51 Ϯ 0.05 G314E 3-4 3.72 Ϯ 0.56 1.65 ؎ 0.09* 1.35 ؎ 0.03* 0.49 Ϯ 0.04 0.53 Ϯ 0.04 G314Q 3-4 3.89 Ϯ 0.37 1.62 Ϯ 0.11 1.27 Ϯ 0.04 0.36 Ϯ 0.03 0.62 Ϯ 0.05 K335R 3-5 3.44 Ϯ 0.29 1.35 Ϯ 0.04 1.22 Ϯ 0.03 0.40 Ϯ 0.05 0.41 Ϯ 0.07 K335A 5-6 5.34 ؎ 0.58* 1.48 Ϯ 0.06 1.28 Ϯ 0.04 0.37 Ϯ 0.03 0.60 Ϯ 0.06 K335D 4-6 3.02 Ϯ 0.19 1.50 Ϯ 0.03 1.10 ؎ 0.02* 0.54 ؎ 0.04* 0.65 ؎ 0.06* K335E 5-8 3.64 Ϯ 0.21 1.48 Ϯ 0.06 1.29 Ϯ 0.03 0.46 Ϯ 0.04 1.10 ؎ 0.04* B. Conductance Ratios Wild type 4-9 0.14 Ϯ 0.02 0.75 Ϯ 0.02 0.64 Ϯ 0.02 0.52 Ϯ 0.03 0.18 Ϯ 0.03 G91A 3-6 0.14 Ϯ 0.01 0.77 Ϯ 0.02 0.61 Ϯ 0.02 0.47 Ϯ 0.02 0.19 Ϯ 0.02 G91E 3-7 0.15 Ϯ 0.03 0.73 Ϯ 0.02 0.60 Ϯ 0.01 0.50 Ϯ 0.04 0.30 Ϯ 0.02 G91R 3-4 0.14 Ϯ 0.00 0.84 Ϯ 0.01 0.63 Ϯ 0.01 0.32 ؎ 0.01* 0.14 Ϯ 0.01 G314A 3-4 0.30 Ϯ 0.09 0.89 ؎ 0.01* 0.66 Ϯ 0.01 0.48 Ϯ 0.09 0.24 Ϯ 0.01 G314D 4 0.28 Ϯ 0.05 0.82 Ϯ 0.01 0.70 Ϯ 0.02 0.49 Ϯ 0.06 0.27 Ϯ 0.03 G314E 3-4 0.62 ؎ 0.07* 1.18 ؎ 0.04* 0.84 ؎ 0.05* 0.42 Ϯ 0.05 0.29 Ϯ 0.09 G314Q 3-4 0.63 ؎ 0.02* 1.01 ؎ 0.04* 0.82 ؎ 0.03* 0.50 Ϯ 0.02 0.42 ؎ 0.02* K335R 3-5 0.14 Ϯ 0.01 0.76 Ϯ 0.03 0.61 Ϯ 0.02 0.59 Ϯ 0.06 0.16 Ϯ 0.03 K335A 6 0.20 Ϯ 0.03 0.77 Ϯ 0.02 0.61 Ϯ 0.02 0.45 Ϯ 0.03 0.21 Ϯ 0.02 K335D 4-6 0.65 ؎ 0.04* 1.25 ؎ 0.02* 0.89 ؎ 0.02* 0.61 Ϯ 0.06 0.58 ؎ 0.06* K335E 5-8 0.50 ؎ 0.06* 1.19 ؎ 0.03* 0.89 ؎ 0.02* 0.53 Ϯ 0.03 0.48 ؎ 0.03* (A) The apparent permeability ratios (PS/PCl) for each substitute anion were calculated from the shift in reversal potential using the Goldman-Hodgkin-Katz relation (noted in Materials and Methods). Login to comment 138 ABCC7 p.Gly314Glu ABCC7 p.Gly314Glu ABCC7 p.Lys335Glu ABCC7 p.Lys335Glu ABCC7 p.Gly91Arg ABCC7 p.Gly91Arg ABCC7 p.Lys335Ala ABCC7 p.Lys335Ala ABCC7 p.Gly91Glu ABCC7 p.Gly91Glu ABCC7 p.Gly314Ala ABCC7 p.Gly314Ala ABCC7 p.Gly314Gln ABCC7 p.Gly314Gln ABCC7 p.Lys335Asp ABCC7 p.Lys335Asp ABCC7 p.Gly91Ala ABCC7 p.Gly91Ala ABCC7 p.Gly314Asp ABCC7 p.Gly314Asp ABCC7 p.Lys335Arg ABCC7 p.Lys335Arg Permeability Ratios Wild type 4-9 3.42 afe; 0.28 1.42 afe; 0.04 1.22 afe; 0.02 0.39 afe; 0.01 0.44 afe; 0.03 G91A 3-6 3.24 afe; 0.26 1.53 afe; 0.04 1.27 afe; 0.02 0.37 afe; 0.04 0.40 afe; 0.04 G91E 3-7 3.50 afe; 0.54 1.59 afe; 0.04 1.27 afe; 0.01 0.35 afe; 0.01 0.51 afe; 0.04 G91R 3-4 5.26 d1e; 0.46* 1.60 afe; 0.03 1.40 d1e; 0.01* 0.32 afe; 0.04 0.64 d1e; 0.04* G314A 3-4 2.87 afe; 0.17 1.45 afe; 0.03 1.19 afe; 0.02 0.31 afe; 0.03 0.33 afe; 0.03 G314D 4 3.42 afe; 0.34 1.44 afe; 0.05 1.25 afe; 0.04 0.33 afe; 0.03 0.51 afe; 0.05 G314E 3-4 3.72 afe; 0.56 1.65 d1e; 0.09* 1.35 d1e; 0.03* 0.49 afe; 0.04 0.53 afe; 0.04 G314Q 3-4 3.89 afe; 0.37 1.62 afe; 0.11 1.27 afe; 0.04 0.36 afe; 0.03 0.62 afe; 0.05 K335R 3-5 3.44 afe; 0.29 1.35 afe; 0.04 1.22 afe; 0.03 0.40 afe; 0.05 0.41 afe; 0.07 K335A 5-6 5.34 d1e; 0.58* 1.48 afe; 0.06 1.28 afe; 0.04 0.37 afe; 0.03 0.60 afe; 0.06 K335D 4-6 3.02 afe; 0.19 1.50 afe; 0.03 1.10 d1e; 0.02* 0.54 d1e; 0.04* 0.65 d1e; 0.06* K335E 5-8 3.64 afe; 0.21 1.48 afe; 0.06 1.29 afe; 0.03 0.46 afe; 0.04 1.10 d1e; 0.04* B. Conductance Ratios Wild type 4-9 0.14 afe; 0.02 0.75 afe; 0.02 0.64 afe; 0.02 0.52 afe; 0.03 0.18 afe; 0.03 G91A 3-6 0.14 afe; 0.01 0.77 afe; 0.02 0.61 afe; 0.02 0.47 afe; 0.02 0.19 afe; 0.02 G91E 3-7 0.15 afe; 0.03 0.73 afe; 0.02 0.60 afe; 0.01 0.50 afe; 0.04 0.30 afe; 0.02 G91R 3-4 0.14 afe; 0.00 0.84 afe; 0.01 0.63 afe; 0.01 0.32 d1e; 0.01* 0.14 afe; 0.01 G314A 3-4 0.30 afe; 0.09 0.89 d1e; 0.01* 0.66 afe; 0.01 0.48 afe; 0.09 0.24 afe; 0.01 G314D 4 0.28 afe; 0.05 0.82 afe; 0.01 0.70 afe; 0.02 0.49 afe; 0.06 0.27 afe; 0.03 G314E 3-4 0.62 d1e; 0.07* 1.18 d1e; 0.04* 0.84 d1e; 0.05* 0.42 afe; 0.05 0.29 afe; 0.09 G314Q 3-4 0.63 d1e; 0.02* 1.01 d1e; 0.04* 0.82 d1e; 0.03* 0.50 afe; 0.02 0.42 d1e; 0.02* K335R 3-5 0.14 afe; 0.01 0.76 afe; 0.03 0.61 afe; 0.02 0.59 afe; 0.06 0.16 afe; 0.03 K335A 6 0.20 afe; 0.03 0.77 afe; 0.02 0.61 afe; 0.02 0.45 afe; 0.03 0.21 afe; 0.02 K335D 4-6 0.65 d1e; 0.04* 1.25 d1e; 0.02* 0.89 d1e; 0.02* 0.61 afe; 0.06 0.58 d1e; 0.06* K335E 5-8 0.50 d1e; 0.06* 1.19 d1e; 0.03* 0.89 d1e; 0.02* 0.53 afe; 0.03 0.48 d1e; 0.03* (A) The apparent permeability ratios (PS/PCl) for each substitute anion were calculated from the shift in reversal potential using the Goldman-Hodgkin-Katz relation (noted in Materials and Methods). Login to comment 144 ABCC7 p.Lys335Glu ABCC7 p.Lys335Ala ABCC7 p.Lys335Asp ABCC7 p.Lys335Arg Similar negative results were obtained for the K335R and K335A constructs, but in the K335D and K335E mutants maximal attenuation by [SCN]o was reduced by nearly 50%, although the apparent binding affinity was unaffected. Login to comment 145 ABCC7 p.Lys335Glu ABCC7 p.Lys335Glu ABCC7 p.Lys335Ala ABCC7 p.Lys335Asp ABCC7 p.Lys335Asp ABCC7 p.Lys335Arg The result is consistent with the notion that the affinity of anion binding was not diminished in the K335D and K335E constructs, but the impact of binding on conduction was reduced. Login to comment 146 ABCC7 p.Lys1250Ala ABCC7 p.Lys335Glu ABCC7 p.Lys335Asp Sensitivity to [SCN]o was identical to wtCFTR in a construct bearing a mutation in NBF2, K1250A (data not shown), which exhibits severely altered activation in the form of a highly stabilized active state (Wilkinson et al., 1996). Login to comment 147 ABCC7 p.Gly314Glu ABCC7 p.Lys1250Ala ABCC7 p.Gly314Gln The data in Table 3 show that for wtCFTR and G314Q and G314E, two of the most severely affected constructs, PSCN/PCl calculated from the shift in Vr was independent of the fractional abundance of [SCN]o. Login to comment 148 ABCC7 p.Gly314Glu ABCC7 p.Gly314Gln The data in Table 3 show that for wtCFTR and G314Q and G314E, two of the most severely affected constructs, PSCN/PCl calculated from the shift in Vr was independent of the fractional abundance of [SCN]o. Login to comment 150 ABCC7 p.Gly314Glu ABCC7 p.Gly91Arg ABCC7 p.Gly91Glu Of the three substitutions for G91, the arginine (G91R) altered the RR most dramatically, increasing it nearly sevenfold, although the negatively charged glutamate (G91E) FIGURE 2 The effect of replacement of [Cl- ]o by [SCN- ]o is shown for wtCFTR (A) and G314E (B). Login to comment 151 ABCC7 p.Gly314Glu ABCC7 p.Gly91Arg ABCC7 p.Gly91Glu Of the three substitutions for G91, the arginine (G91R) altered the RR most dramatically, increasing it nearly sevenfold, although the negatively charged glutamate (G91E) FIGURE 2 The effect of replacement of [Clafa; ]o by [SCNafa; ]o is shown for wtCFTR (A) and G314E (B). Login to comment 158 ABCC7 p.Lys335Glu ABCC7 p.Lys335Asp At the K335 locus, substitution by either glutamate (K335E) or aspartate (K335D) significantly reduced the mean value of RR by 25% and 38%, respectively. Login to comment 163 ABCC7 p.Gly91Arg Finally, the mutant that exhibited the most striking shape change (G91R) did not differ from wild type in its dose-dependent activation by IBMX (see below). Login to comment 165 ABCC7 p.Gly91Arg ABCC7 p.Gly91Glu ABCC7 p.Gly91Ala Concentration-dependent activation of G91A, G91E, and G91R CFTR was not discernibly different from that of wtCFTR. Login to comment 166 ABCC7 p.Lys335Ala ABCC7 p.Lys335Arg Similarly, neither the alanine (K335A) nor the arginine (K335R) substituted K335 constructs exhibited an alteration in sensitivity to activating conditions. Login to comment 167 ABCC7 p.Lys335Glu ABCC7 p.Lys335Asp In contrast, values for the K1/2(IBMX) for both K335D and K335E were significantly greater than that for wtCFTR, an indication of diminished sensitivity to activating conditions. Login to comment 169 ABCC7 p.Gly314Glu ABCC7 p.Gly314Glu ABCC7 p.Lys335Glu ABCC7 p.Lys335Glu ABCC7 p.Gly91Arg ABCC7 p.Gly91Arg ABCC7 p.Lys335Ala ABCC7 p.Lys335Ala ABCC7 p.Gly91Glu ABCC7 p.Gly91Glu ABCC7 p.Gly314Ala ABCC7 p.Gly314Ala ABCC7 p.Gly314Gln ABCC7 p.Gly314Gln ABCC7 p.Lys335Asp ABCC7 p.Lys335Asp ABCC7 p.Gly91Ala ABCC7 p.Gly91Ala ABCC7 p.Gly314Asp ABCC7 p.Gly314Asp ABCC7 p.Lys335Arg ABCC7 p.Lys335Arg TABLE 4 Quantitative analyses of the macroscopic I-V shape changes Mutant ⌬ Net charge n RR g(ϩ30)/g(-30) RR/RRWT Wild type 5 1.220 Ϯ 0.06 1.00 G91A 0 4 1.293 Ϯ 0.06 1.06 G91E -1 5 1.512 ؎ 0.10* 1.24 G91R 1 4 8.041 ؎ 0.87* 6.59 G314A 0 4 1.201 Ϯ 0.09 0.98 G314D -1 4 1.362 Ϯ 0.08 1.12 G314E -1 7 1.405 >e; 0.08 1.15 G314Q 0 5 1.376 Ϯ 0.10 1.13 K335R 0 4 1.209 Ϯ 0.06 0.99 K335A -1 4 1.295 Ϯ; 0.07 1.06 K335D -2 5 0.762 ؎ 0.02* 0.62 K335E -2 4 0.919 ؎ 0.02* 0.75 The slope conductance was measured at ϩ30 mV and -30 mV with respect to the reversal potential. Login to comment 171 ABCC7 p.Gly314Glu ABCC7 p.Gly314Ala ABCC7 p.Gly314Gln ABCC7 p.Gly314Asp TABLE 3 The permeability ratio (PSCN/PCl) is independent of the mole fraction of [SCN]0 for wtCFTR and the G314 variants [SCN]/{[SCN]ϩ[Cl]} n PSCN/PCl 0.02 0.05 0.10 0.20 0.50 0.90 Wild type 12 3.82 Ϯ 0.50 4.43 Ϯ 0.57 4.58 Ϯ 0.48 4.69 Ϯ 0.43 4.66 Ϯ 0.38 4.44 Ϯ 0.35 G314A 9 4.32 Ϯ 0.73 3.78 Ϯ 0.53 3.81 Ϯ 0.47 3.79 Ϯ 0.34 3.82 Ϯ 0.29 3.72 Ϯ 0.25 G314D 3 2.99 Ϯ 0.26 2.56 Ϯ 1.05 2.82 Ϯ 1.07 2.68 Ϯ 0.97 2.87 Ϯ 0.65 2.89 Ϯ 0.43 G314E 6 4.48 Ϯ 1.05 4.01 Ϯ 0.69 4.17 Ϯ 0.62 4.15 Ϯ 0.59 3.96 Ϯ 0.41 3.82 Ϯ 0.40 G314Q 3 5.39 Ϯ 0.57 4.49 Ϯ 0.58 4.69 Ϯ 1.26 4.05 Ϯ 1.26 3.86 Ϯ 1.47 3.68 Ϯ 1.51 The permeability ratios were calculated from the shift in reversal potential using the Goldman-Hodgkin-Katz equation. Login to comment 173 ABCC7 p.Gly314Glu ABCC7 p.Gly314Glu ABCC7 p.Lys335Glu ABCC7 p.Lys335Glu ABCC7 p.Gly91Arg ABCC7 p.Gly91Arg ABCC7 p.Lys335Ala ABCC7 p.Lys335Ala ABCC7 p.Gly91Glu ABCC7 p.Gly91Glu ABCC7 p.Gly314Ala ABCC7 p.Gly314Ala ABCC7 p.Gly314Gln ABCC7 p.Gly314Gln ABCC7 p.Lys335Asp ABCC7 p.Lys335Asp ABCC7 p.Gly91Ala ABCC7 p.Gly91Ala ABCC7 p.Gly314Asp ABCC7 p.Gly314Asp ABCC7 p.Lys335Arg ABCC7 p.Lys335Arg TABLE 5 Concentration-dependent activation of wtCFTR, G91, G314, and K335 variants by IBMX in the presence of 10 ␮M forskolin Mutant n K1/2(IBMX) (mM) Wild type 15 0.35 Ϯ 0.04 G91A 5 0.42 Ϯ 0.06 G91E 8 0.51 ؎ 0.06* G91R 5 0.49 Ϯ 0.09 G314A 10 1.21 ؎ 0.11* G314D 3 1.35 ؎ 0.16* G314E 8 6.39 ؎ 1.35* G314Q 4 14.26 ؎ 6.64* K335R 4 0.46 Ϯ 0.04 K335A 2 0.35 Ϯ 0.15 K335D 7 0.87 ؎ 0.13* K335E 3 0.95 ؎ 0.07* The steady-state slope conductance was measured at -60 mV as increasing concentrations of IBMX (0.02-5.0 mM) were added to the perfusate in the continued presence of 10 mM forskolin. Login to comment 175 ABCC7 p.Gly314Glu ABCC7 p.Gly314Gln Note that this fitting procedure allows us to estimate K1/2 values for even the most insensitive constructs, G314E and G314Q, despite the fact that these values were greater than the highest concentration of IBMX used in this study. Login to comment 178 ABCC7 p.Gly551Asp ABCC7 p.Gly314Glu ABCC7 p.Gly314Gln The K1/2 seen with either G314E or G314Q was comparable to that seen with nucleotide-binding mutations such as G551D that are associated with severe CF (Wilkinson et al., 1996). Login to comment 179 ABCC7 p.Gly551Ser The alanine and aspartic acid substitutions resulted in less dramatic but nevertheless significant decreases in sensitivity comparable to that produced by G551S (Wilkinson et al., 1996), a CF mutation associated with a pancreatic sufficient phenotype (Strong et al., 1991). Login to comment 180 ABCC7 p.Gly314Asp It is noteworthy that, although the activation of G314D CFTR was significantly compromised, SCN binding was virtually identical to that seen with wtCFTR. Login to comment 194 ABCC7 p.Gly314Asp Finally, in at least one mutant, G314D, the effect of [SCN]o on conductance was unaffected despite the fact that sensitivity to activation by IBMX was severely compromised. Login to comment 195 ABCC7 p.Gly314Glu ABCC7 p.Gly314Gln The pattern of the effect of anion substitution was identical for Br, NO3, and SCN, and the conductance ratio for all three ions was increased in G314E and G314Q CFTR channels. Login to comment 198 ABCC7 p.Gly314Glu ABCC7 p.Lys335Glu ABCC7 p.Gly91Arg ABCC7 p.Lys335Ala ABCC7 p.Gly91Glu ABCC7 p.Gly314Ala ABCC7 p.Gly314Gln ABCC7 p.Lys335Asp ABCC7 p.Gly91Ala ABCC7 p.Gly314Asp ABCC7 p.Lys335Arg The results presented here are consistent with the notion that the binding of anions within the CFTR pore is a sensitive indicator of changes in pore structure whereas permeability ratios appear to be rather insensitive to similar TABLE 6 Qualitative summary of the functional consequences of mutations at G91, G314, and K335 Property G91 (TM1) K335 (TM6) G314 (TM5) G91A G91E G91R K335R K335A K335D K335E G314A G314D G314E G314Q I-V shape - - ϩϩϩ - - ϩϩ ϩ - - - - Psub/PCl - - - - - - ϩϩ - - - - gsub/gCl - - - - - ϩϩϩ ϩϩϩ ϩϩ - ϩϩϩ ϩϩϩ SCN- binding - - - - - ϩϩϩ ϩϩϩ ϩϩ - ϩϩϩϩ ϩϩϩϩ Activation - - - - - ϩϩ ϩϩ ϩϩϩ ϩϩϩ ϩϩϩϩ ϩϩϩϩ Results are expressed as follows: -, function of the CFTR construct with the indicated substitution was indistinguishable from wild type; ϩ to ϩϩϩϩ, semiquantitative indication of the magnitude of the change in the function compared with wild type. Login to comment 229 ABCC7 p.Arg347His ABCC7 p.Arg347Glu ABCC7 p.Lys335Glu ABCC7 p.Arg334His ABCC7 p.Arg334Glu ABCC7 p.Lys335His Hipper et al. (1995) reported that the mutations R334E, R334H, K335E, K335H, R347E, and R347H did not alter CFTR conduction properties, but careful inspection of the data presented revealed that the level of CFTR expression was very low so that altered properties of mutant CFTRs might have been easily obscured. Login to comment 233 ABCC7 p.Gly314Glu ABCC7 p.Gly314Ala ABCC7 p.Gly314Gln The increased conductance ratios seen in G314E, G314Q, and to a lesser extent, G314A channels are compatible with the hypothesis that substitution for G314 distorted an anion binding site such that the affinities for Br, NO3, and SCN were all reduced relative to Cl. Login to comment 236 ABCC7 p.Gly314Asp Simply introducing a negative charge at G314 (G314D) was not sufficient to reduce SCN binding, suggesting that the destabilizing effect was not electrostatic in nature, but rather that the role of TM5 may be to provide some element of conformation necessary for the tight binding of anions to another residue, such as R347 in TM6 (Tabcharani et al., 1993). Login to comment 237 ABCC7 p.Gly314Gln Furthermore, the dramatic reduction in SCN binding seen in G314Q CFTR demonstrates that the presence of arginines, such as R347, is not sufficient to induce tight anion binding in the pore. Login to comment 238 ABCC7 p.Gly314Arg As reported previously (Nasr et al., 1996), low expression of the G314R construct made it impossible to assess its properties. Login to comment 245 ABCC7 p.Lys335Glu ABCC7 p.Lys335Asp The fact that the apparent affinity of SCN binding was not affected by the addition of a negative charge but the maximal effect was reduced suggests that SCN binding in K335D or K335E CFTR is similar to that seen with wtCFTR but that the bound anion is not as effective in obstructing the conduction path. Login to comment 253 ABCC7 p.Lys335Glu We found, as did Anderson et al. (1991), that the sequence for Cl and I was reversed in K335E CFTR. Login to comment 254 ABCC7 p.Lys335Glu In the present study, however, the sequence for the mutant was not that predicted solely by hydration energies, suggesting that I interacts in a unique way with both wtCFTR and K335E. Login to comment 262 ABCC7 p.Gly91Arg One of the most interesting mutants surveyed in this study was the G91R CFTR, which exhibited a dramatic, nearly sevenfold, increase in outward rectification (Table 4) but was identical to wtCFTR with regard to the apparent binding of SCN and dose-dependent activation by IBMX. Login to comment 263 ABCC7 p.Gly314Glu In contrast, G314E CFTR exhibited a dramatic decrease in anion binding and sensitivity to activation by IBMX but no change in I-V shape. Login to comment 270 ABCC7 p.Gly91Cys In fact, the data presented by Akabas et al. (1994) suggest that the activation of the G91C mutant was markedly slowed compared with that of wtCFTR. Login to comment 273 ABCC7 p.Gly314Asp In G314D CFTR, however, apparent SCN binding was similar to that seen with wtCFTR, but activation was significantly impaired, suggesting that 1) sensitivity to activation by IBMX is, if anything, even more sensitive than anion binding to alterations in the structure of TM5 and 2) there is no obligatory relation between anion binding and activation. Login to comment