ABCMdb

ABCC7 p.Glu1474*

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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
184 We eliminated the PDZ binding domain of CFTR (E1474X-CFTR) and gradually increased stimulation of the oocytes as shown in Fig. 5D. Login to comment
187 In marked contrast 3 mM 8Br- -cAMP alone was unable to promote full activation of E1474X-CFTR but instead required costimulation by 8Br- -cAMP, forskolin, and IBMX, and compound C further augmented whole cell conductance (Fig. 5D) suggesting AMPK sensitivity was retained by this mutant. Login to comment

[hide] CFTR induces extracellular acid sensing in Xenopus... Pflugers Arch. 2011 Sep;462(3):479-87. Epub 2011 Jun 7. Kongsuphol P, Schreiber R, Kraidith K, Kunzelmann K
CFTR induces extracellular acid sensing in Xenopus oocytes which activates endogenous Ca(2)-activated Cl conductance.
Pflugers Arch. 2011 Sep;462(3):479-87. Epub 2011 Jun 7., [PMID:21647592]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) produces a cyclic adenosine monophosphate (cAMP)-dependent Cl conductance of distinct properties that is essential for electrolyte secretion in human epithelial tissues. However, the functional consequences of CFTR expression are multifaceted, encompassing much more than simply supplying a cellular cAMP-regulated Cl conductance. When we expressed CFTR in Xenopus oocytes, we found that extracellular acidic pH activates a Ca(2)-dependent outwardly rectifying Cl conductance that does not reflect CFTR activity. The proton-activated Cl conductance showed biophysical and pharmacological features of a Ca(2)-dependent Cl conductance, most likely mediated by Xenopus TMEM16A. In contrast to the effects of extracellular acidification, intracellular acidification did not activate an endogenous Cl conductance. Proton/CFTR-mediated activation of human TMEM16A was also detected in HEK293 cells. The gating mutant G551D-CFTR conferred proton sensitivity, while deltaF508-CFTR enabled proton activation of TMEM16A only in Xenopus oocytes, which, unlike HEK293 cells, allow deltaF508-CFTR to be trafficked to the cell membrane. Activation of TMEM16A by lysophosphatidic acid was enhanced in the presence of CFTR but was additive with activation by extracellular protons. Because expression of CFTR-E1474X did not confer proton sensitivity, we propose that CFTR translocates a proton receptor to the plasma membrane via its PDZ-binding domain.

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8 Because expression of CFTR-E1474X did not confer proton sensitivity, we propose that CFTR translocates a proton receptor to the plasma membrane via its PDZ-binding domain. Login to comment
121 In order to further support the concept of membrane translocation of a proton receptor by CFTR, we expressed a C-terminally truncated CFTR that lacks of the PDZBD (CFTR-E1474X) and that should no longer be able Fig. 6 The H+ -activated current is blocked by inhibitors of CaCC. a Original recording obtained from an oocyte expressing wtCFTR and exposed to extracellular pH 5.5 in the absence or presence of DIDS (100 μM) or NFA (100 μM). Login to comment
133 When expressed in Xenopus oocytes, CFTR-E1474X generated a cAMP-activated whole cell conductance of 23±3.2 μS (n=6), indicating membrane expression of functional CFTR. Login to comment
134 However, in the presence of CFTR-E1474X, Ca2+ - activated Cl- currents-conductance were no longer activated by extracellular acidic pH (ΔG=0±0.1 μS; n=6). Login to comment

[hide] Role of CFTR's PDZ1-binding domain, NBF1 and Cl(-)... Biochim Biophys Acta. 2001 Nov 1;1515(1):64-71. Boucherot A, Schreiber R, Kunzelmann K
Role of CFTR's PDZ1-binding domain, NBF1 and Cl(-) conductance in inhibition of epithelial Na(+) channels in Xenopus oocytes.
Biochim Biophys Acta. 2001 Nov 1;1515(1):64-71., [PMID:11597353]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) inhibits epithelial Na(+) channels (ENaC). Evidence has accumulated that both Cl(-) transport through CFTR Cl(-) channels and the first nucleotide binding domain (NBF1) of CFTR are crucial for inhibition of ENaC. A PDZ binding domain (PDZ-BD) at the C-terminal end links CFTR to scaffolding and cytoskeletal proteins, which have been suggested to play an important role in activation of CFTR and eventually inhibition of ENaC. We eliminated the PDZ-BD of CFTR and coexpressed Na(+)/H(+)-exchange regulator factors together with CFTR and ENaC. The results do not support a role of PDZ-BD in inhibition of ENaC by CFTR. However, inhibition of ENaC was closely linked to Cl(-) currents generated by CFTR and was observed in the presence of Cl(-), I(-) or Br(-) but not gluconate. Therefore, functional NBF1 and Cl(-) transport are required for inhibition of ENaC in Xenopus oocytes, while the PDZ-BD is not essential.

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72 In addition, we generated a CFTR mutant which only lacks the last six amino acids, encoding the PDZ-BD (E1474X-CFTR). Login to comment
77 E1474X-CFTR generated a signi'cantly larger current than wtCFTR and largely downregulated GAmil (Fig. 2). Login to comment
78 In fact, downregulation of ENaC was signi'cantly enhanced when compared to wtCFTR, which is likely due to the large Cl3 conductance generated by E1474X-CFTR. Login to comment
91 Coexpression of wtCFTR, L1480V-CFTR, E831X-CFTR and E1474X with ENaC. Login to comment
94 Stimulation of either wtCFTR, L1480V-CFTR, E831X-CFTR or E1474X signi'cantly enhanced CFTR whole cell Cl3 conductance. Login to comment
151 It was a surprising and rather unexpected result that E1474X-CFTR caused a very large Cl3 conductance which was signi'cantly enhanced compared to wtCFTR. Login to comment