ABCMdb

ABCC7 p.Arg334Lys

ClinVar:	c.1000C>T , p.Arg334Trp D , Pathogenic c.1001G>T , p.Arg334Leu ? , not provided c.1001G>A , p.Arg334Gln ? , not provided
CF databases:	c.1000C>T , p.Arg334Trp D , CF-causing ; CFTR1: This mutation has been found in two Spanish CF chromosomes. One of the patients has the [delta]F508 mutation in the other chromosome and the other patient does not. We have not found this mutation on 30 normal chromosomes with the same haplotype, and in 88 CF chromosomes without the [delta]F508, and in 24 with the [delta]F508. The mutation destroys a MapI site and is easily identified by agarose gel electrophoresis after PCR with intron primers. c.1001G>A , p.Arg334Gln (CFTR1) ? , The above mutation was found by DGGE and direct sequencing in Caucasian patients. c.1001G>T , p.Arg334Leu (CFTR1) D , Missense mutation E334L was detected in a German CBAVD patient who is compound heterozygous for the R334L and I336K mutations.
Predicted by SNAP2:	A: D (91%), C: D (95%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (91%), I: D (95%), K: D (85%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (91%), S: D (91%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: N, C: D, D: N, E: N, F: D, G: N, H: N, I: D, K: N, L: N, M: N, N: N, P: N, Q: N, S: N, T: N, V: D, W: D, Y: D,

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[hide] Molecular determinants and role of an anion bindin... J Physiol. 2003 Jun 1;549(Pt 2):387-97. Epub 2003 Apr 4. Gong X, Linsdell P
Molecular determinants and role of an anion binding site in the external mouth of the CFTR chloride channel pore.
J Physiol. 2003 Jun 1;549(Pt 2):387-97. Epub 2003 Apr 4., 2003-06-01 [PMID:12679372]

Abstract [show]
Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is blocked by highly lyotropic permeant anions which bind tightly within the pore. Here we show that several different substitutions of a positively charged amino acid residue, arginine R334, in the putative outer mouth of the CFTR pore, greatly reduce the block caused by lyotropic Au(CN)2- ions applied to the intracellular side of the channel. Fixed positive charge at this site appears to play a role in Au(CN)2- binding, as judged by multiple substitutions of differently charged amino acid side chains and also by the pH dependence of block conferred by the R334H mutant. However, non-charge-dependent effects also appear to contribute to Au(CN)2- binding. Mutation of R334 also disrupts the apparent electrostatic interaction between intracellular Au(CN)2- ions and extracellular permeant anions, an interaction which normally acts to relieve channel block. All six mutations studied at R334 significantly weakened this interaction, suggesting that arginine possesses a unique ability to coordinate ion-ion interactions at this site in the pore. Our results suggest that lyotropic anions bind tightly to a site in the outer mouth of the CFTR pore that involves interaction with a fixed positive charge. Binding to this site is also involved in coordination of multiple permeant anions within the pore, suggesting that anion binding in the outer mouth of the pore is an important aspect in the normal anion permeation mechanism.

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No. Sentence Comment
53 Block of wild-type, R334C-, R334E-, R334H-, R334K-, R334L- and R334Q-CFTR by 100 mM and 1 mM intracellular Au(CN)2 _ are compared in Fig. 4B. Login to comment
54 Block was affected in all mutants, depending on the ionic conditions used, but was particularly weakened in R334C, R334E and R334K (Fig. 5A-C). Login to comment
93 As noted by Smith et al. (2001), this effect was clearly charge dependent, being strongest in R334E and weak (but still significant) in R334K. Login to comment
119 R334K greatly decreased Au(CN)2 _ affinity (Figs B and 5), increasing Kd(0) 4-7-fold under conditions of high extracellular Cl_ (Fig. 5A), and 14-24-fold with the impermeant gluconate ion in the extracellular solution (Fig. 5B), conditions under which Au(CN)2 _ binding is studied in relative isolation from interactions with other anions. Login to comment
141 With either Cl_ or gluconate in the extracellular solution, Au(CN)2 _ block was most dramatically weakened in the mutants R334C, R334E and R334K, which involve replacement of the positively charged arginine side chain with one neutral side chain (cysteine), one negatively charged side chain (glutamate) and one positively charged side chain Anion binding site in the CFTR pore outer mouthJ Physiol 549.2 395 (lysine). Login to comment

[hide] Direct comparison of the functional roles played b... J Biol Chem. 2004 Dec 31;279(53):55283-9. Epub 2004 Oct 25. Ge N, Muise CN, Gong X, Linsdell P
Direct comparison of the functional roles played by different transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Biol Chem. 2004 Dec 31;279(53):55283-9. Epub 2004 Oct 25., 2004-12-31 [PMID:15504721]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel contains 12 transmembrane (TM) regions that are presumed to form the channel pore. However, little is known about the relative functional contribution of different TM regions to the pore. We have used patch clamp recording to investigate the functional consequences of point mutations throughout the six transmembrane regions in the N-terminal part of the CFTR protein (TM1-TM6). A range of specific functional assays compared the single channel conductance, anion binding, and anion selectivity properties of different channel variants. Overall, our results suggest that TM1 and -6 play dominant roles in forming the channel pore and determining its functional properties, with TM5 perhaps playing a lesser role. In contrast, TM2, -3, and -4 appear to play only minor supporting roles. These results define transmembrane regions 1 and 6 as major contributors to the CFTR channel pore and have strong implications for emerging structural models of CFTR and related ATP-binding cassette proteins.

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No. Sentence Comment
45 Previously we showed that the TM6 mutant R334A did not express but characterized several other Arg-334 mutants (10, 28); in the present study we have used the charge conservative R334K mutant. Login to comment
76 However, the unitary conductance was drastically reduced by some mutations in TM1 (K95Q, Q98A, P99A) and TM6 (R334K, F337A) (Figs. 2-4). Login to comment
82 In most cases macroscopic I-V relationships were linear or weakly inwardly rectifying in the presence of symmetrical high Cl- concentra- tions (as quantified in Fig. 6), although particularly strong inward rectification was observed in Q98A, P99A, and R334K. Login to comment
102 The weakest block by Au(CN)2 - was observed in K95Q, T338A, R334K, and Q98A, consistent with these residues perhaps being associated with permeant anion binding sites inside the pore. Login to comment
119 Some mutants that significantly affect both unitary conductance and Au(CN)2 - block were found to be without effect on SCN- permeability (Q98A, V318A, R334K, K335A). Login to comment

[hide] Direct and indirect effects of mutations at the ou... J Membr Biol. 2007 Apr;216(2-3):129-42. Epub 2007 Aug 3. Zhou JJ, Fatehi M, Linsdell P
Direct and indirect effects of mutations at the outer mouth of the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Membr Biol. 2007 Apr;216(2-3):129-42. Epub 2007 Aug 3., [PMID:17673962]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel pore is thought to contain multiple binding sites for permeant and impermeant anions. Here, we investigate the effects of mutation of different positively charged residues in the pore on current inhibition by impermeant Pt(NO(2)) (4) (2-) and suramin anions. We show that mutations that remove positive charges (K95, R303) influence interactions with intracellular, but not extracellular, Pt(NO(2))(4)(2-) ions, consistent with these residues being situated within the pore inner vestibule. In contrast, mutation of R334, supposedly located in the outer vestibule of the pore, affects block by both extracellular and intracellular Pt(NO(2))(4)(2-). Inhibition by extracellular Pt(NO(2))(4)(2-) requires a positive charge at position 334, consistent with a direct electrostatic interaction resulting in either open channel block or surface charge screening. In contrast, inhibition by intracellular Pt(NO(2))(4)(2-) is weakened in all R334-mutant forms of the channel studied, inconsistent with a direct interaction. Furthermore, mutation of R334 had similar effects on block by intracellular suramin, a large organic molecule that is apparently unable to enter deeply into the channel pore. Mutation of R334 altered interactions between intracellular Pt(NO(2))(4)(2-) and extracellular Cl(-) but not those between intracellular Pt(NO(2))(4)(2-) and extracellular Pt(NO(2))(4)(2-). We propose that while the positive charge of R334 interacts directly with extracellular anions, mutation of this residue also alters interactions with intracellular anions by an indirect mechanism, due to mutation-induced conformational changes in the protein that are propagated some distance from the site of the mutation in the outer mouth of the pore.

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23 However, all mutations studied, including the charge-conservative R334K, lead to significant weakening of Au(CN)2 À binding (Gong & Linsdell, 2003a), which is inconsistent with an electrostatic interaction. Login to comment
28 However, single-channel recording showed that all mutations at this site, including the charge-conservative R334K, lead to a dramatic decrease in the amplitude of unitary currents carried by ClÀ efflux through the pore (Gong & Linsdell, 2004). Login to comment
85 Figure 3 shows the blocking effects of internally applied Pt(NO2)4 2À in six different channel mutants (R334C, R334E, R334H, R334K, R334L, R334Q) under conditions of both low (Fig. 3a) and high (Fig. 3b) extracellular ClÀ concentration. Login to comment
91 With elevated extracellular ClÀ , the Kd(0) was significantly increased only in R334C and R334E; not significantly altered in R334K, R334L and R334Q; and significantly decreased in R334H (Fig. 5b). Login to comment
106 Comparison of the mean Kd estimated for suramin (at 0 mV) shows that R334C, R334E, R334K, R334L and R334Q were all associated with weakened suramin block, with only R334H failing to significantly affect suramin block (Fig. 7). Login to comment
129 Mean of data from three to eight patches. Fitted lines are to equation 1 as described in Figure 1 for wild type and R334Q and with the following parameters for other channel variants: R334C 4 mM external ClÀ , Kd(0) = 1362 lM, zd = À0.295; R334C 154 mM external ClÀ , Kd(0) = 836 lM, zd = À0.219; R334E 4 mM external ClÀ , Kd(0) = 759 lM, zd = À0.376; R334E 154 mM external ClÀ , Kd(0) = 564 lM, zd = À0.173; R334H 4 mM external ClÀ , Kd(0) = 140 lM, zd = À0.166; R334H 154 mM external ClÀ , Kd(0) = 119 lM, zd = À0.149; R334K 4 mM external ClÀ , Kd(0) = 143 lM, zd = À0.314; R334K 154 mM external ClÀ , Kd(0) = 317 lM, zd = À0.374; R334L 4 mM external ClÀ , Kd(0) = 176 lM, zd = À0.258; R334L 154 mM external ClÀ , Kd(0) = 284 lM, zd = À0.366 extracellular Pt(NO2)4 2À by normalizing current amplitude at the hyperpolarized extreme of the voltage range studied, -80 mV (Fig. 10b). Login to comment
139 However, it can be seen that for all R334 mutations except the charge-conservative R334K, current-voltage relationship shape was not strongly altered by the presence of Pt(NO2)4 2À in the extracellular solution. Login to comment
142 Considering only the data at +80 mV (Fig. 11d), where block of wild-type CFTR is strongest (Figs. 9, 10), the blocking effects of Pt(NO2)4 2À are slightly (but significantly) weakened in K95Q, R303Q and R334K (p < 0.05) but practically abolished in all other R334 mutants (p < 0.0005). Login to comment
143 In fact, only wild type, R334K, K95Q and R303Q - those mutants that retain a positive charge at position 334 - were significantly affected by 10 mM Pt(NO2)4 2À according to this analysis (as illustrated by the daggers in Fig. 11d, p < 0.001), whereas all mutants associated with removal of the positive charge at R334 showed no significant differences in the absence or presence of external Pt(NO2)4 2À (p > 0.15). Login to comment
159 These plots represent mean data from four to seven patches. Fitted lines are to equation 1 with the following parameters: wild type, Kd(0) = 2.51 lM, zd = À0.042; R334C, Kd(0) = 18.5 lM, zd = À0.056; R334E, Kd(0) = 25.0 lM, zd = À0.107; R334H, Kd(0) = 3.10 lM, zd = À0.085; R334K, Kd(0) = 6.31 lM, zd = À0.232; R334L, Kd(0) = 4.08 lM, zd = À0.061; R334Q, Kd(0) = 6.64 lM, zd = À0.239 with our previous suggestion that intracellular Au(CN)2 À blocks the channel by interacting directly with R334, several reasons prompt us to suggest that Pt(NO2)4 2À does not interact directly with the arginine side chain at this position. Login to comment
161 Second, Pt(NO2)4 2À block is significantly weakened in all of the six R334-mutated variants studied, including the charge-conservative R334K (Fig. 5a). Login to comment
185 In contrast, the charge-conservative R334K mutant showed only very slightly weakened block by external Pt(NO2)4 2À (Fig. 11d). Login to comment
200 Our results are also entirely consistent with extracellular Pt(NO2)4 2À ions screening the surface charge contributed by R334 (or by the substituted lysine in R334K), resulting in loss of electrostatic attractive forces on extracellular ClÀ ions and reduced ClÀ entry into the pore at depolarized voltages. Login to comment
218 As a result, these direct effects show a strong dependence on side chain charge, being strongly disrupted in all mutants except R334K. Login to comment
226 Note the change in current rectification induced by extracellular Pt(NO2)4 2À in wild type, R334K, K95Q and R303Q but not other R334 mutants. Login to comment
228 ), R334E (5), R334H (j), R334K (), R334L (h), R334Q (u); c wild type (d), K95Q (m), R303Q (Å). Login to comment

[hide] Maximization of the rate of chloride conduction in... Arch Biochem Biophys. 2004 Jun 1;426(1):78-82. Gong X, Linsdell P
Maximization of the rate of chloride conduction in the CFTR channel pore by ion-ion interactions.
Arch Biochem Biophys. 2004 Jun 1;426(1):78-82., [PMID:15130785]

Abstract [show]
Multi-ion pore behaviour has been identified in many Cl(-) channel types but its biophysical significance is uncertain. Here, we show that mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel that disrupt anion-anion interactions within the pore are associated with drastically reduced single channel conductance. These results are consistent with models suggesting that rapid Cl(-) permeation in CFTR results from repulsive ion-ion interactions between Cl(-) ions bound concurrently inside the pore. Naturally occurring mutations that disrupt these interactions can result in cystic fibrosis.

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No. Sentence Comment
35 Results and discussion Previously we characterized the properties of six different R334 mutants (R334C, R334E, R334H, R334K, R334L, and R334Q) [19]. Login to comment
43 Each of these traces shows the activity of two active CFTR channels, except for R334K and R334W (one active channel). Login to comment
50 Comparison of unitary current amplitude at )100 mV showed that, compared to wild type, mean current was reduced by between 53 (in R334K) and 91% (in R334H) (Fig. 2B). Login to comment
65 (A) Unitary current-voltage relationships for each of the channel variants shown in Fig. 1: (d) wild type, (r) R334C, (j) R334E, (}) R334H, (s) R334K, () R334L, (.) Login to comment