ABCMdb

ABCC7 p.Thr665Ala

CF databases:	c.1993A>T , p.Thr665Ser (CFTR1) ? , A novel mutation was identified by DGGE and direct sequencing. This mutation was identified on a CF chromosome of Tunisian origin. The mutation was found once while screning 57 non[delta]F508 CF chromosomes.
Predicted by SNAP2:	A: D (71%), C: D (85%), D: D (85%), E: D (80%), F: D (85%), G: D (80%), H: D (75%), I: D (80%), K: D (80%), L: D (80%), M: D (91%), N: D (71%), P: D (80%), Q: D (71%), R: D (80%), S: N (82%), V: D (75%), W: D (91%), Y: D (80%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: D, I: N, K: N, L: N, M: N, N: N, P: D, Q: N, R: N, S: N, V: N, W: D, Y: D,

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Publications
[hide] PKC-mediated stimulation of amphibian CFTR depends... J Gen Physiol. 2001 May;117(5):457-68. Button B, Reuss L, Altenberg GA
PKC-mediated stimulation of amphibian CFTR depends on a single phosphorylation consensus site. insertion of this site confers PKC sensitivity to human CFTR.
J Gen Physiol. 2001 May;117(5):457-68., [PMID:11331356]

Abstract [show]
Mutations of the CFTR, a phosphorylation-regulated Cl(-) channel, cause cystic fibrosis. Activation of CFTR by PKA stimulation appears to be mediated by a complex interaction between several consensus phosphorylation sites in the regulatory domain (R domain). None of these sites has a critical role in this process. Here, we show that although endogenous phosphorylation by PKC is required for the effect of PKA on CFTR, stimulation of PKC by itself has only a minor effect on human CFTR. In contrast, CFTR from the amphibians Necturus maculosus and Xenopus laevis (XCFTR) can be activated to similar degrees by stimulation of either PKA or PKC. Furthermore, the activation of XCFTR by PKC is independent of the net charge of the R domain, and mutagenesis experiments indicate that a single site (Thr665) is required for the activation of XCFTR. Human CFTR lacks the PKC phosphorylation consensus site that includes Thr665, but insertion of an equivalent site results in a large activation upon PKC stimulation. These observations establish the presence of a novel mechanism of activation of CFTR by phosphorylation of the R domain, i.e., activation by PKC requires a single consensus phosphorylation site and is unrelated to the net charge of the R domain.

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No. Sentence Comment
70 The following primers were used (only sense primer is shown, with mutations underlined): 5Ј-TAATAACTGAGGCCCTGAGACGATGCT-3Ј (Thr665 to Ala, T A B L E I Conductances of Xenopus Oocytes Expressing CFTR Unstimulated cAMP-stimulated 24 h 48 h 72 h Mock 2.9 Ϯ 1.7 (11) 2.7 Ϯ 1.4 (4) 2.9 Ϯ 1.6 (4) 3.2 Ϯ 2.1 (3) hCFTR 4.2 Ϯ 3.8 (35) 426 Ϯ 60 (16) 685 Ϯ 131 (13) 1,147 Ϯ 217 (6) XCFTR 3.1 Ϯ 2.1 (34) 214 Ϯ 50 (14) 1,009 Ϯ 268 (14) 2,028 Ϯ 217 (6) Xenopus laevis oocytes were injected with water (mock), hCFTR cRNA, or XCFTR cRNA, and their membrane conductances were measured at 24, 48, or 72 h after injection with and without stimulation with cAMP cocktail. Login to comment
207 Thr665 Is Essential for the Response to PKC Activation of XCFTR To investigate the roles of the unique PKC consensus sites (Thr665 and Ser694), we expressed the mutant lacking both sites (T665A/S694A-XCFTR) in Xenopus oocytes. Login to comment
215 (B) I-V plot from a cell expressing the double knockout of unique PKC consensus phosphorylation sites (T665A/S694A-XCFTR). Login to comment
221 In contrast, in oocytes expressing T665A-XCFTR, PMA failed to produce full current activation (Fig. 8, B and C). Login to comment
223 Thus, it is possible that the reduced activation by PMA of XCFTR mutants containing the Thr665 to Ala mutation is not absolute, but relative to the activation by PKA stimulation (i.e., the mutation increases the activation of CFTR by cAMP). Login to comment
224 The data in Fig. 9 indicate that this is not the case because the cAMP-activated currents are not different among oocytes expressing wild-type XCFTR, S686A/ S790A-XCFTR, or T665A/S694A-XCFTR. Login to comment
245 The mutation Thr665 to Ala does not increase cAMP-activated XCFTR currents. Login to comment
246 Time course of the currents after stimulation with the cAMP cocktail in oocytes injected with wild-type XCFTR, S686A/S790A-XCFTR, or T665A/S694A-XCFTR cRNAs. Login to comment
248 The data show that the Thr665 to Ala mutation has no significant effect on the level of cAMP-activated currents. Login to comment

[hide] Mechanism of activation of Xenopus CFTR by stimula... Am J Physiol Cell Physiol. 2004 Nov;287(5):C1256-63. Epub 2004 Jun 30. Chen Y, Altenberg GA, Reuss L
Mechanism of activation of Xenopus CFTR by stimulation of PKC.
Am J Physiol Cell Physiol. 2004 Nov;287(5):C1256-63. Epub 2004 Jun 30., [PMID:15229107]

Abstract [show]
PKA-mediated phosphorylation of the regulatory (R) domain plays a major role in the activation of the human cystic fibrosis transmembrane conductance regulator (hCFTR). In contrast, the effect of PKC-mediated phosphorylation is controversial, smaller than that of PKA, and dependent on the cell type. In the present study, we expressed Xenopus CFTR (XCFTR) and hCFTR in Xenopus oocytes and examined their responses (i.e., macroscopic membrane conductance) to maximal stimulation by PKC and PKA agonists. With XCFTR, the average response to PKC was approximately sixfold that of PKA stimulation. In contrast, with hCFTR, the response to PKC was approximately 90% of the response to PKA stimulation. The reason for these differences was the small response of XCFTR to PKA stimulation. Using the substituted cysteine accessibility method, we found no evidence for insertion of functional CFTR channels in the plasma membrane in response to PKC stimulation. The increase in macroscopic conductance in response to PKC stimulation of XCFTR was due to an approximately fivefold increase in single-channel open probability, with a minor (approximately 30%) increase in single-channel conductance. The responses of XCFTR to PKC stimulation and of hCFTR to PKA stimulation were mediated by similar increases in Po. In both instances, there were no changes in the number of channels in the membrane. We speculate that in animals other than humans, PKC stimulation may be the dominant mechanism for activation of CFTR.

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No. Sentence Comment
211 A nonspecific stimulatory effect of high PMA concentration can be ruled out because the same concentration of 4␣-PMA, an inactive form of PMA, had no effect, and the mutation T665A reduced the activation of XCFTR by PMA by ϳ75% (6). Login to comment

[hide] Potentiation of effect of PKA stimulation of Xenop... Am J Physiol Cell Physiol. 2004 Nov;287(5):C1436-44. Epub 2004 Jul 28. Chen Y, Button B, Altenberg GA, Reuss L
Potentiation of effect of PKA stimulation of Xenopus CFTR by activation of PKC: role of NBD2.
Am J Physiol Cell Physiol. 2004 Nov;287(5):C1436-44. Epub 2004 Jul 28., [PMID:15282191]

Abstract [show]
Activity of the human (h) cystic fibrosis transmembrane conductance regulator (CFTR) channel is predominantly regulated by PKA-mediated phosphorylation. In contrast, Xenopus (X)CFTR is more responsive to PKC than PKA stimulation. We investigated the interaction between the two kinases in XCFTR. We expressed XCFTR in Xenopus oocytes and maximally stimulated it with PKA agonists. The magnitude of activation after PKC stimulation was about eightfold that without pretreatment with PKC agonist. hCFTR, expressed in the same system, lacked this response. We name this phenomenon XCFTR-specific PKC potentiation effect. To ascertain its biophysical mechanism, we first tested for XCFTR channel insertion into the plasma membrane by a substituted-cysteine-accessibility method. No insertion was detected during kinase stimulation. Next, we studied single-channel properties and found that the single-channel open probability (Po) with PKA stimulation subsequent to PKC stimulation was 2.8-fold that observed in the absence of PKC preactivation and that single-channel conductance (gamma) was increased by approximately 22%. To ascertain which XCFTR regions are responsible for the potentiation, we constructed several XCFTR-hCFTR chimeras, expressed them in Xenopus oocytes, and tested them electrophysiologically. Two chimeras [hCFTR NH2-terminal region or regulatory (R) domain in XCFTR] showed a significant decrease in potentiation. In the chimera in which XCFTR nucleotide-binding domain (NBD)2 was replaced with the hCFTR sequence there was no potentiation whatsoever. The converse chimera (hCFTR with Xenopus NBD2) did not exhibit potentiation. These results indicate that potentiation by PKC involves a large increase in Po (with a small change in gamma) without CFTR channel insertion into the plasma membrane, that XCFTR NBD2 is necessary but not sufficient for the effect, and that the potentiation effect is likely to involve other CFTR domains.

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Sentences [show]
No. Sentence Comment
207 We found that the activation by PKC stimulation was abolished in the mutant T665A XCFTR and transferred to the hCFTR mutant TLHR 3 TLRR (7). Login to comment