ABCMdb

ABCC7 p.Arg352Lys

ClinVar:	c.1054C>T , p.Arg352Trp D , Likely pathogenic c.1055G>A , p.Arg352Gln D , Pathogenic
CF databases:	c.1055G>A , p.Arg352Gln D , CF-causing ; CFTR1: This missense mutation, at nucleotide position 1187 (G to A) in exon 7, has been detected in an Italian CF patient through DGGE and direct sequencing. The mutation generates an Arg to Gln substitution (R352Q) and creates a novel DdeI restriction site in the mutated allele. This mutation has been detected in a PS patient (paternal chromosome), associated with the haplotype A; the maternal chromosome carries a still uncharacterized mutation. It was found in one of 60 non-[delta] Italian CF chromosomes. c.1054C>G , p.Arg352Gly (CFTR1) ? , c.1054C>T , p.Arg352Trp (CFTR1) ? , The mutation was detected by SSCP/heteroduplex analysis and identified by direct DNA sequencing. The mutation was seen in a boy referred by West Midlands Regional Genetics Service, and whose other CF mutation was [delta]F508. We have seen it only once in over 150 samples tested.
Predicted by SNAP2:	A: D (91%), C: D (95%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (91%), K: D (85%), L: D (91%), M: D (95%), N: D (95%), P: D (95%), Q: D (59%), S: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, S: N, T: N, V: N, W: N, Y: N,

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Publications
[hide] Positive charges at the intracellular mouth of the... J Gen Physiol. 2006 Nov;128(5):535-45. Epub 2006 Oct 16. Aubin CN, Linsdell P
Positive charges at the intracellular mouth of the pore regulate anion conduction in the CFTR chloride channel.
J Gen Physiol. 2006 Nov;128(5):535-45. Epub 2006 Oct 16., [PMID:17043152]

Abstract [show]
Many different ion channel pores are thought to have charged amino acid residues clustered around their entrances. The so-called surface charges contributed by these residues can play important roles in attracting oppositely charged ions from the bulk solution on one side of the membrane, increasing effective local counterion concentration and favoring rapid ion movement through the channel. Here we use site-directed mutagenesis to identify arginine residues contributing important surface charges in the intracellular mouth of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel pore. While wild-type CFTR was associated with a linear current-voltage relationship with symmetrical solutions, strong outward rectification was observed after mutagenesis of two arginine residues (R303 and R352) located near the intracellular ends of the fifth and sixth transmembrane regions. Current rectification was dependent on the charge present at these positions, consistent with an electrostatic effect. Furthermore, mutagenesis-induced rectification was more pronounced at lower Cl(-) concentrations, suggesting that these mutants had a reduced ability to concentrate Cl(-) ions near the inner pore mouth. R303 and R352 mutants exhibited reduced single channel conductance, especially at negative membrane potentials, that was dependent on the charge of the amino acid residue present at these positions. However, the very low conductance of both R303E and R352E-CFTR could be greatly increased by elevating intracellular Cl(-) concentration. Modification of an introduced cysteine residue at position 303 by charged methanethiosulfonate reagents reproduced charge-dependent effects on current rectification. Mutagenesis of arginine residues in the second and tenth transmembrane regions also altered channel permeation properties, however these effects were not consistent with changes in channel surface charges. These results suggest that positively charged arginine residues act to concentrate Cl(-) ions at the inner mouth of the CFTR pore, and that this contributes to maximization of the rate of Cl(-) ion permeation through the pore.

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

[hide] Mutations at arginine 352 alter the pore architect... J Membr Biol. 2008 Mar;222(2):91-106. Epub 2008 Apr 18. Cui G, Zhang ZR, O'Brien AR, Song B, McCarty NA
Mutations at arginine 352 alter the pore architecture of CFTR.
J Membr Biol. 2008 Mar;222(2):91-106. Epub 2008 Apr 18., [PMID:18421494]

Abstract [show]
Arginine 352 (R352) in the sixth transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) previously was reported to form an anion/cation selectivity filter and to provide positive charge in the intracellular vestibule. However, mutations at this site have nonspecific effects, such as inducing susceptibility of endogenous cysteines to chemical modification. We hypothesized that R352 stabilizes channel structure and that charge-destroying mutations at this site disrupt pore architecture, with multiple consequences. We tested the effects of mutations at R352 on conductance, anion selectivity and block by the sulfonylurea drug glipizide, using recordings of wild-type and mutant channels. Charge-altering mutations at R352 destabilized the open state and altered both selectivity and block. In contrast, R352K-CFTR was similar to wild-type. Full conductance state amplitude was similar to that of wild-type CFTR in all mutants except R352E, suggesting that R352 does not itself form an anion coordination site. In an attempt to identify an acidic residue that may interact with R352, we found that permeation properties were similarly affected by charge-reversing mutations at D993. Wild-type-like properties were rescued in R352E/D993R-CFTR, suggesting that R352 and D993 in the wild-type channel may interact to stabilize pore architecture. Finally, R352A-CFTR was sensitive to modification by externally applied MTSEA+, while wild-type and R352E/D993R-CFTR were not. These data suggest that R352 plays an important structural role in CFTR, perhaps reflecting its involvement in forming a salt bridge with residue D993.

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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

[hide] Evolutionary and functional divergence between the... Proc Natl Acad Sci U S A. 2008 Dec 2;105(48):18865-70. Epub 2008 Nov 19. Jordan IK, Kota KC, Cui G, Thompson CH, McCarty NA
Evolutionary and functional divergence between the cystic fibrosis transmembrane conductance regulator and related ATP-binding cassette transporters.
Proc Natl Acad Sci U S A. 2008 Dec 2;105(48):18865-70. Epub 2008 Nov 19., 2008-12-02 [PMID:19020075]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily, an ancient family of proteins found in all phyla. In nearly all cases, ABC proteins are transporters that couple the hydrolysis of ATP to the transmembrane movement of substrate via an alternating access mechanism. In contrast, CFTR is best known for its activity as an ATP-dependent chloride channel. We asked why CFTR, which shares the domain architecture of ABC proteins that function as transporters, exhibits functional divergence. We compared CFTR protein sequences to those of other ABC transporters, which identified the ABCC4 proteins as the closest mammalian paralogs, and used statistical analysis of the CFTR-ABCC4 multiple sequence alignment to identify the specific domains and residues most likely to be involved in the evolutionary transition from transporter to channel activity. Among the residues identified as being involved in CFTR functional divergence, by virtue of being both CFTR-specific and conserved among all CFTR orthologs, was R352 in the sixth transmembrane helix (TM6). Patch-clamp experiments show that R352 interacts with D993 in TM9 to stabilize the open-channel state; D993 is absolutely conserved between CFTRs and ABCC4s. These data suggest that CFTR channel activity evolved, at least in part, by converting the conformational changes associated with binding and hydrolysis of ATP, as are found in true ABC Transporters, into an open permeation pathway by means of intraprotein interactions that stabilize the open state. This analysis sets the stage for understanding the evolutionary and functional relationships that make CFTR a unique ABC transporter protein.

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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