ABCMdb

ABCC7 p.Ser660Ala

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PMID: 10792060 [PubMed] Ostedgaard LS et al: "A functional R domain from cystic fibrosis transmembrane conductance regulator is predominantly unstructured in solution."

No. Sentence Comment

26 An example is CFTR-⌬R͞ S660A, in which residues 708-835 are deleted and Ser-660 is mutated to alanine (12, 19, 23). Login to comment
46 10 ͉ 5657-5662 S660A in lipid bilayers (13, 14). Login to comment

PMID: 10962022 [PubMed] Csanady L et al: "Severed channels probe regulation of gating of cystic fibrosis transmembrane conductance regulator by its cytoplasmic domains."

No. Sentence Comment

332 Based on these findings, and the effect of phosphorylated R-domain peptide on ⌬R(708-835)-S660A channels, the phosphorylated R domain was proposed to stimulate channel activity by enhancing the affinity of CFTR for ATP (Winter and Welsh, 1997). Login to comment
357 However, in the absence of the phosphopeptide, the opening rate of ⌬R(708-835)-S660A, with or without PKA, KrCO KPo ( ) was only ‫%03ف‬ that of phosphorylated WT, and increased only to ‫%54ف‬ of the latter, even in the presence of the phosphopeptide (Winter and Welsh, 1997; compare Ma et al., 1997). Login to comment
363 This activation is intriguing, because the effect of PKA on CFTR has generally been attributed to phosphorylation of serine residues within the R domain, based on both biochemical and functional evidence: CNBr cleavage and peptide mapping experiments on CFTR protein prephosphorylated by PKA with ␥-32P ATP found no evidence for phosphorylation outside the R domain (Cheng et al., 1991; Picciotto et al., 1992; Seibert et al., 1995), and PKA no longer stimulated ⌬R(708-835) channels after mutation of serine 660 [⌬R(708-835)-S660A, Rich et al., 1993; compare Ma et al., 1997]. Login to comment
365 Also, lack of response of ⌬R(708-835)-S660A channels to PKA could reflect steric constraints introduced by linking residue 707 to 836. Login to comment
372 Interestingly, a likely ␣-helical section of CFTR`s NH2 terminus (within residues 46-60) was recently reported to bind to the R domain, and enhance channel activity (Naren et al., 1999): point mutations in that stretch of amino acids impaired the gating of WT (an effect apparently on channel closing rate), but not of ⌬R(708-835)-S660A, channels. Login to comment

PMID: 11053017 [PubMed] Baldursson O et al: "Contribution of R domain phosphoserines to the function of CFTR studied in Fischer rat thyroid epithelia."

No. Sentence Comment

56 In each of the serine to alanine mutants, S660A, S737A, S795A, and S813A, alanine replaced serine at the designated residue. Login to comment
107 The S660A and S813A variants generated small but significant cAMP-stimulated Cl- currents. Login to comment
112 The S660A and S813A variants generated currents, but they were no greater than those obtained with SQuad-A. Login to comment
186 Mutation of either residue alone significantly decreased current; with maximal stimulation by cAMP agonists, neither the S660A nor the S813A mutant gave more current than the S-Quad-A Fig. 5. Login to comment
195 With submaximal cAMP agonists, S660A- and S813A-generated current was also reduced, but it was slightly greater than that obtained with S-Quad-A. Login to comment

PMID: 12502786 [PubMed] Naren AP et al: "A macromolecular complex of beta 2 adrenergic receptor, CFTR, and ezrin/radixin/moesin-binding phosphoprotein 50 is regulated by PKA."

No. Sentence Comment

198 Deleting most of the R domain of CFTR (S660A ⌬RCFTR; lacking amino acids 708-835; ref. 32) abolished the PKA sensitivity of CFTR binding in the complex (Fig. 4 E and F). Login to comment

PMID: 15155835 [PubMed] Ai T et al: "Capsaicin potentiates wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride-channel currents."

No. Sentence Comment

142 We further explored the action of capsaicin using CFTR mutants whose eight major PKA consensus serines are substituted with alanine (S660A, S686A, S700A, S712A, S737A, S768A, S795A, and S813A), so called S-oct-A or 8SA. Login to comment

PMID: 15304546 [PubMed] Sugita M et al: "Molecular dissection of the butyrate action revealed the involvement of mitogen-activated protein kinase in cystic fibrosis transmembrane conductance regulator biogenesis."

No. Sentence Comment

39 To construct the pEGFP-⌬R-CFTR mammalian expression vector, an AflII and SacI fragment from pTM1-CFTR⌬R-S660A containing the C-terminal region of CFTR was swapped with the wild-type cassette present in pEGFP-C1-CFTR. Login to comment
104 In contrast, the B form of the deletion mutant GFP-⌬R-CFTR, which lacks much of the R domain and replaces Ser660 with alanine (Rich et al., 1993), was detected as an approximately 15-kDa increase in electrophoresis mobility (ϳ158 kDa) (Fig. 1B). Login to comment
303 Acknowledgments We thank Dr. K. Foskett for providing pEGFP-CFTR, Dr. M. Welsh for CFTR⌬R-S660A, and Dr. E. Nishida for LA-SDSE-MAPKK and SASA-MAPKK. Login to comment

PMID: 15596536 [PubMed] Csanady L et al: "Functional roles of nonconserved structural segments in CFTR's NH2-terminal nucleotide binding domain."

No. Sentence Comment

204 Indeed, the single point mutation S660A was found to have a negligible effect on the gating of phosphorylated CFTR channels in excised patches exposed to saturating [ATP] (Winter and Welsh, 1997). Login to comment
206 These results on gating of strongly phosphorylated CFTR channels in excised patches do not contradict the finding (Wilkinson et al., 1997) of a Յ2-fold reduction in sensitivity to activation by IBMX (hence presumably by PKA) of S660A or S670A CFTR channels in intact oocytes. Login to comment

PMID: 15657297 [PubMed] Wang W et al: "Reversible silencing of CFTR chloride channels by glutathionylation."

No. Sentence Comment

43 The S660A/⌬R-CFTR mutant and all but one of the alanine-substituted cysteine mutants were provided by M. Welsh (University of Iowa, Iowa City, IA) (Rich et al., 1991; Cotton and Welsh, 1997) and were subcloned into the pCDNA3 expression vector (Invitrogen). Login to comment
51 HEK-293T cells that were transfected with S660A/⌬R-CFTR or with the cys-free constructs were grown overnight at 27ЊC because these mutants are temperature-sensitive ER processing mutants as determined by immunoblot analysis. Login to comment
203 Fig. 9 (A and B) shows that S660A/⌬R-CFTR, which was previously shown to have moderate channel activity in the absence of PKA (Rich et al., 1991), was substantially and reversibly inhibited by diamide/GSH in the absence of added kinase. Login to comment
205 Interestingly, Grx could rescue completely the currents mediated by S660A/⌬R-CFTR, which indicates that the Grx-insensitive oxidation previously observed for the WT channel (Fig. 4) requires an intact R domain. Login to comment
259 (A) Inhibitory effect of 20 ␮M diamide/ GSH on macroscopic current mediated by ⌬R-S660A-CFTR in patch excised from transfected HEK-293T cell. Login to comment
260 (B) Mean data for ⌬R-S660A-CFTR showing inhibition by diamide/GSH (20 ␮M) and recovery by E. coli Grx1 (4 ␮M) plus 1 mM GSH. Login to comment

PMID: 15857825 [PubMed] Wang W et al: "Activating cystic fibrosis transmembrane conductance regulator channels with pore blocker analogs."

No. Sentence Comment

27 Cells expressing ⌬F508-CFTR or ⌬R-S660A-CFTR (where "R" is the regulatory domain) were grown for 1-2 days at 27 °C (low temperature-corrected) because these mutants are temperature-sensitive endoplasmic reticulum processing mutants that exhibit low surface expression when cells are cultured at 37 °C. Login to comment
105 Second, NPPB-AM markedly stimulated the currents mediated by a regulatory domain deletion mutant (⌬R-S660A-CFTR) (Fig. 3A) that exhibits low constitutive activity in the absence of PKA (23). Login to comment
157 A, NPPB-AM and NPPB markedly stimulate ⌬R-S660A-CFTR currents minus PKA across an excised HEK-293T patch. Login to comment
203 NPPB-AM and NPPB are capable of stimulating CFTR opening without affecting channel phosphorylation by PKA, i.e. these compounds enhance wild-type or ⌬R-S660A-CFTR activity in the absence of active kinase. Login to comment
208 In this regard, the ⌬R-S660A-CFTR construct used here (23) has much lower channel activity than highly phosphorylated wild-type channels (Po estimated at Ͻ0.05) (29), although this activity is not dependent on PKA. Login to comment
234 Michael Welsh and John Wakefield for sharing the ⌬R-S660A-CFTR construct and the ⌬F508-CFTR-transfected CFBE41o-cells, respectively. Login to comment

PMID: 17178710 [PubMed] Wang W et al: "Curcumin opens cystic fibrosis transmembrane conductance regulator channels by a novel mechanism that requires neither ATP binding nor dimerization of the nucleotide-binding domains."

No. Sentence Comment

50 The ⌬R/S660A-CFTR mutant was provided by M. Welsh (University of Iowa) (23), subcloned into the pCDNA3 vector, and truncated by PCR mutagenesis to make ⌬1198/⌬R/S660A-CFTR. Login to comment
204 D, curcumin activates ⌬1198/⌬R-S660A-CFTR channels in the absence of ATP and PKA. Login to comment
211 We also tested the effect of curcumin on channels that lack a large portion of the R domain in addition to NBD2 (⌬1198/⌬R/S660A), because previous work has shown that ⌬R/S660A-CFTR channels exhibit constitutive activity in the absence of added kinase (23). Login to comment
267 Acknowledgments-We thank J. P. Clancy for the HeLa cells stably transfected with W1282X-CFTR and M. J. Welsh for the ⌬R/S660A-CFTR construct. Login to comment

PMID: 17660831 [PubMed] Baker JM et al: "CFTR regulatory region interacts with NBD1 predominantly via multiple transient helices."

No. Sentence Comment

19 The R region may have an additional stimulatory role, as shown by CFTR channels lacking much of the R region (D708À835/S660A), which gate independently of PKA yet are further stimulated by the addition in trans of phosphorylated R region (residues 645À835)15. Login to comment

PMID: 18989824 [PubMed] Hughes LK et al: "Potentiation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- currents by the chemical solvent tetrahydrofuran."

No. Sentence Comment

7 Similar results were obtained with the DR-S660A-CFTR Cl( channel that is not regulated by PKA-dependent phosphorylation and using 2?deoxy-ATP, which gates wild-type CFTR more effectively than ATP. Login to comment
28 Materials and methods Cells and cell culture For this study, we used Fischer rat thyroid (FRT) epithelial cells expressing wild-type, F508del- and G551D- human CFTR [8] and mouse mammary epithelial (C127) cells stably expressing either wild-type human CFTR or the variant DR-S660A-CFTR Figure 1. Login to comment
179 THF potentiates the DR-S660A-CFTR Cl( channel and wild-type CFTR gated by 2?-deoxy-ATP The lack of effect of THF on the CFTR Cl( channel after phosphorylation by PKA, suggests that the RD might be a target for the actions of THF. Login to comment
180 To test this hypothesis, we used the CFTR construct DR-S660A [9], performing the same experiments on this CFTR variant as those with wild-type CFTR. Login to comment
181 The CFTR mutant DR-S660A lacks much of the RD (residues 708Á835) and disables the dibasic phosphorylation site at serine 660. Login to comment
183 Supplementary Information (SI) Figure 1 (online version only) demonstrates that THF (100 mM) augments the activity of DR-S660A-CFTR Cl( channels by increasing the frequency of channel openings. Login to comment
227 Our observation that THF potentiates the DR-S660A-CFTR Cl( channel argues that the solvent interacts with a site distinct from the RD. Login to comment

PMID: 19837660 [PubMed] Chen JH et al: "Direct sensing of intracellular pH by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel."

No. Sentence Comment

46 These included (i) mouse mammary epithelial cells (C127 cells) expressing wild-type human CFTR, the CFTR variant ⌬R-S660A (13) or the CF mutant G1349D (14), (ii) Fischer rat thyroid epithelial cells expressing the CF mutant G551D (15), and (iii) NIH-3T3 cells expressing the CFTR construct K1250M (16). Login to comment
158 To test this hypothesis, we adopted two strategies; first, we employed the CFTR construct ⌬R-S660A that deletes a large part of the RD and is not regulated by PKA-dependent phosphorylation (13) to explore separately how pHi influences the function of the RD and NBDs. Login to comment
161 Fig. 5A shows representative recordings of a single ⌬R-S660A-CFTR Cl-channel at different pHi, whereas Fig. 5, B-D quantifies the effects of pHi on channel gating. Consistent with previous studies (13), at pHi 7.3 the Po of ⌬R-S660A-CFTR was attenuated markedly compared with that of wild-type CFTR FIGURE 4. Login to comment
176 Acidic pHi potentiates the activity of ⌬R-S660A-CFTR. Login to comment
177 A, representative recordings show the effects of pHi on the activity of a single ⌬R-S660A-CFTR Cl-channel. Login to comment
178 Dotted lines indicate where the channel is closed, and downward deflections correspond to channel openings. B, C, and D, effects of pHi on the Po, MBD, and IBI of ⌬R-S660A (columns) and wild-type CFTR (circles). Login to comment
179 Data are means Ϯ S.E. (B, n ϭ 6 for all data; C and D, ⌬R-S660A-CFTR, n ϭ 3; wild-type-CFTR, n Ն 6); asterisks indicate ⌬R-S660A-CFTR values that are significantly different from the pHi 7.3 control (p Ͻ 0.05). Login to comment
181 Acidifying to pHi 6.3 potentiated the Po of wild-type CFTR 0.4-fold, but that of ⌬R-S660A-CFTR 3.3-fold (Fig. 5B). Login to comment
182 Fig. 5, C and D, demonstrates that the marked potentiation of ⌬R-S660A-CFTR Po at pHi 6.3 was primarily caused by a 0.9-fold decrease in IBI, but it was also enhanced by a 1.5-fold increase of MBD. Login to comment
183 Alkalinizing to pHi 8.3 decreased the Po of wild-type CFTR 0.3-fold, but was without effect on the Po of ⌬R-S660A-CFTR (Fig. 5B). Login to comment
184 An explanation for this striking difference between wild-type and ⌬R-S660A-CFTR is shown in Fig. 5D. Login to comment
185 Between pHi 7.3 and 8.3, the IBI of wild-type CFTR increased 0.3-fold, whereas that of ⌬R-S660A-CFTR decreased 0.5-fold (Fig. 5D). Login to comment
186 Fig. 5D also reveals that the relationship between IBI and pHi is linear for wild-type CFTR, but bell-shaped for ⌬R-S660A-CFTR over the pHi range 6.3-8.3. Login to comment
187 We interpret the failure of pHi 8.3 to attenuate ⌬R-S660A-CFTR channel gating to suggest that alkaline pHi might inhibit CFTR channel gating, at least in part, by modulating RD function. Login to comment
188 However, the robust potentiation of ⌬R-S660A-CFTR channel gating at pHi 6.3 argues that acidic pHi likely enhances CFTR channel gating by acting on sites distinct from the RD. Login to comment
254 Second, studies of the CFTR construct ⌬R-S660A suggest that OH- ions might inhibit CFTR channel gating, at least in part, by modulating RD function. Login to comment
283 In contrast to the effects of strong alkaline pHi on wild-type CFTR, our studies of the ⌬R-S660A-CFTR Cl-channel at pHi 8.3 argue that alkaline pHi inhibits CFTR channel gating, at FIGURE 8. Login to comment
290 The reason why ⌬R-S660A-CFTR is not inhibited at pHi 8.3 is that its prolonged IBI is attenuated, not extended, at this pHi value. Login to comment
292 However, it is interesting to note that for both ⌬R-S660A-CFTR and the ATP-binding site 1 mutant D572N-CFTR, the relationship between pHi and IBI is bell-shaped, not linear, between pHi 6.3 and 8.3 (Figs. 5D and 8B). Login to comment

PMID: 20133716 [PubMed] Wang W et al: "ATP-independent CFTR channel gating and allosteric modulation by phosphorylation."

No. Sentence Comment

213 ΔR-S660A-CFTR (29) was provided by Michael Welsh (University of Iowa, Iowa City, IA).Cells transfected with cysteine-free or ΔR constructs were cultured at 26-28 °C for 24-48 h before patch clamping to increase expression of these mutants, which mature less efficiently than WT-CFTR (16, 17, 30). Login to comment

PMID: 20952391 [PubMed] Wang G et al: "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."

No. Sentence Comment

45 In addition, removal of a segment 760-783 or 817-838 (NEG2) or much of the R domain (⌬708-835/ S660A) from CFTR eliminates the PKA dependence of channel activity (26-28). Login to comment

PMID: 21059651 [PubMed] Wang G et al: "The inhibition mechanism of non-phosphorylated Ser768 in the regulatory domain of cystic fibrosis transmembrane conductance regulator."

No. Sentence Comment

42 In addition, removal of residues 760-783 or 817-838 (NEG2) or much of the R domain (⌬708-835/S660A) from CFTR eliminates PKA dependence of channel activity (26-28). Login to comment
62 ⌬R-S660A-CFTR was provided by Michael Welsh (University of Iowa) (31). Login to comment

PMID: 21658632 [PubMed] Becq F et al: "Pharmacological therapy for cystic fibrosis: from bench to bedside."

No. Sentence Comment

244 5-nitro-2-(3-phenyl propylamino)benzoate (NPPB) analogues [142] BHK, HEK293, CFBE41o- transfected WT, F508del, R-S660A-CFTR Electrophysiology NPPB analogues stimulate WT and F508del-CFTR channel activity at submicromolar concentrations without blocking the channel pore. Login to comment

PMID: 9769419 [PubMed] Lansdell KA et al: "Regulation of murine cystic fibrosis transmembrane conductance regulator Cl- channels expressed in Chinese hamster ovary cells."

No. Sentence Comment

248 However, recent data suggest that the effect of fluoride may be independent of phosphatase inhibition, because fluoride stimulated the human CFTR mutant CFTRÄR-S660A (Berger et al. 1998). Login to comment

PMID: 9922375 [PubMed] Sheppard DN et al: "Structure and function of the CFTR chloride channel."

No. Sentence Comment

521 When S660 in CFTRDR was mutated to alanine dan (42) proposed a two-domain model of the R domain: RD1 (amino acids 587-672) and RD2 (amino acids 679-(CFTRDR-S660A), PKA failed to stimulate channel activity. Login to comment
526 In addition, deletion studies of portions of the R domain suggest thatout that PKA cannot interact with CFTRDR-S660A, even though it does interact with CFTRDR. there are two halves to the R domain with the first conserved in other ABC transporters and the second uniqueLike CFTRDR, the amino-terminal portion of CFTR (D836X, which contains MSD1, NBD1, and the R domain) to CFTR (102, 103). Login to comment
533 Alternatively, the PKA-independent activity of D836X could be due to As described above, phosphorylation by PKA, deletion of FIG. 11 Effect of a recombinant R domain (R1, residues 645-834) on CFTRDR/S660A channel in presence of PKA. Login to comment
535 B and C: effect of R1 on rates of entry into and exit from burst for CFTR-DR/S660A channels in presence of PKA. Login to comment

PMID: 9922377 [PubMed] Gadsby DC et al: "Control of CFTR channel gating by phosphorylation and nucleotide hydrolysis."

No. Sentence Comment

133 Along the same lines, coimmunoprecipitation experiments suggested that phosphorylation of functional decrements attending incorporation of addi- P33-8/ 9j0e$$ja10 01-13-99 16:19:50 prsa APS-Phys Rev tional mutations at Ser-422 to yield a 10SA mutant (31), single-channel currents in mutant CFTR missing R-domain residues 708-835 (CFTRDR), with (163) or withoutat Ser-753 to give the 11SA mutant (178), and at four remaining R-domain serines and threonines (S670, T690, (122, but cf. Ref. 164) additional mutation of Ser-660 to alanine (CFTRDR-S660A), does not constitute proof thatT787, and S790) mutated together to yield a (11/4)SA mutant (177). Login to comment

PMID: 23060444 [PubMed] Wang G et al: "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."

No. Sentence Comment

37 R-S660A-CFTR was provided by Michael Welsh (University of Iowa, Iowa City, IA). Login to comment
41 èc;R-S660A-CFTR was provided by Michael Welsh (University of Iowa, Iowa City, IA). Login to comment

PMID: 9530164 [PubMed] Berger HA et al: "Fluoride stimulates cystic fibrosis transmembrane conductance regulator Cl- channel activity."

No. Sentence Comment

134 Effect of Fon CFTR⌬R-S660A current. Because CFTR requires phosphorylation for activation and because Fis known to inhibit several protein phosphatases (34), we asked whether F- might stimulate CFTR activity by inhibiting a phosphatase. Login to comment
135 To test this possibility, we studied a CFTR variant (CFTR⌬R-S660A) in which part of the R domain (amino acids 708-835) is deleted and a remaining phosphorylation site (serine 660) is mutated to alanine (30). Login to comment
137 Figure 5 shows that addition of F- increased the current in CFTR⌬R-S660A (n ϭ 3), suggesting that F- did not stimulate the channel by inhibiting a phosphatase. Login to comment
157 Effect of Fon Cl- current in CFTR⌬R-S660A. Login to comment
158 Data show time course of current in an excised, inside-out membrane patch from NIH/3T3 cells expressing CFTR⌬R-S660A. Login to comment
182 The effect of F- was likely independent of phosphatase inhibition, since F- increased current in CFTR⌬R-S660A. Login to comment
201 It seemed unlikely that F- stimu- lated through an interaction with the R domain because F- stimulated CFTR⌬R-S660A. Login to comment
127 Effect of F2 on CFTRDR-S660A current. Because CFTR requires phosphorylation for activation and because F2 is known to inhibit several protein phosphatases (34), we asked whether F2 might stimulate CFTR activity by inhibiting a phosphatase. Login to comment
128 To test this possibility, we studied a CFTR variant (CFTRDR-S660A) in which part of the R domain (amino acids 708-835) is deleted and a remaining phosphorylation site (serine 660) is mutated to alanine (30). Login to comment
130 Figure 5 shows that addition of F2 increased the current in CFTRDR-S660A (n 5 3), suggesting that F2 did not stimulate the channel by inhibiting a phosphatase. Login to comment
150 Effect of F2 on Cl2 current in CFTRDR-S660A. Login to comment
151 Data show time course of current in an excised, inside-out membrane patch from NIH/3T3 cells expressing CFTRDR-S660A. Login to comment
175 The effect of F2 was likely independent of phosphatase inhibition, since F2 increased current in CFTRDR-S660A. Login to comment
193 It seemed unlikely that F2 stimulated through an interaction with the R domain because F2 stimulated CFTRDR-S660A. Login to comment

PMID: 9463368 [PubMed] Sugita M et al: "CFTR Cl- channel and CFTR-associated ATP channel: distinct pores regulated by common gates."

No. Sentence Comment

128 We first examined the deletion mutant CFTR∆R-S660A, Fig. 8. Login to comment
131 The residues altered in CFTR∆R-S660A, CFTR S-oct-D, K464A and K1250A mutants are shown. Login to comment
132 (B) Current traces from a MDCK cell expressing CFTR∆R-S660A in the presence or absence of PKA at various membrane potentials (representative of five independently observed channels). Login to comment
137 which lacks much of the R domain and replaces Ser-660 with alanine (Figure 8A). Login to comment
139 The CFTR∆R-S660A Cl- channels were active in MDCK cells (Po ϭ 0.05 Ϯ 0.003, nϭ3) requiring the presence of 903 cytosolic ATP alone (Figure 8B), and the gating activity did not increase following addition of PKA, in agreement with the previous results. Login to comment
140 However, the CFTR∆R-S660A mutant eliminated CFTR-associated ATP channel activities, even in the presence of both PKA and ATP (nϭ5; Figure 8B). Login to comment
272 Acknowledgements We thank M.Welsh for providing the CFTR∆R-S660A and CFTR S-oct-D mutants, R.Kopito for providing the K1250A and K464A mutants, J.Engelhardt for providing the R347E mutant, U.Patel for her precious technical help and D.Mak for helpful discussions. Login to comment
135 We first examined the deletion mutant CFTRƊR-S660A, Fig. 8. Login to comment
138 The residues altered in CFTRƊR-S660A, CFTR S-oct-D, K464A and K1250A mutants are shown. Login to comment
145 which lacks much of the R domain and replaces Ser-660 with alanine (Figure 8A). Login to comment
147 The CFTRƊR-S660A Cl-channels were active in MDCK cells (Po afd; 0.05 afe; 0.003, nafd;3) requiring the presence of 903 cytosolic ATP alone (Figure 8B), and the gating activity did not increase following addition of PKA, in agreement with the previous results. Login to comment
148 However, the CFTRƊR-S660A mutant eliminated CFTR-associated ATP channel activities, even in the presence of both PKA and ATP (nafd;5; Figure 8B). Login to comment
282 Acknowledgements We thank M.Welsh for providing the CFTRƊR-S660A and CFTR S-oct-D mutants, R.Kopito for providing the K1250A and K464A mutants, J.Engelhardt for providing the R347E mutant, U.Patel for her precious technical help and D.Mak for helpful discussions. Login to comment

PMID: 9305845 [PubMed] Winter MC et al: "Stimulation of CFTR activity by its phosphorylated R domain."

No. Sentence Comment

124 Here we test this by adding an R-domain peptide to a CFTR variant in which much of the R domain had been deleted (CFTR-DR/S660A): in contrast to predictions, we found that adding an unphosphorylated R domain to CFTR-DR/S660A did not inhibit activity, whereas a phosphorylated R-domain peptide stimulated activity. Login to comment
125 To investigate how phosphorylation controls activity, we studied channel gating and found that phosphorylation of the R domain increases the rate of channel opening by enhancing the sensitivity to ATP. Our results indicate that CFTR is regulated by a new mechanism in which phosphorylation of one domain stimulates the interaction of ATP with another domain, thereby increasing activity. We previously studied a CFTR variant, CFTR-DR/S660A, in which much of the R domain (residues 708-835) was deleted and the phosphorylation site was removed at Ser 660 by mutation to alanine4 . Login to comment
128 This model predicts that the activity of CFTR-DR/S660A should be similar to that of phosphorylated wild-type CFTR. Login to comment
129 However, when we examined the single-channel properties of CFTR-DR/ S660A, we found that Po was 0:13 Ϯ 0:01 (1 mM ATP, n ¼ 7), much lower than that of phosphorylated wild-type CFTR (Po ¼ 0:46 Ϯ 0:02; 1 mM ATP, n ¼ 17, P Ͻ 0:001). Login to comment
131 But deletion of the R domain in CFTR-DR/S660A could still have additional nonspecific effects on channel structure which might reduce activity. We considered, therefore, that the phosphorylated R domain might stimulate the channel and that the defective functioning of CFTR-DR/S660A is due to its lacking a phosphorylated R domain. Login to comment
132 To test this idea, we added a recombinant R domain (R1, residues 645-834, similar to a peptide reported previously10 ) to the cytosolic surface of CFTR-DR/S660A and measured activity by using the excised, inside-out patch-clamp technique. Login to comment
136 Additional evidence that the effect of phosphorylation was on R1 rather than on CFTR-DR/S660A came from our finding that addition of phosphorylated R1 in the presence of protein-kinase-inhibitor peptide under conditions that block the effect of PKA11 gave similar results (43:2 Ϯ 3:4% increase; n ¼ 3). Login to comment
141 Our results also suggest that the low Po found for CFTR-DR/S660A results at least in part from a lack of stimulation by a phosphorylated R domain, rather than from nonspecific structural changes. Login to comment
142 The fact that R1 did not increase the Po of CFTR-DR/S660A to wild-type CFTR values may be because the endogenous R domain is constrained in the correct orientation by the site of interaction; the interaction of exogenously applied R1 with CFTR-DR/S660A is probably less efficient. Login to comment
143 Our results contrast with a report that addition of an in vitro translation mixture containing a different unphosphorylated R domain (residues letters to nature 294 NATURE |VOL 389 |18 SEPTEMBER 1997 a -20 -10 0 I(pA) 5 min R1 PKA b-30 -20 -10 0 5 min R1 ∆R/S660A Wild type 0 10 20 30 40 50 - + - + c * PKA I(pA)ChangewithR1addition(%) Figure 1 Effect of R1 on CFTR-DR/S660A and wild-type CFTR. Login to comment
144 a, b, Time course of Cl- current in excised, inside-out patches containing many CFTR-DR/S660A channels during addition of PKA and R1 (50 nM), as indicated by bars. Login to comment
148 c, Effect of R1 in the presence and absence of PKA on current measured from CFTR-DR/S660A and wild-type CFTR. Login to comment
152 For CFTR-DR/S660A, n is 10 and 9; for wild-type CFTR, n is 4 and 5, in the absence or presence of PKA, respectively. Login to comment
156 To find out how the phosphorylated R domain stimulates CFTR-DR/S660A, we investigated channel gating. Login to comment
158 Figure 2 shows that phosphorylated R1 increases the rate at which CFTR-DR/S660A opened into bursts, without affecting the rate of closure from bursts. Login to comment
166 Thus addition of the phosphorylated R domain to CFTR-DR/ S660A and mutation of phosphorylation sites in wild-type CFTR had opposite effects on the same kinetic step in channel gating, the rate of channel opening. Login to comment
170 Figure 4 shows that the mutations reduced Po at low concentrations of ATP; Po was letters to nature NATURE |VOL 389 |18 SEPTEMBER 1997 295 Figure 2 Effect of R1 on CFTR-DR/S660A channels in the presence of PKA. Login to comment
172 b, c, Effect of R1 on the rates of entry into and exit from a burst for CFTR-DR/S660A channels in the presence of PKA. Login to comment
185 Number of experiments for b/c and d were: 17/6 for wild-type (WT), 8 for wild-type (-PKA), 8/7 for S660A, 5/5 for S737A, 8/6 for S795A, 9/7 for S813A, and 11/4 for S-Quad-A. Login to comment
188 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Po 0 0.2 0.4 0.6 0.8 1 10 [ATP] (mM) S813A S795A S737A S660A WT (-PKA) WT Figure 4 Effect of ATP concentration on Po of CFTR containing phosphorylation site mutations. Login to comment
193 This might also be true for CFTR-DR/S660A. Login to comment
200 When phosphorylation modifies the R domain, it might have two effects: the first could be permissive, releasing a steric inhibition, consistent with our finding that deletion of the R domain produces a channel that is partially active; the second effect might be stimulatory, facilitating interaction of the NBDs with ATP, which would be consistent with results we obtained when a phosphorylated R domain was added to CFTR-DR/S660A and with the effects of CFTR-containing mutations in the endogenous R domain. Login to comment
126 To investigate how phosphorylation controls activity, we studied channel gating and found that phosphorylation of the R domain increases the rate of channel opening by enhancing the sensitivity to ATP. Our results indicate that CFTR is regulated by a new mechanism in which phosphorylation of one domain stimulates the interaction of ATP with another domain, thereby increasing activity. We previously studied a CFTR variant, CFTR-DR/S660A, in which much of the R domain (residues 708-835) was deleted and the phosphorylation site was removed at Ser 660 by mutation to alanine4 . Login to comment
130 However, when we examined the single-channel properties of CFTR-DR/ S660A, we found that Po was 0:13 6 0:01 (1 mM ATP, n &#bc; 7), much lower than that of phosphorylated wild-type CFTR (Po &#bc; 0:46 6 0:02; 1 mM ATP, n &#bc; 17, P , 0:001). Login to comment
133 To test this idea, we added a recombinant R domain (R1, residues 645-834, similar to a peptide reported previously10 ) to the cytosolic surface of CFTR-DR/S660A and measured activity by using the excised, inside-out patch-clamp technique. Login to comment
137 Additional evidence that the effect of phosphorylation was on R1 rather than on CFTR-DR/S660A came from our finding that addition of phosphorylated R1 in the presence of protein-kinase-inhibitor peptide under conditions that block the effect of PKA11 gave similar results (43:2 6 3:4% increase; n &#bc; 3). Login to comment

PMID: 9252549 [PubMed] Wilkinson DJ et al: "CFTR activation: additive effects of stimulatory and inhibitory phosphorylation sites in the R domain."

No. Sentence Comment

85 Activation constants for wild-type and single serine-to-alanine mutant CFTRs PICA Phosphorylation CFTR K4, n-m n Class In vivo In vitro Wild type 0.65 t 0.08 26 S64lA 0.6520.12 14 N No concensus S660A 1.21+ 0.19* 11 S ++ ++ S670A 0.99t0.12* 12 s ? Login to comment
107 Symbols show averaged IBMX dose-response data for wild-type CFTR and several single-site serine-to-alanine mutants (S660A, S737A, S768A, S795A, and S813A). Login to comment
189 Gadsby and Nairn (10) called attention to the fact that the open probability of constructs like CFTRAR-S660A is well below that of wild-type channels. Login to comment

PMID: 8631756 [PubMed] Ma J et al: "Phosphorylation-dependent block of cystic fibrosis transmembrane conductance regulator chloride channel by exogenous R domain protein."

No. Sentence Comment

159 However, CFTR⌬R-S660A, a molecule with amino acids 708-835 deleted (to render it constitutively open) with the remaining consensus PKA site mutated, is insensitive to activation by PKA (9), suggesting that activation by phosphorylation takes place in the R domain. Login to comment

PMID: 7532021 [PubMed] Carson MR et al: "Phosphate stimulates CFTR Cl- channels."

No. Sentence Comment

187 To determine whether the R domain is necessary for Pi stimulation of CFITR Cl- current, we examined two mutant CFTRs; CFTRAR and CFTRAR-S660A. Login to comment
189 In CFTRAR-S660A, amino acids 708-835 are deleted and serine 660 (a PKA phosphorylation site) is mutated to alanine. Login to comment
191 Stimulation was not dependent on the presence of serine 660, because we observed similar stimulation in the mutant CFTRAR-S660A (n = 6, not shown). Login to comment
186 To determine whether the R domain is necessary for Pi stimulation of CFITR Cl-current, we examined two mutant CFTRs; CFTRAR and CFTRAR-S660A. Login to comment
188 In CFTRAR-S660A, amino acids 708-835 are deleted and serine 660 (a PKA phosphorylation site) is mutated to alanine. Login to comment
190 Stimulation was not dependent on the presence of serine 660, because we observed similar stimulation in the mutant CFTRAR-S660A (n = 6, not shown). Login to comment

PMID: 7690753 [PubMed] Rich DP et al: "Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by negative charge in the R domain."

No. Sentence Comment

8 The additional sites are likelylocated within the R domain: CFTRAR-S660A, which lacksmuch of the R domain (residues 708-835)and replaces Ser-660 withan alanine,was no longer regulated byPKA. Login to comment
66 In vivo phosphorylation ofwild-typeand mutant CFTR CFTR S-Quad-A (S66OA,S737A,S795A,S813A), or CFTR S-Oct-A (S660A,S686A,S700A,S712A,S737A,S768A,S795A,S813A) weretran- siently expressed in HeLa cells. Login to comment
94 Single-channelanalysis of gle-channel traces from an excised, in-cell expressing CFTR S-Quad-A side-out membrane patch from a HeLa (S660A,S737A,S795A,S813A).Each trace is about 14s long. Login to comment
109 RESULTS Serine-to-Alanine Substitutionsin the R Domain Do NotAbolish CFTR Cl- Channel Activity-& a firststep to address the possibility that additional PKA phosphorylation sites might regulate CFTR, we asked whether the S-Quad-A (S660A,S737A,S795A,S813A) mutant could still bephosphorylated in vivofollowing CAMPstimulation. Login to comment
124 CFTR S-Oct-A (S660A,S686A,S700A, CFTR A 795 737 4+B5 -813 B," -02 -B3 sion was high, we expected the mutant channels to be less active than wild-type CFTR C1-channels. Login to comment
139 CFTR (A) or CFTR S-Quad-A (S660A,S737A,S795A,S813A)( B )were transiently expressed inCOS-7 cells. Login to comment
161 CFTRAR-S660A is similar toCFTRAR except that Ser-660, the sole remaining in vivo PKA phosphorylation site (13), is mutatedto alanine. Login to comment
166 CFTRAR-S660A C1-channels had biophysical properties similar towild-type CFTR C1-channels: single-channel slope conductance was 9.2 *0.4 pS (n = 3).The poof CFTRARS660ACl- channels was0.15 *0.04 (n= 6) in the presence ofATPalone and did not increasefollowingaddition of PKA (n = 4) (Fig. lOB). Login to comment
174 Fig. 1lA shows that simultaneous substitutionof the four in vivo PKA sites with aspartates (in the S-Quad-D mutant) did not generate constitutively active CFTR C1-channels as assessed by the SPQ fluorescence assay: the mutant channels opened only after stimulation by CAMP.We observed similar results withCFTR S-Quint-D which contained substitutionsof A MockS-Hept-A C S-OCt-A transfected (A) or were transfected with pMT-CFTR S-Hept-A (S660A,S686A,S700A,S712A,S737A,S768A,S795A) (B)or pMT-CFl`R S-Oct-A FIG.9. Login to comment
176 COS-7 cells were mock- (S660A,S686A,S700A,S712A,S737A,S768A,S795A,S813A)(C). Login to comment
232 CFTRAR-S660A contains only one "nonclassic" (R-X-X-Sor R-X-S) PKA phosphorylation site (Ser-753)in thedeleted segment; its consensus sequence is R-I-S.Alternatively, if PKAphosphorylated "nonclassic"sites within other partsof CFTR which interacted with theR domain, then deletion of part of the R domainwould disrupt those interactions whether or not the additional sites were phosphorylated. Login to comment
234 Finally, we cannot rule out thepossi- bility that PKA simply cannot interact with CFTRAR-S660A, even though it does interact withCFTRAR (14). Login to comment
7 The additional sites are likelylocated within the R domain: CFTRAR-S660A, which lacksmuch of the R domain (residues 708-835)and replaces Ser-660 with an alanine,was no longer regulated byPKA. Login to comment
65 In vivo phosphorylation ofwild-typeand mutant CFTR CFTR S-Quad-A (S66OA,S737A,S795A,S813A), or CFTR S-Oct-A (S660A,S686A,S700A,S712A,S737A,S768A,S795A,S813A) were transiently expressed in HeLa cells. Login to comment
95 Single-channelanalysis of gle-channel traces from an excised, in-cell expressing CFTR S-Quad-A side-out membrane patch from a HeLa (S660A,S737A,S795A,S813A).Each trace is about 14s long. Login to comment
111 RESULTS Serine-to-Alanine Substitutions in the R Domain Do NotAbolish CFTR Cl-Channel Activity-& a firststep to address the possibility that additional PKA phosphorylation sites might regulate CFTR, we asked whether the S-Quad-A (S660A,S737A,S795A,S813A) mutant could still be phosphorylated in vivofollowing CAMPstimulation. Login to comment
126 CFTR S-Oct-A (S660A,S686A,S700A, CFTR A 795 737 4+B5 - 8 1 3 B," -02 -B3 sion was high, we expected the mutant channels to be less active than wild-type CFTR C1-channels. Login to comment
142 CFTR (A) or CFTR S-Quad-A (S660A,S737A,S795A,S813A) ( B )were transiently expressed inCOS-7 cells. Login to comment
165 CFTRAR-S660A is similar toCFTRAR except that Ser-660, the sole remaining in vivo PKA phosphorylation site (13), is mutatedto alanine. Login to comment
170 CFTRAR-S660A C1-channels had biophysical properties similar towild-type CFTR C1-channels: single-channel slope conductance was 9.2 *0.4 pS (n = 3).The poof CFTRARS660ACl- channels was 0.15 *0.04 (n= 6) in the presence ofATPalone and did not increasefollowingaddition of PKA (n = 4) (Fig. lOB). Login to comment
178 Fig. 1lA shows that simultaneous substitution of the four in vivo PKA sites with aspartates (in the S-Quad-D mutant) did not generate constitutively active CFTR C1-channels as assessed by the SPQ fluorescence assay: the mutant channels opened only after stimulation by CAMP.We observed similar results withCFTR S-Quint-D which contained substitutionsof A Mock S-Hept-A C S-OCt-A transfected (A) or were transfected with pMT-CFTR S-Hept-A (S660A,S686A,S700A,S712A,S737A,S768A,S795A) (B) or pMT-CFl`R S-Oct-A FIG. 9. Login to comment
180 COS-7 cells were mock- (S660A,S686A,S700A,S712A,S737A,S768A,S795A,S813A) (C). Login to comment
235 CFTRAR-S660A contains only one "nonclassic" (R-X-X-Sor R-X-S) PKA phosphorylation site (Ser-753)in thedeleted segment; its consensus sequence is R-I-S.Alternatively, if PKAphosphorylated "nonclassic"sites within other parts of CFTR which interacted with theR domain, then deletion of part of the R domainwould disrupt those interactions whether or not the additional sites were phosphorylated. Login to comment
237 Finally, we cannot rule out thepossibility that PKA simply cannot interact with CFTRAR-S660A, even though it does interact withCFTRAR (14). Login to comment

PMID: 7684377 [PubMed] Chang XB et al: "Protein kinase A (PKA) still activates CFTR chloride channel after mutagenesis of all 10 PKA consensus phosphorylation sites."

No. Sentence Comment

7 A previous investigationconcluded thatactivationby PKA is crit- icallydependent on phosphorylation at four of the nine predicted PKA sites in the R domain (S660A, S737A, S795A,SS13A), becausea "Quad"mutant lacking these sites couldnotbeactivated. Login to comment
37 The following mutations were introduced into CFTR, S422A (TCT to GCT), S660A (TCA to GCA), S686A (TCT to GCT), S700A (TCT toGCT), S712A (TCC to GCC), S737A (TCC to GCC), S768A (TCT toGCT), T788A (ACAto GCA),S795A (TCA to GCA), S813A (TCA to GCA), S660E (TCA to GAA),S737E (TCC to GAG), S795E (TCA to GAA), and S813E (TCA to GAA). Login to comment
39 The counterparts of CFTR cDNA in pUCF2.5 were replaced by PCR- mutated versions by interchange of the following fragments, S660A, DraIIIIEcoRI fragment, S737A, EcoRIIHpaIfragment, S795A and S813A,StyIIStyI fragment(Fig. 1A). Login to comment
40 A mutant containing S660/737/ 795/813A (pUCF2.5/4SA) was assembled by replacing the counterparts of a plasmid containing S795/813A with the DraIII/EcoRI fragment (S660A) and EcoRI/HpaI fragment (S737A). Login to comment
69 For example, S660A means that serine residue 660 was changed to alanine. Login to comment

PMID: 1375035 [PubMed] Welsh MJ et al: "Cystic fibrosis transmembrane conductance regulator: a chloride channel with novel regulation."

No. Sentence Comment

136 This point is illustrated by consideration of two mutants: in S737/795/813A, phosphorylation of Ser-660 ap- Review: The CFTR Cl-Channel pears to be sufficient to open the channel; yet in S660A, which lacks Ser-660 but retains Ser-737, -795, and -813, CAMP activates the channel. Login to comment
143 However, CFTRAR retains Ser-660, and when a combined mutant, CFTRAR-S660A, was created, only constitutive channel activity was found; CAMP no longer caused a further increase in activity (Cheng et al., 1991). Login to comment
206 Similar results were obtained with CFTRAR-S660A channels (unpublished data). Login to comment

PMID: 23760269 [PubMed] Billet A et al: "Role of tyrosine phosphorylation in the muscarinic activation of the cystic fibrosis transmembrane conductance regulator (CFTR)."

No. Sentence Comment

102 Carbachol Stimulates CFTR through PKA and Non-PKA Signaling Pathways-To explore PKA-independent regulation of CFTR without using inhibitors that might have confounding effects on other pathways, we studied the activation of 15SA-CFTR (S422A/S660A/S670A/S686A/T690A/S700A/S712A/ S737A/S753A/S768A/T787A/T788A/S790A/S795A/S813A). Login to comment