ABCMdb

ABCC7 p.Ser768Asp

Predicted by SNAP2:	A: N (61%), C: N (61%), D: N (57%), E: N (61%), F: N (57%), G: N (66%), H: N (61%), I: D (59%), K: N (53%), L: N (53%), M: D (63%), N: N (72%), P: N (53%), Q: N (53%), R: N (53%), T: N (66%), V: N (66%), W: D (66%), Y: D (53%),
Predicted by PROVEAN:	A: N, C: D, D: N, E: N, F: D, G: N, H: N, I: D, K: N, L: D, M: D, N: N, P: D, Q: N, R: N, T: N, V: D, W: D, Y: D,

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[hide] Mechanistic insight into control of CFTR by AMPK. J Biol Chem. 2009 Feb 27;284(9):5645-53. Epub 2008 Dec 18. Kongsuphol P, Cassidy D, Hieke B, Treharne KJ, Schreiber R, Mehta A, Kunzelmann K
Mechanistic insight into control of CFTR by AMPK.
J Biol Chem. 2009 Feb 27;284(9):5645-53. Epub 2008 Dec 18., 2009-02-27 [PMID:19095655]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP and protein kinase A (PKA)-regulated Cl(-) channel in the apical membrane of epithelial cells. The metabolically regulated and adenosine monophosphate-stimulated kinase (AMPK) is colocalized with CFTR and attenuates its function. However, the sites for CFTR phosphorylation and the precise mechanism of inhibition of CFTR by AMPK remain obscure. We demonstrate that CFTR normally remains closed at baseline, but nevertheless, opens after inhibition of AMPK. AMPK phosphorylates CFTR in vitro at two essential serines (Ser(737) and Ser(768)) in the R domain, formerly identified as "inhibitory" PKA sites. Replacement of both serines by alanines (i) reduced phosphorylation of the R domain, with Ser(768) having dramatically greater impact, (ii) produced CFTR channels that were partially open in the absence of any stimulation, (iii) significantly augmented their activation by IBMX/forskolin, and (iv) eliminated CFTR inhibition post AMPK activation. Attenuation of CFTR by AMPK activation was detectable in the absence of cAMP-dependent stimulation but disappeared in maximally stimulated oocytes. Our data also suggest that AMP is produced by local phosphodiesterases in close proximity to CFTR. Thus we propose that CFTR channels are kept closed in nonstimulated epithelia with high baseline AMPK activity but CFTR may be basally active in tissues with lowered endogenous AMPK activity.

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43 EXPERIMENTAL PROCEDURES cRNAs for CFTR and Double Electrode Voltage Clamp-Oocytes were injected with cRNA (10 ng, 47 nl of double-distilled water) encoding wtCFTR, L1430A/L1431A, S573A, S1248A, F508del-CFTR, G551D-CFTR, S768A, S737A, S768D, S737D, E1474X, and AMPK␣1. Login to comment
129 In contrast, the S768D mutation, mimicking phosphorylation at Ser768 , produced a whole cell conductance that was significantly smaller than even wtCFTR (note that conductance not lowered with S737D; see also Fig. 3C for phenformin sensitivity of these mutants). Login to comment
147 In contrast the residual CFTR conductances generated by S768D (but not S737D) were not only further inhibited by phenformin, but neither phospho-mimic mutant could be augmented by the AMPK inhibitor compound C. Login to comment

[hide] AMP-activated protein kinase phosphorylation of th... Am J Physiol Cell Physiol. 2009 Jul;297(1):C94-101. Epub 2009 May 6. King JD Jr, Fitch AC, Lee JK, McCane JE, Mak DO, Foskett JK, Hallows KR
AMP-activated protein kinase phosphorylation of the R domain inhibits PKA stimulation of CFTR.
Am J Physiol Cell Physiol. 2009 Jul;297(1):C94-101. Epub 2009 May 6., [PMID:19419994]

Abstract [show]
The metabolic sensor AMP-activated protein kinase (AMPK) has emerged as an important link between cellular metabolic status and ion transport activity. We previously found that AMPK binds to and phosphorylates CFTR in vitro and inhibits PKA-dependent stimulation of CFTR channel gating in Calu-3 bronchial serous gland epithelial cells. To further characterize the mechanism of AMPK-dependent regulation of CFTR, whole cell patch-clamp measurements were performed with PKA activation in Calu-3 cells expressing either constitutively active or dominant-negative AMPK mutants (AMPK-CA or AMPK-DN). Baseline CFTR conductance in cells expressing AMPK-DN was substantially greater than controls, suggesting that tonic AMPK activity in these cells inhibits CFTR under basal conditions. Although baseline CFTR conductance in cells expressing AMPK-CA was comparable to that of controls, PKA stimulation of CFTR was completely blocked in AMPK-CA-expressing cells, suggesting that AMPK activation renders CFTR resistant to PKA activation in vivo. Phosphorylation studies of CFTR in human embryonic kidney-293 cells using tetracycline-inducible expression of AMPK-DN demonstrated AMPK-dependent phosphorylation of CFTR in vivo. However, AMPK activity modulation had no effect on CFTR in vivo phosphorylation in response to graded doses of PKA or PKC agonists. Thus, AMPK-dependent CFTR phosphorylation renders the channel resistant to activation by PKA and PKC without preventing phosphorylation by these kinases. We found that Ser768, a CFTR R domain residue considered to be an inhibitory PKA site, is the dominant site of AMPK phosphorylation in vitro. Ser-to-Ala mutation at this site enhanced baseline CFTR activity and rendered CFTR resistant to inhibition by AMPK, suggesting that AMPK phosphorylation at Ser768 is required for its inhibition of CFTR. In summary, our findings indicate that AMPK-dependent phosphorylation of CFTR inhibits CFTR activation by PKA, thereby tuning the PKA-responsiveness of CFTR to metabolic and other stresses in the cell.

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144 It was reported previously that CFTR-S768A exhibits a very high open probability, whereas the phospho-mimic Ser-to-Asp S768D CFTR mutant has a very low open probability relative to wild-type CFTR following PKA stimulation (8, 27, 28). Login to comment
143 It was reported previously that CFTR-S768A exhibits a very high open probability, whereas the phospho-mimic Ser-to-Asp S768D CFTR mutant has a very low open probability relative to wild-type CFTR following PKA stimulation (8, 27, 28). 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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177 Regulation of CFTR by Fe3؉ 40442 not reverse Fe3ϩ inhibition found in S768D, which is equivalent to phosphorylated Ser-768 (Fig. 6, C and D). Login to comment
237 Macroscopic currents across inside-out membrane patches excised from transfected HEK293T cells expressing the hCFTR (A), S768A (B), and S768D (C) constructs. 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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156 Thus, if primary phosphorylation of Ser768 inhibits channel activation by curcumin in the presence of ATP, S768D, which is equivalent to phosphorylated Ser768 , should also dampen channel activation by curcumin. Login to comment
157 However, Fig. 6A demonstrates that S768D was also activated by curcumin with ATP. Login to comment
159 In addition, because S768D is negatively charged, an electrostatic attraction between S768D and Lys946 or Lys951 may be impossible because modification of S768C with MTSCE (negatively charged) or MTSET (positively charged) failed to change channel activity (supplemental Fig. S1, A and B). Login to comment
160 On the other hand, S768D is a strong H-bond acceptor (Table 1). 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
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
210 It is interesting that S768D also promoted channel opening by ATP (Fig. 8, D and E). Login to comment
211 Because S768D is equivalent to phosphorylated Ser768 , this finding suggests that phosphorylated Ser768 should not form an inhibitory H-bond. 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
223 However, the activation time became significantly shorter for H950A, S768A, and S768D (Fig. 9, B-E). Login to comment
225 It is very interesting that apparent basal activity of S768A was not so high and was comparable with that seen with S768D (Fig. 9, C and D). 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
236 For S768A or S768D, a basal open probability was only a little higher (Po(app) ϭ 0.0004) than that of WT CFTR, and extracellular cAMP failed to increase channel activity significantly. Login to comment
239 Although S768A/D disrupted hydrogen bonding with His950 , the Fe3ϩ binding was still strong, and S768D may enhance the metal binding affinity (30). Login to comment
246 Furthermore, both S768A and S768D increased sensitivity of CFTR activity to ATP, curcumin, and PKA phosphorylation. 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
253 Finally, even if S768D activity is as low as WT CFTR activity under basal conditions (25), functional studies based on the whole-cell recordings cannot distinguish phosphorylated Ser768 from the unphosphorylated residue if both inhibit channel activity. 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
262 Fig. 6 clearly demonstrates that S768D, which is equivalent to phosphorylated Ser768 , is different from WT CFTR in the inside-out patch. Login to comment
263 First, both S768A and S768D mutants could be activated by ATP followed by curcumin, but WT CFTR could not even be activated in the presence of ATP (Figs. 4 and 6). Login to comment
264 Second, both S768A and S768D were more sensitive to PKA phosphorylation than WT CFTR (Fig. 7D). Login to comment
265 Third, the open probabilities of both S768A and S768D were increased by ATP, whereas that of WT CFTR was not (Fig. 8). Login to comment
267 In fact, not all Ser768 may be phosphorylated in the resting cell because the basal in vivo open probability of S768D was a little higher (Po ϭ 0.0004) than that of WT CFTR (Po ϭ 0.00004) (Fig. 9). 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
293 A-D, unitary currents across cell-attached membrane patches of transfected HEK-293T cells expressing WT CFTR (A), H950A (B), S768A (C), and S768D (D). 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
324 Because S768D also reduced PKA dependence of channel activity even without curcumin involvement (Figs. 6 and 7), most of the Ser768 in WT CFTR may not be phosphorylated in the excised patch, and early phosphorylated Ser768 may not attenuate channel activation by prohibiting PKA phosphorylation at some stimulatory sites, as suggested by Csana´dy and co-workers (24). Login to comment
340 This difference may not result from phosphorylation of Ser768 because a basal open probability of S768D was higher (Po ϭ 0.0004) than that of WT CFTR (Fig. 9). Login to comment
344 Unlike H950A, a basal channel open probability of S768A and S768D was still low (Po ϭ 0.0004) (Fig. 9). Login to comment
346 A similar observation would be seen with H950D/S768D. Login to comment
347 Despite this complex involvement, H950A, S768A, and S768D were more sensitive to forskolin than WT CFTR because they were dramatically activated soon after forskolin was introduced (Fig. 9). 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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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
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
139 Second, the curcumin sensitivity was also increased for K946A, K946D and D835R/D836R/E838R (Fig.3). 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
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

[hide] CFTR Cl- channel and CFTR-associated ATP channel: ... EMBO J. 1998 Feb 16;17(4):898-908. Sugita M, Yue Y, Foskett JK
CFTR Cl- channel and CFTR-associated ATP channel: distinct pores regulated by common gates.
EMBO J. 1998 Feb 16;17(4):898-908., [PMID:9463368]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is regulated by phosphorylation of the R domain and ATP hydrolysis at two nucleotide-binding domains (NBDs). It is controversial whether CFTR conducts ATP or whether CFTR might be closely associated with a separate ATP conductance. To characterize ATP channels associated with CFTR, we analyzed Cl- and ATP single channel-currents in excised inside-out membrane patches from MDCK epithelial cells transiently expressing CFTR. With 100 mM ATP in the pipette and 140 mM Cl- in the bath, ATP channels were associated with CFTR Cl- channels in two-thirds of patches that included CFTR. CFTR Cl- channels and CFTR-associated ATP channels had slope conductances of 7.4 pS and 5.2 pS, respectively, and had distinct reversal potentials and sensitivities to channel blockers. CFTR-associated ATP channels exhibited slow gating kinetics that depended on the presence of protein kinase A and cytoplasmic ATP, similar to CFTR Cl- channels. Gating kinetics of the ATP channels as well as the CFTR Cl- channels were similarly affected by non-hydrolyzable ATP analogues and mutations in the CFTR R domain and NBDs. Our results indicate that phosphorylation- and nucleotide-hydrolysis-dependent gating of CFTR is directly involved in gating of an associated ATP channel. However, the permeation pathways for Cl- and ATP are distinct and the ATP conduction pathway is not obligatorily associated with the expression of CFTR.

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142 To examine this further, we expressed CFTR S-oct-D, which contains eight serine-to-aspartate substitutions in the R domain (S660D, S686D, S700D, S712D, S737D, S768D, S795D and S813D) (Figure 8A). Login to comment
150 To examine this further, we expressed CFTR S-oct-D, which contains eight serine-to-aspartate substitutions in the R domain (S660D, S686D, S700D, S712D, S737D, S768D, S795D and S813D) (Figure 8A). Login to comment

[hide] Regulation of the cystic fibrosis transmembrane co... J Biol Chem. 1993 Sep 25;268(27):20259-67. Rich DP, Berger HA, Cheng SH, Travis SM, Saxena M, Smith AE, Welsh MJ
Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by negative charge in the R domain.
J Biol Chem. 1993 Sep 25;268(27):20259-67., [PMID:7690753]

Abstract [show]
Phosphorylation by cAMP-dependent protein kinase (PKA) regulates the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. We previously showed that in vivo PKA phosphorylated 4 serines (Ser-660, Ser-737, Ser-795, and Ser-813) within the R domain. Here we show that a mutant CFTR lacking all 4 serines can still be phosphorylated by PKA to yield an activated Cl- channel, but channel open-state probability was substantially reduced. We also observed phosphorylation and Cl- channel activity in another mutant lacking all 8 consensus PKA serines in the R domain. We were unable to identify the residual phosphorylation sites by tryptic phosphopeptide mapping. These data suggest two possible interpretations: (a) additional, as yet unidentified, phosphorylation sites within CFTR may also open the channel, or (b) the 4 serines, previously identified as in vivo PKA phosphorylation sites, are the primary regulatory sites within CFTR, but in their absence, other sites can be phosphorylated to open the channel. The additional sites are likely located within the R domain: CFTR delta R-S660A, which lacks much of the R domain (residues 708-835) and replaces Ser-660 with an alanine, was no longer regulated by PKA. Substitution of aspartate for consensus PKA phosphorylation sites in the R domain mimicked the effect of phosphorylation. Mutants containing six or more serine-to-aspartate substitutions generated Cl- channels that opened without PKA phosphorylation. These results suggest that the R domain keeps the channel closed and that phosphorylation of the R domain or insertion of the negatively charged aspartate opens the channel, perhaps by electrostatic interactions.

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121 The single-channel open-stateprobabil- ity for wild-type CFTR (n = 14), CFTR S-Quad-A (S600A,S737A, S712A,S737A,S768A,S795A,S813A) (n = 7), or CFTR S-Oct-D (S660D,S686D,S700D,S712D,S737D,S768D,S795D,S813D)(n = 7in ATP alone (-PKA);n = 10 inPKA and ATP (+PkX))C1-channels was determined as described under "Experimental Procedures." Login to comment
123 S795A,S813A) (n = 5). Login to comment
205 Functional analysisof serine-to-aspartate mutantsof CFTR.A, the changes in SPQ fluorescence ofHeLa cells expressing wild-type CFTR (n = 56), CFTR S-Quad-D (S600D,S737D, S795D,S813D) (n = 24), CFTR S-Hex-D (S660D,S686D,S700D, S712D,S737D,S768D,S795D,S813D)( n = 23), or virus only-infected control cells (n = 53) after substitution of NO; for Iat 0 min. Login to comment
209 B, time course of current changes inan excised, inside-outmembrane patchfrom a HeLa cell expressingCFTR S-Oct-D (S660D,S686D,S700D,S712D,S737D,S768D,S795D,S813D) C1- chan- nels.ATP (0.88 mM)and PKA (75 m)were added tothe cytosolic(bath) side of the membrane as indicated by the burs. Login to comment
213 S737D,S795D,S813D) (n = 37), CFTR S-Oct-D (S660D,S686D,S700D, vitro or in vivo (Figs.6 and 9). Login to comment