ABCMdb

ABCC7 p.His950Arg

Predicted by SNAP2:	A: N (82%), C: N (78%), D: N (87%), E: N (93%), F: D (53%), G: N (82%), I: N (53%), K: N (93%), L: N (72%), M: N (66%), N: N (97%), P: N (61%), Q: N (93%), R: N (93%), S: N (97%), T: N (93%), V: N (82%), W: D (63%), Y: N (57%),
Predicted by PROVEAN:	A: N, C: D, D: N, E: N, F: N, G: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N,

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[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
141 Fig. 4, D and E, confirm this possibility because both H950A and H950R profoundly reduced inhibition by Fe3ϩ , and their channel activity was also greatly potentiated by curcumin. Login to comment

[hide] The inhibition mechanism of non-phosphorylated Ser... J Biol Chem. 2011 Jan 21;286(3):2171-82. Epub 2010 Nov 8. Wang G
The inhibition mechanism of non-phosphorylated Ser768 in the regulatory domain of cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 2011 Jan 21;286(3):2171-82. Epub 2010 Nov 8., 2011-01-21 [PMID:21059651]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette transporters but serves as a chloride channel dysfunctional in cystic fibrosis. The activity of CFTR is tightly controlled not only by ATP-driven dimerization of its nucleotide-binding domains but also by phosphorylation of a unique regulatory (R) domain by protein kinase A (PKA). The R domain has multiple excitatory phosphorylation sites, but Ser(737) and Ser(768) are inhibitory. The underlying mechanism is unclear. Here, sulfhydryl-specific cross-linking strategy was employed to demonstrate that Ser(768) or Ser(737) could interact with outwardly facing hydrophilic residues of cytoplasmic loop 3 regulating channel gating. Furthermore, mutation of these residues to alanines promoted channel opening by curcumin in an ATP-dependent manner even in the absence of PKA. However, mutation of Ser(768) and His(950) with different hydrogen bond donors or acceptors clearly changed ATP- and PKA-dependent channel activity no matter whether curcumin was present or not. More importantly, significant activation of a double mutant H950R/S768R needed only ATP. Finally, in vitro and in vivo single channel recordings suggest that Ser(768) may form a putative hydrogen bond with His(950) of cytoplasmic loop 3 to prevent channel opening by ATP in the non-phosphorylated state and by subsequent cAMP-dependent phosphorylation. These observations support an electron cryomicroscopy-based structural model on which the R domain is closed to cytoplasmic loops regulating channel gating.

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No. Sentence Comment
7 More importantly, significant activation of a double mutant H950R/S768R needed only ATP. Login to comment
173 In agreement with a putative H-bond between Ser768 and His950 , H950R was also activated by curcumin upon ATP treatment, but H950D was not (Fig. 6, C and D). Login to comment
176 What is more, curcumin also activated H950R/ S768R and H950D/S768D constructs in the presence of ATP (Fig. 6E) because two strong proton donors or acceptors cannot form an H-bond (Table 1). Login to comment
177 More importantly, even if both H950R and S768D could be activated by curcumin with ATP involvement (Fig. 6, A and C), H950R/S768D was silent in response to curcumin even in the presence of ATP (Fig. 6E), suggesting that a strong electrostatic attraction between H950R and S768D prohibit the channel from activation. Login to comment
184 Fig. 7A shows that H950R/S768R was activated by ATP only, even without curcumin, but H950R or S768R was not (Fig. 7C). Login to comment
185 More importantly, H950R/S768R completely removed PKA dependence of channel activity (Fig. 7, A and D) no matter whether K978C, which promotes the channel opening without ATP, was inserted and accelerated channel activation by ATP (Fig. 7B) or not. Login to comment
191 Thus, a strong electrostatic expulsion between H950R/D and S768R/D promoted channel opening by ATP alone. Login to comment
193 Unlike H950R/S768R and H950D/S768D, which exerted an electrostatic interaction between the R domain and CL3, H950A, S768A, S768D, and H950R were not apparently activated by ATP only (Fig. 7C) but more sensitive to PKA than WT CFTR (Fig. 7D). Login to comment
213 Unlike H950A or S768A/D, an apparent open probability of H950R/S768R was higher (Po(app) ϭ 0.0042) than that of WT CFTR even in the absence of ATP, and ATP binding further increased channel opening (Po(app) ϭ 0.198) (Fig. 8, D and E). Login to comment
228 Fig. 9G shows that an open probability of WT CFTR was very low (0.00004) in the resting cells, no FIGURE 6. Effects of curcumin on PKA-dependent activity of His950 and Ser768 mutants with different H-bond donors and acceptors. A and C, macroscopic currents across inside-out membrane patches excised from transfected HEK-293T cells expressing S768D (A) and H950R (C). Login to comment
234 Error bars, S.E. TABLE 1 Potential roles in hydrogen bonding at the CL3-R domain interface Note that mutants whose channel activity was increased by curcumin in the presence of ATP are highlighted in boldface type. Residues Role in H-bond Mutants Arg Strong donor H950R, S768R, H950R/S768R, H950R/S768D, H950D/S768R Asp Strong acceptor H950D, S768D, H950D/S768D, H950R/S768D, H950D/S768R Thr, Gln, Ser, His Donor/Acceptor H950Q, S768T, WT Ala Negative control K946A, H950A, K951A, H954A, S955A, Q958A, S737A, S768A, ⌬R Inhibition of CFTR by Ser768 JANUARY 21, 2011•VOLUME 286•NUMBER 3 JOURNAL OF BIOLOGICAL CHEMISTRY 2177 matter whether cAMP was present or not in the extracellular perfusate. Login to comment
244 Although thiol-specific disulfide cross-linking of S768C to H950C or nearby cysteines inserted in CL3 inhibited channel activity primarily by stopping the channel from opening, an electrostatic expulsion between S768R/D and H950R/D clearly promoted channel opening even in the absence of ATP. Login to comment
248 Finally, both H950R and S768D promoted channel opening by ATP followed by curcumin or PKA phosphorylation, but H950D or S768R could not. Login to comment
255 PKA-dependent activity of His950 and Ser768 mutants with different H-bond donors and acceptors. A and B, activation of H950R/S768R (A) and H950R/S768R/K978C (B) before and after ATP (1. Login to comment
291 In agreement with a proposal of His950 as an H-bond acceptor and Ser768 as an H-bond donor, S768D and H950R mutants were more sensitive to ATP or curcumin or PKA phosphorylation than S768R and H950D (Figs. 6-8). Login to comment
305 Finally, both proton donors cannot form an inhibitory H-bond between H950R and S768R. Login to comment
306 Instead, an electrostatic expulsion between H950R and S768R dramatically increased the ATP-independent open probabilities and sensitivity to ATP and PKA (Figs. 7 and 8). Login to comment
343 It is expected that H950R and H950R/S768R may also exert similar effects on the basal channel opening. Login to comment

[hide] Regulation of Activation and Processing of the Cys... J Biol Chem. 2012 Oct 11. Wang G, Duan DD
Regulation of Activation and Processing of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by a Complex Electrostatic Interaction between the Regulatory Domain and Cytoplasmic Loop 3.
J Biol Chem. 2012 Oct 11., [PMID:23060444]

Abstract [show]
NEG2, a short C-terminal segment (817-838) of the unique regulatory (R) domain of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, has been reported to regulate CFTR gating in response to cAMP-dependent R domain phosphorylation. The underlying mechanism, however, is unclear. Here, K946 of cytoplasmic loop 3 (CL3) is proposed as counter-ion of D835, D836 or E838 of NEG2 to prevent channel activation by PKA. R764 or R766 of the S768 phosphorylation site of the R domain is proposed to promote channel activation possibly by weakening the putative CL3-NEG2 electrostatic attraction. First, not only D835A, D836A and E838A but also K946A reduced the PKA dependent CFTR activation. Second, both K946D and D835R/D836R/E838R mutants were activated by ATP and curcumin to a different extent. Third, R764A and R766A mutants enhanced the PKA-dependent activation. On the other hand, it is very exciting that D835R/D836R/E838R and K946D/H950D and H950R exhibited normal channel processing and activity while D835R/D836R/E838R/K946D/H950D was misprocessed and silent in response to forskolin. Further, D836R and E838R played a critical role in the asymmetric electrostatic regulation of CFTR processing and S768 phosphorylation may not be involved. Thus, a complex interfacial interaction among CL3, NEG2 and the S768 phosphorylation site may be responsible for the asymmetric electrostatic regulation of CFTR activation and processing.

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No. Sentence Comment
12 Second, both K946D and D835R/D836R/E838R mutants were activated by ATP and curcumin to a different extent. Login to comment
14 On the other hand, it is very exciting that D835R/D836R/E838R and K946D/H950D and H950R exhibited normal channel processing and activity while D835R/D836R/E838R/K946D/H950D was misprocessed and silent in response to forskolin. Login to comment
86 A similar case was observed with H950R (Fig. 4B). Login to comment
87 Because the D835R/D836R/E838R mutant exhibited the lower current with forskolin than H950R or WT CFTR, a cell capacitance was measured with WT and D835R/D836R/E838R CFTR constructs before and after forskolin was applied to evaluate the contribution of trafficking (20). Login to comment
90 Although both D835R/D836R/E838R and H950R were activated by forskolin and curcumin greatly, the introduction of K946D and H950D to D835R/D836R/E838R prevented the channel activation by forskolin and curcumin (Fig. 4C and 4D). Login to comment
92 In contrast, D835R/D836R/E838R and H950R failed to exhibit the basic activity (Fig. 4A and B). Login to comment
96 In contrast, D835R/D836R/E838R and K946D/H950D and H950R exhibited comparable current densities (Fig. 4D). Login to comment
99 The putative electrostatic attraction between K946/H950R and D835/D836/E838 failed to inhibit the channel activity but the putative electrostatic attraction of D835R/D836R/E838R with K946D/H950D greatly suppressed the channel activity. Login to comment
102 The similar cases were seen with D835R/D836R/E838R and H950R and K946D/H950D (Fig.5). Login to comment
105 On the other hand, the channel activity of D835R/D836R/E838R/K946D/H950D was still very low (Fig. 4D). Login to comment
108 Therefore, we prepared a control mutant S768D/K946D/H950D to exclude this putative H-bond. Login to comment
110 The introduction of D835R to this mutant failed to change the current density. Login to comment
115 On the other hand, because S768D mimics S768 phosphorylation and H950R/S768D also exhibited the high channel activity (10), S768 phosphorylation failed to change the asymmetric electrostatic regulation of CFTR activation and processing. Login to comment
117 Because R764X and R766M were reported with CF patients (www.genet.sickkids.on.ca/cftr/app), we investigated if missense alanine mutation of R764 and R766 alters the channel processing and activity. Login to comment
139 Second, the curcumin sensitivity was also increased for K946A, K946D and D835R/D836R/E838R (Fig.3). Login to comment
141 However, the putative electrostatic attraction between K946/H950R and D835/D836/E838 failed to exert this inhibitory effect (Fig.4-5). Login to comment
150 Otherwise, a putative electrostatic attraction of CL3 (K946D/H950D) with both NEG2 (D836R or E838R) and the S768 phosphorylation site (R764 or R766) may result in misprocessing and low channel activity (Fig.8B). Login to comment
104 A similar case was observed with H950R (Fig. 4B). Login to comment
114 In contrast, D835R/D836R/E838R and K946D/H950D and H950R exhibited comparable current densities (Fig. 4D). Login to comment
120 Similar cases were seen with D835R/ D836R/E838R and H950R and K946D/H950D (Fig. 5). Login to comment
146 Effects of electrostatic interactions on current densities of CFTR mutants at the R-CL3 interface. A-C, whole cell currents from transfected HEK-293T cells expressing CFTR mutants D835R/D836R/E838R (A), H950R (B), and K946D/H950D/D835R/D836R/E838R (C) recorded by using a ramp protocol (afe;40 mV). Login to comment
170 However, the putative electrostatic attraction between K946/H950R and D835/D836/E838 failed to exert FIGURE 5. Login to comment