ABCMdb

ABCC7 p.His950Asp

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,

[switch to compact view]
Comments [show]

None has been submitted yet.

Publications
[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.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence 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
178 Consistent with this notion, H950D/S768R was also not activated by curcumin with ATP (Fig. 6E). Login to comment
186 In contrast, ATP failed to activate construct H950D/S768D, although an H-bond cannot be formed between two strong proton acceptors (Fig. 7C). Login to comment
187 This result may be due to an endogenous Fe3ϩ binding between S768D and H950D, which also prevented the channel from opening (30). Login to comment
188 Supporting this hypothesis, Fig. 7C and supplemental Fig. S2 clearly demonstrate that the mutant S768D/H950D can be much activated by ATP once 5 mM EDTA was added to remove the endogenous Fe3ϩ in the channel. Login to comment
189 In contrast, H950D and S768D could not be dramatically activated by ATP only even in the presence of 5 mM EDTA (Fig. 7C). Login to comment
190 Fig. 7D and supplemental Fig. S2 further show that the presence of EDTA clearly weakened the PKA dependence of H950D/S768D channel activity. 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
214 Similarly, H950D/S768D also exhibited an increased apparent open probability (Po(app) ϭ 0.0132) in the presence of 5 mm EDTA, and ATP continued to promote channel opening (Po(app) ϭ 0.0452) (Fig. 8, D and E). 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
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
259 For the H950D/S768D construct in the presence of 5 mM EDTA (n ϭ 6-7); **, p Ͻ 0.05 compared with the absence of EDTA. 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
307 In contrast, channel activity was not potentiated by an electrostatic expulsion between H950D and S768D until EDTA was added to remove potential endogenous Fe3ϩ binding to S768D and H950D, although both proton acceptors cannot form an inhibitory H-bond (Fig. 7 and 8). Login to comment
346 A similar observation would be seen with H950D/S768D. 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.

Comments [show]

None has been submitted yet.

Sentences [show]
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
74 In order to support this argument, K946D/H950D was employed to weaken both the electrostatic attractions and the Fe3+ bridge between CL3 and the R domain. Login to comment
75 Fig. 3D demonstrates that ATP and curucmin completely activated the K946D/H950D mutant. Login to comment
76 This high activity may not be due to the H950D mutation because it cannot be activated by ATP and curcumin (10). 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
88 Table 1 demonstrates that the cell capacitances of both WT and D835R/D836R/E838R were stable upon activation by forskolin. 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
95 Upon normalization of the channel current to the cell capacitance, the insertion of K946D/H950D to D835R/D836R/E838R dramatically reduced the CFTR current density (Fig.4D). 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
100 The asymmetric effects of R-CL3 electrostatic attractions on CFTR processing - To further determine if the reduction in the current density of K946D/H950D/D835R/D836R/E838R originates from the decrease in the channel expression or the single channel conductance, we carried out western-blotting analysis. Login to comment
102 The similar cases were seen with D835R/D836R/E838R and H950R and K946D/H950D (Fig.5). Login to comment
103 However, the insertion of K946D/H950D to D835R/D836R/E838R clearly reduced the fractional mature Band C by 50%. 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
107 The effects of S768 phosphorylation on the asymmetric NEG2-CL3 electrostatic regulation of the CFTR activity and processing- Although previous studies demonstrated that S768 phosphorylation had no electrostatic contribution to the channel gating, non-phosphorylated S768 may form a strong putative H-bond with H950D to affect the CL3-NEG2 electrostatic interaction (10). Login to comment
108 Therefore, we prepared a control mutant S768D/K946D/H950D to exclude this putative H-bond. Login to comment
109 Fig. 4D indicates a high current density of S768D/K946D/H950D based on a whole-cell patch recording. Login to comment
111 However, the insertion of D836R or E838R to S768D/K946D/H950D dramatically reduced the CFTR current density (Fig.4D). Login to comment
112 Fig.5 further demonstrates that the fractions of the mature Band C of S768D/K946D/H950D/D836R and S768D/K946D/H950D/E838R were also significantly decreased by 45%. 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
118 Fig. 4D demonstrates that both R764A and R766A exhibited a normal channel density. Login to comment
121 The K1/2 for PKA activation of R764A and R766A increased from 10 units/ml to 25 and 44 units/ml, respectively. Login to comment
123 To support this hypothesis, we determined if S832C is closed enough to S768C to form a detectable disulfide-bond crosslinking based on the Cys-free CFTR construct. Login to comment
140 Finally, it is very exciting that the putative electrostatic attraction between K946D/H950D and D835R/D836R/E838R dramatically suppressed the channel activity by stopping the channel processing or opening(10). 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
113 Upon normalization of the channel current to the cell capacitance, the insertion of K946D/H950D to D835R/D836R/ E838R dramatically reduced the CFTR current density (Fig. 4D). 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
131 The relative activity of K946D was significantly smaller than that of D835R/D836R/E838R (n afd; 3-4; *, p b0d; 0.05, unpaired t test); error bars, S.E. Asymmetric Electrostatic Regulation of CFTR NOVEMBER 23, 2012ߦVOLUME 287ߦNUMBER 48 JOURNAL OF BIOLOGICAL CHEMISTRY 40487 The Effects of Ser-768 Phosphorylation on the Asymmetric NEG2-CL3 Electrostatic Regulation of the CFTR Activity and Processing-Although previous studies demonstrated that Ser-768 phosphorylation had no electrostatic contribution to the channel gating, nonphosphorylated Ser-768 may form a strong putative H-bond with H950D to affect the CL3-NEG2 electrostatic interaction (10). Login to comment
132 Therefore, we prepared a control mutant S768D/K946D/H950D to exclude this putative H-bond. Login to comment
133 Fig. 4D indicates a high current density of S768D/K946D/H950D based on a whole cell patch recording. Login to comment
135 However, the insertion of D836R or E838R to S768D/K946D/H950D dramatically reduced the CFTR current density (Fig. 4D). Login to comment
136 Fig. 5 further demonstrates that the fractions of the mature Band C of S768D/K946D/H950D/D836R and S768D/K946D/H950D/ E838R were also significantly decreased by 45%. 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
151 The currents mediated by S768D/K946D/H950D/D836R and S768D/K946D/H950D/E838R were statistically smaller than the S768D/K946D/H950D current (n afd; 3,*, p b0d; 0.05, unpaired t test); error bars, S.E. TABLE 1 WholecellcurrentsIm (picoamperes)andcapacitancesCm (picofarads) of HEK-293T cells expressing CFTR constructs in response to forskolin Constructs Stimulated Im Control Cm Stimulated Cm n WT-CFTR 701.6 afe; 9.0 17.4 afe; 0.9 16.4 afe; 1.1 3 D835R/D836R/E838R 539.5 afe; 5.3 15.0 afe; 1.0 16.2 afe; 0.8 3 Asymmetric Electrostatic Regulation of CFTR 40488 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287ߦNUMBER 48ߦNOVEMBER 23, 2012 at SEMMELWEIS UNIV OF MEDICINE on December , The K1/2 for PKA activation of R764A and R766A increased from 10 units/ml to 25 and 44 units/ml, respectively. Login to comment
169 Finally, it is very exciting that the putative electrostatic attraction between K946D/H950D and D835R/D836R/E838R dramatically suppressed the maximal channel activity by stopping the channel processing or opening (10). Login to comment
189 Otherwise, a putative electrostatic attraction of CL3 (K946D/H950D) with both NEG2 (D836R or E838R) and the Ser-768 phosphorylation site (Arg-764 or Arg-766) may result in misprocessing and low channel activity (Fig. 8B). Login to comment