ABCMdb

ABCC7 p.Lys946Asp

ClinVar:	c.2836A>T , p.Lys946* ? , not provided
Predicted by SNAP2:	A: D (53%), C: N (53%), D: D (75%), E: D (59%), F: D (66%), G: D (71%), H: N (57%), I: D (53%), L: D (59%), M: N (78%), N: D (59%), P: D (80%), Q: N (87%), R: N (87%), S: D (53%), T: N (53%), V: D (59%), W: D (80%), Y: D (63%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: N, F: D, G: D, H: D, I: D, L: D, M: D, N: N, P: D, Q: N, R: N, S: D, T: D, V: D, W: D, Y: D,

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Publications
[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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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
65 If this effect is due to a disruption of the putative electrostatic interaction between CL3 and NEG2, K946D or D835R/D836R/E838R should exhibit the similar effect in the presence of ATP. Login to comment
66 To support this argument, ATP and curcumin partially increased the K946D activity and PKA (6 units/ml) completely activated this mutant (Fig. 3B). Login to comment
70 It is interesting that ATP and curucmin only activated the K946D mutant by 40% but activated the D835R/D836R/E838R mutant completely (Fig. 3D). Login to comment
73 However, for K946D, H950 or H954 may still form the inhibitive Fe3+ bridge with the R domain. 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
80 However, neither K946D nor D835R/D836R/E838R was fully activated by ATP alone (Fig. 3B and 3C). 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
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
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
139 Second, the curcumin sensitivity was also increased for K946A, K946D and D835R/D836R/E838R (Fig.3). 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
10 Second, both K946D and D835R/D836R/ E838R mutants were activated by ATP and curcumin to a different extent. Login to comment
69 If this effect is due to a disruption of the putative electrostatic interaction between CL3 and NEG2, K946D or D835R/D836R/ E838R should exhibit a similar effect in the presence of ATP. Login to comment
82 It is interesting that ATP and curcumin only activated the K946D mutant by 40% but activated the D835R/ D836R/E838R mutant completely (Fig. 3D). Login to comment
85 However, for K946D, His-950 or His-954 may still form the inhibitive Fe3af9; bridge with the R domain. 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
127 Macroscopic currents across inside-out membrane patches excised from transfected HEK-293T cells expressing CFTR mutants K946A (A), K946D (B) and D835R/D836R/E838R (C) by using a ramp protocol (afe;80 mV) are shown. 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
168 Second, the curcumin sensitivity was also increased for K946A, K946D, and D835R/ D836R/E838R (Fig. 3). 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