ABCMdb

ABCC7 p.Arg352Lys

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Publications

PMID: 17043152 [PubMed] Aubin CN et al: "Positive charges at the intracellular mouth of the pore regulate anion conduction in the CFTR chloride channel."

No. Sentence Comment

67 Charge-conservative R303K and R352K mutations were associated with wild type like linear I-V relationships (Fig. 3 A). Login to comment

PMID: 18421494 [PubMed] Cui G et al: "Mutations at arginine 352 alter the pore architecture of CFTR."

No. Sentence Comment

5 In contrast, R352K-CFTR was similar to wild-type. Login to comment
120 R352K-CFTR showed single-channel properties very similar to those of WT-CFTR: Transitions to the s1 and s2 conductance states were rare events in this mutant (Fig. 1D). Login to comment
122 0 0.0 -0.4 -0.8 #ofevents 4000 -1.2 0.0 -0.4 -0.8 6000 #ofevents 0 -1.2 0.0 -0.4 -0.8 3000 #ofevents 0 -1.2 2500 #ofevents 0.0 -0.4 -0.8 0 -1.2 Current (pA) fc s1 s2 s1 s2 B C D A 0.4 pA 2 s c s1 s2 f R352A 0.4 pA 2 s 0.4 pA 2 s c s1 s2 f c f 0.4 pA 2 s c f WT R352Q R352K 00 s1 s2 s1 s2 Fig. 1 Sample traces of WT-CFTR and indicated R352 mutants from excised inside-out membrane patches with symmetrical 150 mM Cl- solution (left) and their all-points amplitude histograms (right). Login to comment
134 Figure 4 shows the i-V relationship for the f conductance states of WT-, R352A-, R352Q- and R352K-CFTR (Fig. 4B) and for the subconductance states of R352A- and R352Q-CFTR (Fig. 4C), at potentials ranging between VM = -100 and +100 mV. Login to comment
138 In contrast, the slope conductances of R352K-CFTR were very similar to those of WT-CFTR (Table 1). Login to comment
146 Anion Selectivity of R352A-, R352E- and R352K-CFTR To determine whether mutations at R352 affected the ability of CFTR channels to select between ions of similar charge, we studied the anion selectivity patterns of R352A-, R352E- and R352K-CFTR using inside-out macropatches and compared them to WT-CFTR. Login to comment
152 For calculation of Gx/GCl, we compared R352A-, R352E- and R352K-CFTR with WT-CFTR as well as R352E- and R352K-CFTR with R352A-CFTR. Login to comment
156 The normalization of relative conductances between the different anions tested likely f 0.2 pA 2 s -100mV -80mV -60mV -40mV 0.2 pA 2 s -100mV -80mV - - pA - - s1 s2 c s1 s2 f c s1 s2 f c f cA B C WT -CFTR R352Q R352A R352K WT -CFTR R352Q R352A R352K mV -100 -50 50 100 -0.8 -0.4 0.4 0.8 pA mV -100 -50 50 100 0.4 0.2 -0.2 -0.4 pA0.6 -0.6 -100 -50 50 100 0.4 0.2 -0.2 -0.4 0.6 -0.6 -100 -50 50 100 0.4 0.2 -0.2 -0.4 0.6 -0.6 R352Q s1 R352Q s2 R352A s1 R352A s2 R352Q s1 R352Q s2 f 0.2 pA 2 s -100mV -80mV -60mV -40mV - - pA - - 0.2 pA 2 s -100mV -80mV - - Fig. 4 Sample traces of R352A-CFTR and i-V relationships of the conducting states of WT-CFTR and R352 mutants. Login to comment
159 (B) Single-channel i-V relationships for f conductance states of R352A-, R352Q- and R352K-CFTR, with WT-CFTR for comparison. Login to comment
162 Slope conductances are summarized in Table 1 Table 1 Slope conductancea (in pS) of the f state of WT-CFTR and multiple single and double mutants CFTR n Negative VM Positive VM WT 7 6.82 ± 0.03 6.97 ± 0.06 R352A 6 6.80 ± 0.06 7.85 ± 0.07*, ** R352Q 6 5.29 ± 0.02* 6.28 ± 0.05*, ** R352K 5 6.87 ± 0.03 6.86 ± 0.01 R352E 5 3.78 ± 0.01* 6.03 ± 0.01*, ** R352E/E873R 6 3.84 ± 0.01* 5.64 ± 0.01*, ** R352E/ E1104R 6 4.36 ± 0.01* 5.86 ± 0.02*, ** R352E/D993R 5 5.90 ± 0.02* 6.44 ± 0.01*, ** D993R 7 8.27 ± 0.05* 7.13 ± 0.07** a Slope conductance indicates single-channel conductance calculated from 0 to +100 mV (positive VM) or to -100 mV (negative VM) by linear regression * P B 0.001 compared to the equivalent slope conductance in WT-CFTR, ** P B 0.001 compared to the slope conductance in the same mutant at negative VM reflects the loss of anion binding properties within the core of the permeation pathway, which contributes to the tight binding of SCN (Smith et al. 1999). Login to comment
166 Our present results suggest -300 -50 300 50 Br -100 100 NO3 Cl SCN pA mVBr NO3 SCN Cl -300 -50 300 50 Br -100 100 NO3 Cl SCNC pA mVBr NO3 SCN Cl R352E -4000 -50 4000 50 -100 100 -800 -50 800 50 -100 100 -6000 -50 6000 50 -100 100 A pA -50 800 50 -100 100 A SCN Br Cl NO3 NO3 Br Cl SCN Br NO3 SCN Cl Br NO3 SCN Cl -800 mV pA mV pA mV pA mV NO3 Br Cl SCN Br NO3 SCN Cl Br NO3 SCN Cl Br NO3 SCN Cl -4000 -50 4000 50 -100 100 D -800 -50 800 50 -100 100 E D993R -6000 -50 6000 50 -100 100 WT pA -50 800 50 -100 100 B SCN Br Cl NO3 NO3 Br Cl SCN Br NO3 SCN Cl Br NO3 SCN Cl -800 mV pA mV pA mV pA mV NO3 Br Cl SCN Br NO3 SCN Cl Br NO3 SCN Cl Br NO3 SCN Cl R352A R352K R352E/ Fig. 5 Mutations at R352 alter anion selectivity. Login to comment
167 Representative inside-out macropatches, recorded in the presence of cytoplasmic Cl- or Cl- plus substitute anions, with voltage ramps between -100 and +100 mV, are shown for (A) WT-CFTR, (B) R352A-CFTR, (C) R352E-CFTR, (D) R352K-CFTR and (E) the double mutant R352E/D993R-CFTR. Login to comment
171 Solutions were at pH 7.45 and are labeled as follows: 150 mM Cl- (black), 130 mM Cl- plus 20 mM NO3 - (purple), 130 mM Cl- plus 20 mM Br- (green) and 130 mM Cl- plus 20 mM SCN- (red) Table 2 Relative permeabilities of some anions in WT-CFTR and R352-CFTR mutants * Significant difference compared with WT-CFTR, P \ 0.05; ** Significant difference compared with R352A, P \ 0.05 CFTR n SCN Br NO3 WT 6 4.11 ± 0.17 1.45 ± 0.04 1.51 ± 0.02 R352A 10 4.18 ± 0.65 1.35 ± 0.21 1.70 ± 0.29 R352E 6 5.18 ± 0.32* 1.47 ± 0.08 1.64 ± 0.43 R352K 7 4.05 ± 0.12 1.52 ± 0.01 1.59 ± 0.03** R352E/D993R 6 3.62 ± 0.06* 1.48 ± 0.04 1.59 ± 0.02** Table 3 Relative conductances of some anions in WT-CFTR and R352-CFTR mutants CFTR n SCN Br NO3 WT 6 0.16 ± 0.02 0.67 ± 0.04 0.84 ± 0.04 R352A 10 1.59 ± 0.12* 1.31 ± 0.08* 1.59 ± 0.14* R352E 6 2.73 ± 0.31*, ** 1.49 ± 0.22* 1.54 ± 0.12* R352K 7 1.12 ± 0.08*, ** 0.99 ± 0.02*, ** 1.73 ± 0.26* R352E/ D993R 7 0.61 ± 0.05*, ** 0.98 ± 0.03*, ** 1.26 ± 0.13* Relative conductance was measured at VM = Vrev -25 mV * Significant difference compared with WT-CFTR, P\0.05; ** Significant difference compared with R352A, P\0.05 that loss of positive charge at position 352 destroyed the overall pore architecture, which subsequently changed the anion selectivity characteristics as seen in R352A- and R352E-CFTR. Login to comment
173 Furthermore, the finding that relative permeability values are nearly identical in R352A-, R352E- and R352K-CFTR suggests that the role of this site in determining anion selectivity is only indirect. Login to comment
182 In contrast, the average fractional block of R352K-CFTR by 200 lM glipizide was not significantly different from the block of WT-CFTR at this concentration (0.52 ± 0.02, n = 6, P = 0.119) (Fig. 6G). Login to comment
183 Figure 6B, D, F, H shows the macroscopic i-V relationships for WT-, R352A-, R347A- and R352K-CFTR in representative experiments, indicating that glipizide blocked the currents primarily at negative membrane potentials in WTand R352K-CFTR. Login to comment
185 Finally, R352A- and R347A-CFTR, but not R352K-CFTR, exhibited outward rectification of macroscopic currents in the absence of blocker, consistent with the outward rectification of single-channel amplitudes (Fig. 4, Table 1) (Cotten and Welsh 1999). Login to comment
187 R352K-CFTR, in contrast, maintained the positive charge and most characteristics of WT-CFTR. This strongly suggests that R352 may serve a critical role in preserving the gross structure of the channel pore, perhaps by contributing to an interfacial pair with a negatively charged amino acid at another position in CFTR. Login to comment
189 100 ms 200 pA 100 ms 2 nA 20 pA 100 ms -100 -600 -400 -200 200 400 600 ATP ATP + Glip 200 50 100 mV -50 pA -100 pA mV 50 100 -60 20 40 ATP + Glip 200 ATP-40 60 -20 -50 mV -100 -50 50 100 -4 -2 2 4 nA ATP ATP + Glip 200 mV -100 -50 50 100 -400 -200 200 400 ATP ATP + Glip 200 pA mV -100 -50 50 100 -400 -200 200 400 ATP ATP + Glip 200 pA 200 pA 100 ms 200 pA 100 ms 100 ms 200 pA 100 ms 200 pA 100 ms 2 nA 100 ms 2 nA 20 pA 100 ms 20 pA 100 ms -100 -600 -400 -200 200 400 600 ATP ATP + Glip 200 50 100 mV -50 pA -600 -400 -200 200 400 600 ATP ATP + Glip 200 50 100 mV -50 pA -100 pA mV 50 100 -60 20 40 ATP + Glip 200 ATP-40 60 -20 -50-100 pA mV 50 100 -60 20 40 ATP + Glip 200 ATP-40 60 -20 -50-100 pA mV 50 100 -60 20 40 ATP + Glip 200 ATP-40 60 -20 -50 mV -100 -50 50 100 -4 -2 2 4 nA ATP ATP + Glip 200 mV -100 -50 50 100 -4 -2 2 4 nA ATP ATP + Glip 200 mV -100 -50 50 100 -400 -200 200 400 ATP ATP + Glip 200 pA mV -100 -50 50 100 -400 -200 200 400 ATP ATP + Glip 200 pA 200 pA 100 ms 200 pA 100 ms R347A-CFTR WT-CFTR R352K-CFTR R352A-CFTR 100 ms 200 pA 100 ms 2 nA 20 pA 100 ms -100 -600 -400 -200 200 400 600 ATP ATP + Glip 200 50 100 mV -50 pA -100 pA mV 50 100 -60 20 40 ATP + Glip 200 ATP-40 60 -20 -50 mV -100 -50 50 100 -4 -2 2 4 nA ATP ATP + Glip 200 mV -100 -50 50 100 -400 -200 200 400 ATP ATP + Glip 200 pA mV -100 -50 50 100 -400 -200 200 400 ATP ATP + Glip 200 pA 200 pA 100 ms 200 pA 100 ms 100 ms 200 pA 100 ms 200 pA 100 ms 2 nA 100 ms 2 nA A B D E F 20 pA 100 ms 20 pA 100 ms -100 -600 -400 -200 200 400 600 ATP ATP + Glip 200 50 100 mV -50 pA -600 -400 -200 200 400 600 ATP ATP + Glip 200 50 100 mV -50 pA G H -100 pA mV 50 100 -60 20 40 ATP + Glip 200 ATP-40 60 -20 -50-100 pA mV 50 100 -60 20 40 ATP + Glip 200 ATP-40 60 -20 -50-100 pA mV 50 100 -60 20 40 ATP + Glip 200 ATP-40 60 -20 -50 mV -100 -50 50 100 -4 -2 2 4 nA ATP ATP + Glip 200 mV -100 -50 50 100 -4 -2 2 4 nA ATP ATP + Glip 200 C mV -100 -50 50 100 -400 -200 200 400 ATP ATP + Glip 200 pA mV -100 -50 50 100 -400 -200 200 400 ATP ATP + Glip 200 pA 200 pA 100 ms 200 pA 100 ms R347A-CFTR WT-CFTR R352K-CFTR R352A-CFTR Fig. 6 Mutations at R352 alter pore pharmacology. Login to comment
190 Left Block of CFTR macropatch currents by glipizide (glip) was time-dependent in WT-CFTR (A) and R352K-CFTR (G) but not in R352A-CFTR (C) or R347A-CFTR (E). Login to comment
192 Right i-V relationships for WT-CFTR (B), R352A-CFTR (D), R347A-CFTR (F) and R352K-CFTR (H) were constructed from voltage ramps performed in the absence (black) and in the presence of 200 lM glipizide (red). Login to comment
202 R352E/D993R-CFTR, in contrast, exhibited stability of the full conductance state similar to that seen in WT-CFTR and R352K-CFTR (Fig. 1); transitions to the s1 and s2 states were rare events in this double mutant. Login to comment
248 In contrast, channels bearing the charge-conserving mutation R352K showed characteristics similar to WT-CFTR. Login to comment
295 Stability of the open state was retained in the case of a charge-conserving mutation, R352K, and in the double mutant R352E/D993R-CFTR. Login to comment
298 The fact that differences in anion selectivity remain between R352K- and WT-CFTR is consistent with the notion that interactions between lysine and the aspartic acid at D993 are not the same as the interactions between arginine and D993. Login to comment
300 We also note that while the relative conductance values for SCN- , Brand NO3 - are shifted in the same direction in R352K-CFTR as they are in R352A- or R352E-CFTR, the shifts for SCN- and Br- are smaller in R352K-CFTR than in the charge-destroying mutants. Login to comment

PMID: 19020075 [PubMed] Jordan IK et al: "Evolutionary and functional divergence between the cystic fibrosis transmembrane conductance regulator and related ATP-binding cassette transporters."

No. Sentence Comment

131 In contrast, approximately wild-type channel behavior is retained in R352K-CFTR and the charge-swapping double mutant, R352E/D993R-CFTR (Fig. 3). Login to comment