ABCMdb

PMID: 23060444

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., [PubMed]

Sentences

No. Mutations Sentence Comment

9 ABCC7 p.Asp836Ala ABCC7 p.Asp835Ala ABCC7 p.Lys946Ala ABCC7 p.Glu838Ala First, not only D835A, D836A, and E838A but also K946A reduced the PKA-dependent CFTR activation. Login to comment 10 ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp Second, both K946D and D835R/D836R/ E838R mutants were activated by ATP and curcumin to a different extent. Login to comment 11 ABCC7 p.Asp836Ala ABCC7 p.Asp835Ala ABCC7 p.Lys946Ala ABCC7 p.Glu838Ala ABCC7 p.Arg764Ala ABCC7 p.Arg766Ala First, not only D835A, D836A and E838A but also K946A reduced the PKA dependent CFTR activation. Login to comment 12 ABCC7 p.His950Arg ABCC7 p.His950Asp ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Asp836Arg ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Asp835Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Glu838Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp ABCC7 p.Lys946Asp ABCC7 p.Lys946Asp Second, both K946D and D835R/D836R/E838R mutants were activated by ATP and curcumin to a different extent. Login to comment 13 ABCC7 p.Asp836Arg ABCC7 p.Glu838Arg ABCC7 p.Arg764Ala ABCC7 p.Arg766Ala Third, R764A and R766A mutants enhanced the PKA-dependent activation. Login to comment 14 ABCC7 p.His950Arg ABCC7 p.His950Asp ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp ABCC7 p.Lys946Asp 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 15 ABCC7 p.Asp836Arg ABCC7 p.Glu838Arg Further, D836R and E838R played a critical role in the asymmetric electrostatic regulation of CFTR processing and S768 phosphorylation may not be involved. Login to comment 37 ABCC7 p.Ser660Ala R-S660A-CFTR was provided by Michael Welsh (University of Iowa, Iowa City, IA). Login to comment 41 ABCC7 p.Ser660Ala èc;R-S660A-CFTR was provided by Michael Welsh (University of Iowa, Iowa City, IA). Login to comment 60 ABCC7 p.Asp835Ala ABCC7 p.Glu838Ala Similar observations with D835A and E838A were summarized in Fig. 2D. The K1/2 for PKA activation reduced from 10 units/ml to 5 units/ml once K946 from the CL3, and D835, D836 and E838 from NEG2 were mutated to alanines (Fig.2D). Login to comment 61 ABCC7 p.Lys951Ala However, K951A failed to change the PKA-dependent channel activity (Fig.2D). Login to comment 62 ABCC7 p.Asp836Ala ABCC7 p.Lys946Ala In contrast, mutation of Lys-946 or Asp-836 to alanine clearly accelerated the channel activation and reduced the PKA dependence (Fig. 2, B and C). Login to comment 63 ABCC7 p.Asp835Ala ABCC7 p.Glu838Ala Similar observations with D835A and E838A are summarized in Fig. 2D. Login to comment 64 ABCC7 p.Lys946Ala In the presence of 1.5mM ATP, 50µM curcumin greatly potentiated the K946A mutant activity, which was further increased by PKA (Fig. 3A). Login to comment 65 ABCC7 p.Lys951Ala ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp 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 ABCC7 p.Lys946Asp 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 67 ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg In contrast, ATP and curcumin dramatically activated the D835R/D836R/E838R mutant. Login to comment 68 ABCC7 p.Lys946Ala In the presence of 1.5 mM ATP, 50 òe;M curcumin greatly potentiated the K946A mutant activity, which was further increased by PKA (Fig. 3A). Login to comment 69 ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp 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 70 ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp ABCC7 p.Lys946Asp 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 ABCC7 p.Lys946Asp However, for K946D, H950 or H954 may still form the inhibitive Fe3+ bridge with the R domain. Login to comment 74 ABCC7 p.His950Asp ABCC7 p.Lys946Asp 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 ABCC7 p.His950Asp ABCC7 p.Lys946Asp Fig. 3D demonstrates that ATP and curucmin completely activated the K946D/H950D mutant. Login to comment 76 ABCC7 p.His950Asp This high activity may not be due to the H950D mutation because it cannot be activated by ATP and curcumin (10). Login to comment 79 ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg Asymmetric Electrostatic Regulation of CFTR NOVEMBER 23, 2012ߦVOLUME 287ߦNUMBER 48 JOURNAL OF BIOLOGICAL CHEMISTRY 40485 cally activated the D835R/D836R/E838R mutant. Login to comment 80 ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp However, neither K946D nor D835R/D836R/E838R was fully activated by ATP alone (Fig. 3B and 3C). Login to comment 82 ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp 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 83 ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg Fig. 4A indicates that when the whole-cell current was determined with D835R/D836R/E838R, the application of external forskolin to the bath solution increased the mutant current. Login to comment 85 ABCC7 p.Lys946Asp However, for K946D, His-950 or His-954 may still form the inhibitive Fe3af9; bridge with the R domain. Login to comment 86 ABCC7 p.His950Arg ABCC7 p.His950Asp ABCC7 p.Lys946Asp A similar case was observed with H950R (Fig. 4B). Login to comment 87 ABCC7 p.His950Arg ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp 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 ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg Table 1 demonstrates that the cell capacitances of both WT and D835R/D836R/E838R were stable upon activation by forskolin. Login to comment 90 ABCC7 p.His950Arg ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp 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 ABCC7 p.His950Arg ABCC7 p.Asp836Arg ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp In contrast, D835R/D836R/E838R and H950R failed to exhibit the basic activity (Fig. 4A and B). Login to comment 95 ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp 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 ABCC7 p.His950Arg ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp In contrast, D835R/D836R/E838R and K946D/H950D and H950R exhibited comparable current densities (Fig. 4D). Login to comment 98 ABCC7 p.Asp836Ala ABCC7 p.Lys946Ala The PKA-dependent activity of CFTR mutants at the R-CL3 interface. A-C, macroscopic currents across inside-out membrane patches excised from transfected HEK-293T cells expressing WT CFTR (A) and mutants K946A (B) and D836A (C) by using a ramp protocol (afe;80 mV). Login to comment 99 ABCC7 p.His950Arg ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp 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 ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp 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 ABCC7 p.His950Arg ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp The similar cases were seen with D835R/D836R/E838R and H950R and K946D/H950D (Fig.5). Login to comment 103 ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp However, the insertion of K946D/H950D to D835R/D836R/E838R clearly reduced the fractional mature Band C by 50%. Login to comment 104 ABCC7 p.His950Arg A similar case was observed with H950R (Fig. 4B). Login to comment 105 ABCC7 p.His950Arg ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Asp836Arg ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Asp835Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Glu838Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp On the other hand, the channel activity of D835R/D836R/E838R/K946D/H950D was still very low (Fig. 4D). Login to comment 106 ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg Table 1 demonstrates that the cell capacitances of both WT and D835R/D836R/E838R were stable upon activation by forskolin. Login to comment 107 ABCC7 p.His950Asp 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 ABCC7 p.Ser768Asp ABCC7 p.His950Arg ABCC7 p.His950Asp ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp ABCC7 p.Lys946Asp Therefore, we prepared a control mutant S768D/K946D/H950D to exclude this putative H-bond. Login to comment 109 ABCC7 p.Ser768Asp ABCC7 p.His950Asp ABCC7 p.Lys946Asp Fig. 4D indicates a high current density of S768D/K946D/H950D based on a whole-cell patch recording. Login to comment 110 ABCC7 p.His950Arg ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg The introduction of D835R to this mutant failed to change the current density. Login to comment 111 ABCC7 p.Ser768Asp ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp However, the insertion of D836R or E838R to S768D/K946D/H950D dramatically reduced the CFTR current density (Fig.4D). Login to comment 112 ABCC7 p.Ser768Asp ABCC7 p.Ser768Asp ABCC7 p.His950Asp ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp ABCC7 p.Lys946Asp 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 113 ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp 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 ABCC7 p.His950Arg ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp In contrast, D835R/D836R/E838R and K946D/H950D and H950R exhibited comparable current densities (Fig. 4D). Login to comment 115 ABCC7 p.Ser768Asp ABCC7 p.Ser768Asp ABCC7 p.His950Arg 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 116 ABCC7 p.Arg764Ala ABCC7 p.Arg766Ala The effects of R764A and R766A on the PKA-dependent channel activation - Because both S768 and D836 are close to H950 of CL3, it is fitting to ask if R764, R765 and R766 form a salt bridge with the N-terminal of NEG2 to cause the asymmetry of the electrostatic regulation. Login to comment 117 ABCC7 p.Arg764* ABCC7 p.Arg766Met ABCC7 p.His950Arg ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp 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 ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp ABCC7 p.Arg764Ala ABCC7 p.Arg766Ala Fig. 4D demonstrates that both R764A and R766A exhibited a normal channel density. Login to comment 120 ABCC7 p.His950Arg ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp Similar cases were seen with D835R/ D836R/E838R and H950R and K946D/H950D (Fig. 5). Login to comment 121 ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp ABCC7 p.Arg764Ala ABCC7 p.Arg766Ala 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 ABCC7 p.Ser768Cys ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp 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 124 ABCC7 p.Val510Ala ABCC7 p.Val510Ala ABCC7 p.Val510Ala ABCC7 p.Ser768Cys ABCC7 p.Ser768Cys Fig. 7 demonstrates that 10M diamide greatly suppressed the channel activity of S768C/S832C/V510A mutant but failed to affect the channel activities of both S768C/V510A and S832C/V510A. Login to comment 127 ABCC7 p.Lys946Ala ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp 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 ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp 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 ABCC7 p.Ser768Asp ABCC7 p.His950Asp ABCC7 p.Lys946Asp Therefore, we prepared a control mutant S768D/K946D/H950D to exclude this putative H-bond. Login to comment 133 ABCC7 p.Ser768Asp ABCC7 p.His950Asp ABCC7 p.Lys946Asp Fig. 4D indicates a high current density of S768D/K946D/H950D based on a whole cell patch recording. Login to comment 134 ABCC7 p.Asp835Arg The introduction of D835R to this mutant failed to change the current density. Login to comment 135 ABCC7 p.Ser768Asp ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp However, the insertion of D836R or E838R to S768D/K946D/H950D dramatically reduced the CFTR current density (Fig. 4D). Login to comment 136 ABCC7 p.Ser768Asp ABCC7 p.Ser768Asp ABCC7 p.His950Asp ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp ABCC7 p.Lys946Asp 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 138 ABCC7 p.Asp836Ala ABCC7 p.Asp835Ala ABCC7 p.Lys946Ala ABCC7 p.Glu838Ala First, the PKA sensitivity of channel activation was significantly enhanced for K946A, D835A, D836A and E838A mutants (Fig.2). Login to comment 139 ABCC7 p.Ser768Asp ABCC7 p.Ser768Asp ABCC7 p.His950Arg ABCC7 p.Lys946Ala ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp Second, the curcumin sensitivity was also increased for K946A, K946D and D835R/D836R/E838R (Fig.3). Login to comment 140 ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp ABCC7 p.Arg764Ala ABCC7 p.Arg766Ala 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 141 ABCC7 p.Arg764* ABCC7 p.Arg766Met ABCC7 p.His950Arg However, the putative electrostatic attraction between K946/H950R and D835/D836/E838 failed to exert this inhibitory effect (Fig.4-5). Login to comment 142 ABCC7 p.Arg764Ala ABCC7 p.Arg766Ala Fig. 4D demonstrates that both R764A and R766A exhibited a normal channel density. Login to comment 145 ABCC7 p.Arg764Ala ABCC7 p.Arg766Ala Our finding shows that R764A and R766A suppressed not the channel processing and the current density but the channel activation by PKA (Fig. 4-6). Login to comment 146 ABCC7 p.His950Arg ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp 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 150 ABCC7 p.His950Arg ABCC7 p.His950Asp ABCC7 p.His950Asp ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Asp836Arg ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Glu838Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp ABCC7 p.Lys946Asp ABCC7 p.Lys946Asp 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 151 ABCC7 p.Ser768Asp ABCC7 p.Ser768Asp ABCC7 p.Ser768Asp ABCC7 p.His950Asp ABCC7 p.His950Asp ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp ABCC7 p.Lys946Asp ABCC7 p.Lys946Asp ABCC7 p.Arg764Ala ABCC7 p.Arg766Ala 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 152 ABCC7 p.Ser945Leu ABCC7 p.His949Tyr On the other hand, S945L and H949Y, found in patients with cystic fibrosis, are close to K946 and H950 of CL3 and stop maturation of the protein (31). Login to comment 153 ABCC7 p.Ser768Cys To support this hypothesis, we determined whether S832C is closed enough to S768C to form a detectable disulfide bond cross-linking based on the Cys-free CFTR construct. Login to comment 154 ABCC7 p.Asp979Ala ABCC7 p.Val510Ala ABCC7 p.Val510Ala ABCC7 p.Val510Ala ABCC7 p.Ser768Cys ABCC7 p.Ser768Cys Finally, D979A in CF patients causes misprocessing (33). Login to comment 167 ABCC7 p.Asp836Ala ABCC7 p.Asp835Ala ABCC7 p.Lys946Ala ABCC7 p.Glu838Ala First, the PKA sensitivity of channel activation was significantly enhanced for K946A, D835A, D836A, and E838A mutants (Fig. 2). Login to comment 168 ABCC7 p.Lys946Ala ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp Second, the curcumin sensitivity was also increased for K946A, K946D, and D835R/ D836R/E838R (Fig. 3). Login to comment 169 ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Asp835Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp 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 170 ABCC7 p.His950Arg However, the putative electrostatic attraction between K946/H950R and D835/D836/E838 failed to exert FIGURE 5. Login to comment 173 ABCC7 p.Arg764Ala ABCC7 p.Arg766Ala The PKA-dependent activity of CFTR mutants R764A and R766A. Login to comment 174 ABCC7 p.Arg766Ala A, unit channel currents across inside-out membrane patches excised from transfected HEK-293T cells expressing R766A by using a holding potential (af9;60 mV). Login to comment 179 ABCC7 p.Arg764Ala ABCC7 p.Arg766Ala B, PKA titration curves for CFTR mutants R764A and R766A (n afd; 3-5). Login to comment 184 ABCC7 p.Arg764Ala ABCC7 p.Arg766Ala Our finding shows that R764A and R766A suppressed not the channel processing and the current density but the channel activation by PKA (Figs. 4-6). Login to comment 189 ABCC7 p.His950Asp ABCC7 p.Asp836Arg ABCC7 p.Glu838Arg ABCC7 p.Lys946Asp 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 191 ABCC7 p.Ser945Leu ABCC7 p.His949Tyr However, S945L and H949Y, found in patients with CF, are close to Lys-946 and His-950 of CL3 and stop maturation of the protein (31). Login to comment 193 ABCC7 p.Asp979Ala Finally, D979A in CF patients causes misprocessing (33). Login to comment 195 ABCC7 p.Val510Ala ABCC7 p.Val510Ala ABCC7 p.Val510Ala ABCC7 p.Ser768Cys ABCC7 p.Ser768Cys Effects of diamide on CFTR mutants at the R-CL3 interface. A-C, macroscopic currents across inside-out membrane patches excised from transfected HEK-293T cells expressing CFTR mutants S768C/V510A (A), S832C/V510A (B), and S768C/S832C/V510A (C) by using a ramp protocol (afe;80 mV). Login to comment