ABCMdb

ABCC7 p.Gly551Cys

ClinVar:	c.1651G>A , p.Gly551Ser D , Pathogenic c.1652G>A , p.Gly551Asp D , Pathogenic
CF databases:	c.1652G>A , p.Gly551Asp D , CF-causing ; CFTR1: This mutation has been found in six Caucasian CF chromosomes out of 155 eamined for a frequency of 4 %. It has not been found on any Black CF chromosomes. This mutation appears to be associated with a particular ten site haplotype shown on the following pages. We have not detected this mutation on any normal Caucasian chromosomes with similar haplotypes or other haplotypes. c.1651G>A , p.Gly551Ser D , CF-causing ; CFTR1: This mutation can be detected using ASOs: normal 5' GAGTGGAGGTCAACG 3', mutant 5' GAGTGGAAGTCAACG 3' with a final wash at 42 degrees celsius in 40 mM NaHPO4, 1 mM EDTA, 0.5 % SDS for 15 minutes. Two patients were found to be homozygous for this mutation. Their parents are second cousins and each carries the G551S mutation. These patients are remarkable in that they have a mild disease without elevated Na+ levels. One patient had decreased lung function, Pseudomonas infections, chronic pancreatitis, clubbing, and is currently 49 years old. This mutation was not found in 363 non-[delta]F508 CF chromosomes, nor in over 700[delta]F508 chromosomes, nor in a small number of normal chromosomes.
Predicted by SNAP2:	A: D (95%), C: D (95%), D: D (71%), E: D (95%), F: D (95%), H: D (95%), I: D (95%), K: D (95%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: N (61%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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Publications
[hide] Mutations at the signature sequence of CFTR create... J Gen Physiol. 2009 Jan;133(1):69-77. Wang X, Bompadre SG, Li M, Hwang TC
Mutations at the signature sequence of CFTR create a Cd(2+)-gated chloride channel.
J Gen Physiol. 2009 Jan;133(1):69-77., [PMID:19114635]

Abstract [show]
The canonical sequence LSGGQ, also known as the signature sequence, defines the adenosine triphosphate (ATP)-binding cassette transporter superfamily. Crystallographic studies reveal that the signature sequence, together with the Walker A and Walker B motifs, forms the ATP-binding pocket upon dimerization of the two nucleotide-binding domains (NBDs) in a head-to-tail configuration. The importance of the signature sequence is attested by the fact that a glycine to aspartate mutation (i.e., G551D) in cystic fibrosis transmembrane conductance regulator (CFTR) results in a severe phenotype of cystic fibrosis. We previously showed that the G551D mutation completely eliminates ATP-dependent gating of the CFTR chloride channel. Here, we report that micromolar [Cd(2+)] can dramatically increase the activity of G551D-CFTR in the absence of ATP. This effect of Cd(2+) is not seen in wild-type channels or in G551A. Pretreatment of G551D-CFTR with the cysteine modification reagent 2-aminoethyl methane thiosulfonate hydrobromide protects the channel from Cd(2+) activation, suggesting an involvement of endogenous cysteine residue(s) in mediating this effect of Cd(2+). The mutants G551C, L548C, and S549C, all in the signature sequence of CFTR's NBD1, show robust response to Cd(2+). On the other hand, negligible effects of Cd(2+) were seen with T547C, Q552C, and R553C, indicating that a specific region of the signature sequence is involved in transmitting the signal of Cd(2+) binding to the gate. Collectively, these results suggest that the effect of Cd(2+) is mediated by a metal bridge formation between yet to be identified cysteine residue(s) and the engineered aspartate or cysteine in the signature sequence. We propose that the signature sequence serves as a switch that transduces the signal of ligand binding to the channel gate.

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No. Sentence Comment
22 The mutants G551C, L548C, and S549C, all in the signature sequence of CFTR`s NBD1, show robust response to Cd2+ . Login to comment
49 For the G551C and S549C mutants, because they are ATP dependent, we used the current in the presence of 1 mM ATP as control. Login to comment
55 The apparent affinity for Cd2+ was further increased when glycine 551 was converted to cysteine, but the effect of Cd2+ was mostly abolished when the G551 residue was substituted by an alanine. Login to comment
94 On the other hand, 10 μM Cd2+ already generates a maximal response for G551C-CFTR, with a maximal fold increase of 7.4 ± 0.3 (n = 5) compared with the currents generated by 1 mM ATP. Fitting the dose-response relationships with the Hill equation yields a K1/2 of 14.6 ± 6.3 μM and 3.29 ± 0.66 μM for G551D and G551C, respectively. Login to comment
96 Like the G551C mutant, G551A-CFTR remains responsive to ATP. Login to comment
97 However, the effect of Cd2+ on G551A-CFTR is negligibly small compared with that of G551C-CFTR (Fig. 5 A vs. Fig. 4 B). Login to comment
98 This difference between G551A and G551C was quantified in Fig. 5 B, where we compared the current generated by 1 mM ATP with the current generated by 10 μM Cd2+ for these two mutants. Login to comment
107 However, for L548C, S549C, and G551C, the specificity of the ligand is altered so that Cd2+ becomes more effective at gating Cd2+ Is More Potent on G551C than on G551D We considered two possible mechanisms for the effect of Cd2+ on G551D-CFTR. Login to comment
111 To differentiate these two possibilities, we first mutated the glycine at position 551 to cysteine. Login to comment
113 Fig. 4 shows representative traces of G551D (A) and G551C (B) in the presence of different [Cd2+ ]. Login to comment
114 Note that 5 μM Cd2+ induces a higher G551C-CFTR current than 1 mM ATP, despite that this mutation retains responsiveness to ATP. Login to comment
132 The Binding Partner of Cd2+ Is Likely To Be a Cysteine Residue The micromolar affinity of Cd2+ in activating G551C or S549C mutants raises the possibility that some endogenous cysteine(s) or histidine(s) may participate in form- Figure 4. Login to comment
133 Representative current traces of G551D-CFTR (A) and G551C-CFTR (B) in the presence of different [Cd2+ ]. Login to comment
134 The Cd2+ dose-response relationships for G551D-CFTR (C) and G551C-CFTR (D) were fitted with the Hill equation, y = min + (max-min)/ [1+ (K1/2/[x])n )] (smooth curves). Login to comment
135 The fold increase of the current in the presence of Cd2+ was normalized to the maximal fold increase for each mutant (G551D: 21.38 ± 4.19-fold, 100 μM Cd2+ ; G551C: 7.4 ± 0.3, 10 μM Cd2+ ). Login to comment
136 G551D: K1/2 = 14.6 ± 6.3 μM and n = 1.03 ± 0.34; G551C: K1/2 = 3.29 ± 0.66 μM and n = 1.89 ± 0.52. Login to comment
137 Figure 5. Comparison of Cd2+ and ATP-induced currents between G551A and G551C mutants. Login to comment
140 (B) The ratio of currents induced by 10 μM Cd2+ and those with 1 mM ATP for G551C-CFTR and G551A-CFTR. Login to comment
144 For G551C-CFTR, this effect of Cd2+ on the opening rate likely also occurs. Login to comment
145 The open time for G551C-CFTR in the absence of ATP is 202.6 ± 29.6 ms (n = 3). Login to comment
148 D I S C U S S I O N Here, we show that micromolar concentrations of Cd2+ can dramatically increase the activity of G551D-CFTR, a disease-associated mutant, as well as G551C-CFTR, L548C, and S549C-CFTR, in the absence of ATP. Login to comment
216 First, the apparent affinities for G551C and S549C are at low micromolar range, supporting the idea that multiple cysteines are involved in coordinating Cd2+ . Login to comment

[hide] Gating of the CFTR Cl- channel by ATP-driven nucle... J Physiol. 2009 May 15;587(Pt 10):2151-61. Epub 2009 Mar 30. Hwang TC, Sheppard DN
Gating of the CFTR Cl- channel by ATP-driven nucleotide-binding domain dimerisation.
J Physiol. 2009 May 15;587(Pt 10):2151-61. Epub 2009 Mar 30., 2009-05-15 [PMID:19332488]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) plays a fundamental role in fluid and electrolyte transport across epithelial tissues. Based on its structure, function and regulation, CFTR is an ATP-binding cassette (ABC) transporter. These transporters are assembled from two membrane-spanning domains (MSDs) and two nucleotide-binding domains (NBDs). In the vast majority of ABC transporters, the NBDs form a common engine that utilises the energy of ATP hydrolysis to pump a wide spectrum of substrates through diverse transmembrane pathways formed by the MSDs. By contrast, in CFTR the MSDs form a pathway for passive anion flow that is gated by cycles of ATP binding and hydrolysis by the NBDs. Here, we consider how the interaction of ATP with two ATP-binding sites, formed by the NBDs, powers conformational changes in CFTR structure to gate the channel pore. We explore how conserved sequences from both NBDs form ATP-binding sites at the interface of an NBD dimer and highlight the distinct roles that each binding site plays during the gating cycle. Knowledge of how ATP gates the CFTR Cl- channel is critical for understanding CFTR's physiological role, its malfunction in disease and the mechanism of action of small molecules that modulate CFTR channel gating.

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No. Sentence Comment
119 Recently, Wang et al. (2009) demonstrated that cadmium (Cd2+ ) can act as a ligand to gate G551Dand G551C-CFTR by serving as a metal bridge connecting G551D/C to an unknown cysteine residue in CFTR. Login to comment