ABCMdb

ABCC7 p.Cys832Ala

ClinVar:	c.2496C>A , p.Cys832* ? , not provided
Predicted by SNAP2:	A: N (82%), D: N (57%), E: N (61%), F: N (72%), G: N (66%), H: N (78%), I: N (72%), K: N (66%), L: N (72%), M: N (66%), N: N (78%), P: N (53%), Q: N (78%), R: N (66%), S: N (78%), T: N (78%), V: N (87%), W: N (66%), Y: N (72%),
Predicted by PROVEAN:	A: N, D: D, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: D, P: D, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N,

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Publications
[hide] Normal gating of CFTR requires ATP binding to both... Proc Natl Acad Sci U S A. 2005 Jan 11;102(2):455-60. Epub 2004 Dec 27. Berger AL, Ikuma M, Welsh MJ
Normal gating of CFTR requires ATP binding to both nucleotide-binding domains and hydrolysis at the second nucleotide-binding domain.
Proc Natl Acad Sci U S A. 2005 Jan 11;102(2):455-60. Epub 2004 Dec 27., 2005-01-11 [PMID:15623556]

Abstract [show]
ATP interacts with the two nucleotide-binding domains (NBDs) of CFTR to control gating. However, it is unclear whether gating involves ATP binding alone, or also involves hydrolysis at each NBD. We introduced phenylalanine residues into nonconserved positions of each NBD Walker A motif to sterically prevent ATP binding. These mutations blocked [alpha-(32)P]8-N(3)-ATP labeling of the mutated NBD and reduced channel opening rate without changing burst duration. Introducing cysteine residues at these positions and modifying with N-ethylmaleimide produced the same gating behavior. These results indicate that normal gating requires ATP binding to both NBDs, but ATP interaction with one NBD is sufficient to support some activity. We also studied mutations of the conserved Walker A lysine residues (K464A and K1250A) that prevent hydrolysis. By combining substitutions that block ATP binding with Walker A lysine mutations, we could differentiate the role of ATP binding vs. hydrolysis at each NBD. The K1250A mutation prolonged burst duration; however, blocking ATP binding prevented the long bursts. These data indicate that ATP binding to NBD2 allowed channel opening and that closing was delayed in the absence of hydrolysis. The corresponding NBD1 mutations showed relatively little effect of preventing ATP hydrolysis but a large inhibition of blocking ATP binding. These data suggest that ATP binding to NBD1 is required for normal activity but that hydrolysis has little effect. Our results suggest that both NBDs contribute to channel gating, NBD1 binds ATP but supports little hydrolysis, and ATP binding and hydrolysis at NBD2 are key for normal gating.

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47 Cys insertion and the ''WT control`` (Fig. 5) also contained the C832A substitution, which prevents NEM modi- Abbreviations: NBD, nucleotide-binding domain; NEM, N-ethylmaleimide. Login to comment

[hide] ATP-independent CFTR channel gating and allosteric... Proc Natl Acad Sci U S A. 2010 Feb 23;107(8):3888-93. Epub 2010 Feb 3. Wang W, Wu J, Bernard K, Li G, Wang G, Bevensee MO, Kirk KL
ATP-independent CFTR channel gating and allosteric modulation by phosphorylation.
Proc Natl Acad Sci U S A. 2010 Feb 23;107(8):3888-93. Epub 2010 Feb 3., 2010-02-23 [PMID:20133716]

Abstract [show]
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel, an ATP binding cassette (ABC) transporter. CFTR gating is linked to ATP binding and dimerization of its two nucleotide binding domains (NBDs). Channel activation also requires phosphorylation of the R domain by poorly understood mechanisms. Unlike conventional ligand-gated channels, CFTR is an ATPase for which ligand (ATP) release typically involves nucleotide hydrolysis. The extent to which CFTR gating conforms to classic allosteric schemes of ligand activation is unclear. Here, we describe point mutations in the CFTR cytosolic loops that markedly increase ATP-independent (constitutive) channel activity. This finding is consistent with an allosteric gating mechanism in which ligand shifts the equilibrium between inactive and active states but is not essential for channel opening. Constitutive mutations mapped to the putative symmetry axis of CFTR based on the crystal structures of related ABC transporters, a common theme for activating mutations in ligand-gated channels. Furthermore, the ATP sensitivity of channel activation was strongly enhanced by these constitutive mutations, as predicted for an allosteric mechanism (reciprocity between protein activation and ligand occupancy). Introducing constitutive mutations into CFTR channels that cannot open in response to ATP (i.e., the G551D CF mutant and an NBD2-deletion mutant) substantially rescued their activities. Importantly, constitutive mutants that opened without ATP or NBD2 still required R domain phosphorylation for optimal activity. Our results confirm that (i) CFTR gating exhibits features of protein allostery that are shared with conventional ligand-gated channels and (ii) the R domain modulates CFTR activity independent of ATP-induced NBD dimerization.

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211 For the MTS (methanethiosulfonate) experiments (Fig. S2), the cysteine substitutions (e.g., K978C) were introduced into the C832A background because modification of C832 can affect CFTR currents (34). Login to comment

[hide] State-dependent regulation of cystic fibrosis tran... J Biol Chem. 2010 Dec 24;285(52):40438-47. Epub 2010 Oct 15. Wang G
State-dependent regulation of cystic fibrosis transmembrane conductance regulator (CFTR) gating by a high affinity Fe3+ bridge between the regulatory domain and cytoplasmic loop 3.
J Biol Chem. 2010 Dec 24;285(52):40438-47. Epub 2010 Oct 15., 2010-12-24 [PMID:20952391]

Abstract [show]
The unique regulatory (R) domain differentiates the human CFTR channel from other ATP-binding cassette transporters and exerts multiple effects on channel function. However, the underlying mechanisms are unclear. Here, an intracellular high affinity (2.3 x 10(-19) M) Fe(3+) bridge is reported as a novel approach to regulating channel gating. It inhibited CFTR activity by primarily reducing an open probability and an opening rate, and inhibition was reversed by EDTA and phenanthroline. His-950, His-954, Cys-832, His-775, and Asp-836 were found essential for inhibition and phosphorylated Ser-768 may enhance Fe(3+) binding. More importantly, inhibition by Fe(3+) was state-dependent. Sensitivity to Fe(3+) was reduced when the channel was locked in an open state by AMP-PNP. Similarly, a K978C mutation from cytoplasmic loop 3 (CL3), which promotes ATP-independent channel opening, greatly weakened inhibition by Fe(3+) no matter whether NBD2 was present or not. Therefore, although ATP binding-induced dimerization of NBD1-NBD2 is required for channel gating, regulation of CFTR activity by Fe(3+) may involve an interaction between the R domain and CL3. These findings may support proximity of the R domain to the cytoplasmic loops. They also suggest that Fe(3+) homeostasis may play a critical role in regulating pathophysiological CFTR activity because dysregulation of this protein causes cystic fibrosis, secretary diarrhea, and infertility.

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No. Sentence Comment
123 NEM doubled hCFTR activity (Fig. 3A), but C832A clearly stopped potentiation (Fig. 3B). Login to comment
128 Fig. 3, E and F, further supports this proposal because C832A weakened inhibition by Fe3ϩ . Login to comment
129 This weak binding affinity seen with C832A was not due to a high open probability because the C832A mutant channel was further activated by curcumin dramatically (Fig. 3E). Login to comment
145 In support of this proposal, H950A/H954A and D836A/C832A/ H774A completely prevented Fe3ϩ inhibition, which was reversed by EDTA (Fig. 4E). Login to comment
170 Macroscopic currents across inside-out membrane patches excised from transfected HEK-293T cells expressing the hCFTR (A and C) and the C832A mutant (B and E). Login to comment
171 Effects of NEM were tested on the hCFTR channel (A and C) and the C832A mutant (B). Login to comment
174 E, effect of Fe3ϩ on the C832A mutant. Login to comment
175 F, fractional Fe3ϩ inhibition of the current for WT and C832A (n ϭ 4-9). Login to comment

[hide] Covalent modification of the nucleotide binding do... J Biol Chem. 1998 Nov 27;273(48):31873-9. Cotten JF, Welsh MJ
Covalent modification of the nucleotide binding domains of cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 1998 Nov 27;273(48):31873-9., 1998-11-27 [PMID:9822656]

Abstract [show]
The cytosolic nucleotide binding domains of cystic fibrosis transmembrane conductance regulator (NBD1 and NBD2) mediate ATP-dependent opening and closing of the Cl- channel pore. To learn more about NBD structure and function, we introduced a cysteine residue into the Walker A motif or the LSGGQ motif of each NBD and examined modification by N-ethylmaleimide (NEM). Covalent modification of either Walker A motif partially inhibited cystic fibrosis transmembrane conductance regulator channel activity, decreasing the open state probability by prolonging the long closed duration. An increase in cytosolic ATP concentration slowed the rate of modification. The data suggest that both NBDs interact with ATP to influence channel opening and that inhibition by NEM modification was in part due to decreased ATP binding. When cysteine was placed in the NBD2 Walker A motif, it was modified more rapidly than when it was placed in NBD1, suggesting that the NBDs are not structurally or functionally identical. Modification of a cysteine inserted in the LSGGQ motif of either NBD1 or NBD2 also inhibited channel activity. The rate of modification was comparable with that of a thiol in free solution, suggesting that the LSGGQ motif resides in a surface-exposed position in both NBDs.

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No. Sentence Comment
37 1 The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; NEM, N-ethylmaleimide; PKA, catalytic subunit of cAMP-dependent protein kinase; ABC, ATP-binding cassette; NBD, nucleotide binding domain; NBD1-Cys, A462C/C832A; NBD2-Cys, C832A/S1248C; TB, mean burst duration; g, single channel conductance; ␶cs, slow, long closed time interval; ␶o, open time interval; ␶, time constant for rate of NEM modification; Iϱ, percentage of current remaining following complete NEM modification; TES, N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid. Login to comment
73 NEM inhibits NBD1-Cys (CFTR-A462C/C832A) and NBD2-Cys(CFTR-S1248C/C832A) channel activity in an ATP-dependent manner. A, CFTR-C832A; B, CFTR-NBD1-Cys; C, CFTR-NBD2-Cys. Login to comment
85 Both NBD1-Cys and NBD2-Cys mutants also contain the C832A mutation. Login to comment
87 To determine the functional consequences of NEM modification of the NBDs, we applied 100 ␮M NEM to the cytoplasmic side of excised membrane macropatches containing CFTR-C832A (control), NBD1-Cys, or NBD2-Cys mutant channels. Login to comment
88 As we have shown previously (33), 100 ␮M NEM had a slight stimulatory effect on CFTR-C832A channel activity (Fig. 2A). Login to comment
102 The NEM response of the CFTR-C832A mutant is also quantified in Fig. 3B. Login to comment
118 Our earlier work showed that NEM did not alter the single channel properties of CFTR-C832A (33). Login to comment
129 B, percentage of current remaining 5 min after application of 100 ␮M NEM for CFTR-C832A (control) and NBD1-Cys mutants. Login to comment

[hide] Covalent modification of the regulatory domain irr... J Biol Chem. 1997 Oct 10;272(41):25617-22. Cotten JF, Welsh MJ
Covalent modification of the regulatory domain irreversibly stimulates cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 1997 Oct 10;272(41):25617-22., [PMID:9325282]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is regulated by three cytosolic domains, the regulatory domain (R domain) and two nucleotide binding domains. To learn more about how the cytosolic domains regulate channel activity, we used chemical modification to probe their structure. When we applied the sulfhydryl-modifying reagent N-ethylmaleimide (NEM) and other N-substituted maleimides to the cytosolic domains, we found that they rapidly and irreversibly stimulated channel activity. CFTR contains 14 intracellular cysteine residues that might be targets for NEM modification. We identified one, Cys832, that was essential for the response. Cys832 is located in the R domain. Single channel studies showed that NEM stimulated CFTR by increasing the duration of bursts of activity and by shortening the closed interval between bursts. At the single channel level, CFTR in which Cys832 was mutated to alanine behaved identically to wild-type CFTR, except that it failed to respond to NEM. Additional studies showed that NEM modification increased the potency of ATP-mediated stimulation. Previous work has shown that modification of the R domain by phosphorylation, which introduces negative charge, or replacement of multiple serines by negatively charged aspartates stimulates the channel. Our current data show that covalent modification of the R domain with a neutral, hydrophobic adduct at a site that is not phosphorylated can also stimulate CFTR. This finding suggests that an alteration in the conformation of the R domain may be a key feature that regulates channel activity.

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No. Sentence Comment
6 At the single channel level, CFTR in which Cys832 was mutated to alanine behaved identically to wild-type CFTR, except that it failed to respond to NEM. Login to comment
58 Similar results were obtained with a value of 10 or 30 ms for both wild-type CFTR and CFTR-C832A (Ϯ PKA). Login to comment
59 Regions of data from multichannel patches where there were no superimposed openings were used for burst analysis for the CFTR-C832A mutant following PKA washout, as described previously (16). Login to comment
82 However one mutant, CFTR-C832A, showed a marked decrease in its response to NEM (Fig. 4). Login to comment
146 We also examined the effect of NEM on the single channel properties of CFTR-C832A. Login to comment
148 First, if the CFTR-C832A mutation produced a channel with a very high Po (e.g. nearing a value of 1.0), then it might appear to be resistant to NEM because Po was already near maximal. Login to comment
150 The behavior of CFTR-C832A was very similar to that of wild-type protein (Fig. 6B). Login to comment
152 Like wild-type CFTR, Po and burst duration decreased when PKA was removed from CFTR-C832A. Login to comment
154 These data indicate that CFTR-C832A is functionally and structurally similar to wild-type CFTR; however, the failure to respond to NEM identifies Cys832 as an important site of modification. Login to comment
168 Single channel traces of wild-type CFTR (A) and CFTR-C832A (B) studied in excised, inside-out membrane patches. Login to comment
173 TABLE I Single channel data for wild-type CFTR and CFTR-C832A Data were collected with membrane voltage clamped at -80 mV in the presence of 1 mM ATP. Login to comment
176 For wild-type CFTR, the average number of events used to calculate time constants for each experimental condition in each individual experiment was 1039, and for CFTR-C832A it was 775. Login to comment
177 The average number of bursts used at each experimental condition in each experiment to calculate mean burst duration was 337 and 297 for wild-type and CFTR-C832A, respectively. Login to comment
180 n ATP ϩ PKA ATP ATP ϩ NEM Wild-type CFTR Po 11 0.51 Ϯ 0.02 0.38 Ϯ 0.02a 0.57 Ϯ 0.02b g (pS) 8 ND 9.6 Ϯ 0.4 10 Ϯ 0.6 TB (ms) 5 184 Ϯ 16 149 Ϯ 8a 237 Ϯ 17b ␶o (ms) 5 68 Ϯ 7 58 Ϯ 7 56 Ϯ 7 ␶cf (ms) 5 2.16 Ϯ 0.09 2.37 Ϯ 0.11 2.39 Ϯ 0.15 ␶cs (ms) 5 141 Ϯ 25 152 Ϯ 12 118 Ϯ 8b CFTR-C832A Po 7-9 0.55 Ϯ 0.03 0.39 Ϯ 0.02a 0.42 Ϯ 0.04 g (pS) 6-8 ND 10.2 Ϯ 0.3 10.3 Ϯ 0.2 TB (ms) 3-5 194 Ϯ 19 130 Ϯ 10a 133 Ϯ 10 ␶o (ms) 3-5 62 Ϯ 3 51 Ϯ 5 50 Ϯ 4 ␶cf (ms) 3-5 2.07 Ϯ 0.13 2.63 Ϯ 0.37 2.18 Ϯ 0.28 ␶cs (ms) 3 164 Ϯ 46 206 Ϯ 75 185 Ϯ 81 a p Ͻ 0.05 relative to CFTR in the presence of PKA and ATP. Login to comment
83 However one mutant, CFTR-C832A, showed a marked decrease in its response to NEM (Fig. 4). Login to comment
147 We also examined the effect of NEM on the single channel properties of CFTR-C832A. Login to comment
149 First, if the CFTR-C832A mutation produced a channel with a very high Po (e.g. nearing a value of 1.0), then it might appear to be resistant to NEM because Po was already near maximal. Login to comment
151 The behavior of CFTR-C832A was very similar to that of wild-type protein (Fig. 6B). Login to comment
153 Like wild-type CFTR, Po and burst duration decreased when PKA was removed from CFTR-C832A. Login to comment
155 These data indicate that CFTR-C832A is functionally and structurally similar to wild-type CFTR; however, the failure to respond to NEM identifies Cys832 as an important site of modification. Login to comment
169 Single channel traces of wild-type CFTR (A) and CFTR-C832A (B) studied in excised, inside-out membrane patches. Login to comment
174 TABLE I Single channel data for wild-type CFTR and CFTR-C832A Data were collected with membrane voltage clamped at 280 mV in the presence of 1 mM ATP. Login to comment
178 The average number of bursts used at each experimental condition in each experiment to calculate mean burst duration was 337 and 297 for wild-type and CFTR-C832A, respectively. Login to comment
181 n ATP 1 PKA ATP ATP 1 NEM Wild-type CFTR Po 11 0.51 6 0.02 0.38 6 0.02a 0.57 6 0.02b g (pS) 8 ND 9.6 6 0.4 10 6 0.6 TB (ms) 5 184 6 16 149 6 8a 237 6 17b to (ms) 5 68 6 7 58 6 7 56 6 7 tcf (ms) 5 2.16 6 0.09 2.37 6 0.11 2.39 6 0.15 tcs (ms) 5 141 6 25 152 6 12 118 6 8b CFTR-C832A Po 7-9 0.55 6 0.03 0.39 6 0.02a 0.42 6 0.04 g (pS) 6-8 ND 10.2 6 0.3 10.3 6 0.2 TB (ms) 3-5 194 6 19 130 6 10a 133 6 10 to (ms) 3-5 62 6 3 51 6 5 50 6 4 tcf (ms) 3-5 2.07 6 0.13 2.63 6 0.37 2.18 6 0.28 tcs (ms) 3 164 6 46 206 6 75 185 6 81 a p , 0.05 relative to CFTR in the presence of PKA and ATP. Login to comment