ABCMdb

ABCC7 p.Lys335Arg

Predicted by SNAP2:	A: D (66%), C: D (80%), D: D (91%), E: D (75%), F: D (91%), G: D (80%), H: D (80%), I: D (80%), L: D (80%), M: D (80%), N: D (80%), P: D (91%), Q: N (53%), R: N (87%), S: D (66%), T: D (75%), V: D (80%), W: D (91%), Y: D (85%),
Predicted by PROVEAN:	A: N, C: D, D: N, E: N, F: D, G: N, H: N, I: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: D, Y: D,

[switch to compact view]
Comments [show]

None has been submitted yet.

Publications
[hide] Cystic fibrosis transmembrane conductance regulato... Biophys J. 1998 Mar;74(3):1320-32. 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., [PMID:9512029]

Abstract [show]
We compared the effects of mutations in transmembrane segments (TMs) TM1, TM5, and TM6 on the conduction and activation properties of the cystic fibrosis transmembrane conductance regulator (CFTR) to determine which functional property was most sensitive to mutations and, thereby, to develop a criterion for measuring the importance of a particular residue or TM for anion conduction or activation. Anion substitution studies provided strong evidence for the binding of permeant anions in the pore. Anion binding was highly sensitive to point mutations in TM5 and TM6. Permeability ratios, in contrast, were relatively unaffected by the same mutations, so that anion binding emerged as the conduction property most sensitive to structural changes in CFTR. The relative insensitivity of permeability ratios to CFTR mutations was in accord with the notion that anion-water interactions are important determinants of permeability selectivity. By the criterion of anion binding, TM5 and TM6 were judged to be likely to contribute to the structure of the anion-selective pore, whereas TM1 was judged to be less important. Mutations in TM5 and TM6 also dramatically reduced the sensitivity of CFTR to activation by 3-isobutyl 1-methyl xanthine (IBMX), as expected if these TMs are intimately involved in the physical process that opens and closes the channel.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
62 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
107 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 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 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
137 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
144 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 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
166 Similarly, neither the alanine (K335A) nor the arginine (K335R) substituted K335 constructs exhibited an alteration in sensitivity to activating conditions. Login to comment
169 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 Ϯ 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
173 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
198 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
108 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
138 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