ABCC7 p.Glu402*
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[hide] The first-nucleotide binding domain of the cystic-... Proc Natl Acad Sci U S A. 1999 Apr 27;96(9):5310-5. Schreiber R, Hopf A, Mall M, Greger R, Kunzelmann K
The first-nucleotide binding domain of the cystic-fibrosis transmembrane conductance regulator is important for inhibition of the epithelial Na+ channel.
Proc Natl Acad Sci U S A. 1999 Apr 27;96(9):5310-5., 1999-04-27 [PMID:10220462]
Abstract [show]
The cystic-fibrosis transmembrane conductance regulator (CFTR) functions as a cAMP-regulated Cl- channel and as a regulator of other membrane conductances. cAMP-dependent activation of CFTR inhibits epithelial Na+ channels (ENaC). The specificity of interaction between CFTR and ENaC was examined by coexpression of ENaC and ATP-binding cassette (ABC) proteins other than CFTR. In addition, we identified domains within CFTR that are of particular importance for the inhibition of ENaC. To that end, two-electrode voltage-clamp experiments were performed on Xenopus oocytes coexpressing ENaC together with CFTR, the multidrug resistance protein MDR1, the sulfonyl urea receptor SUR1, or the cadmium permease YCF1. Except for CFTR, none of the other ABC proteins were able to inhibit ENaC. Several truncated versions of CFTR were examined for their inhibitory effects on ENaC. In fact, it is shown that C-terminal truncated CFTR is able to inhibit ENaC on activation by intracellular cAMP. Moreover, the data also show that an intact first-nucleotide binding domain (NBF-1) is important for inhibition of ENaC. We conclude that NBF-1 of CFTR contains a CFTR-specific regulatory site that down-regulates ENaC. It is speculated that this regulatory site also is needed for CFTR-mediated interactions with other membrane proteins and that it is not present in NBF-1 of other ABC proteins.
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34 (iii) The N-terminal CFTR truncation including the N terminus and the first six transmembrane helices (E402X) was generated with 5Ј-CCC- GAGGTACCATGCAGAGGTC-3Ј (s) and 5Ј-CTACTCGA- GCTACCAGAAGGCTGTTACATTC-3Ј (as).
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ABCC7 p.Glu402* 10220462:34:103
status: NEW97 Several truncated versions of CFTR were generated to identify CFTR domains essential to the down-regulation of ENaC: (i) the central cytosolic part of CFTR including two transmembrane helices on each site (M284͞I942X); (ii) NBF-1 (W401M͞D651X); (iii) the N-terminal half of CFTR without NBF-1 (E402X); (iv) the N-terminal half of CFTR including NBF-1 (C590X); (v) the N-terminal half of CFTR including an additional stretch of 61 amino acids, as described in a recent study (ref. 17; D651X); (vi) the N-terminal half of CFTR including NBF-1 and the R domain (E831X); and (vii) the C-terminal half including the R domain but not NBF-1 (M595-C).
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ABCC7 p.Glu402* 10220462:97:306
status: NEW98 With the exception of E402X, expression of all of these constructs enhanced baseline whole-cell Cl-conductance when compared with water-injected control oocytes (Fig. 4).
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ABCC7 p.Glu402* 10220462:98:22
status: NEW132 However, the results indicate that, except for M595-C and E402X, all CFTR truncations inhibited ENaC conductances slightly (about 25%) but significantly (P Ͻ 0.01) during stimulation.
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ABCC7 p.Glu402* 10220462:132:58
status: NEW133 Because M595-C and E402X are the only CFTR truncations that do not contain NBF-1, these results suggest that CFTR`s ability to inhibit ENaC relies on the presence of an intact NBF-1.
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ABCC7 p.Glu402* 10220462:133:19
status: NEW160 GAmiloride values measured under control conditions (white bars) were significantly smaller after stimulation of the oocytes with IBMX and forskolin (black bars), except for E402X and M959-C. The numbers of experiments are shown in parentheses.
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ABCC7 p.Glu402* 10220462:160:174
status: NEW[hide] Synergistic effects of cystic fibrosis transmembra... Biol Reprod. 2003 May;68(5):1505-10. Epub 2002 Nov 27. Cheung KH, Leung CT, Leung GP, Wong PY
Synergistic effects of cystic fibrosis transmembrane conductance regulator and aquaporin-9 in the rat epididymis.
Biol Reprod. 2003 May;68(5):1505-10. Epub 2002 Nov 27., [PMID:12606488]
Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) and aquaporin-9 (AQP-9) are present in the luminal membrane of the epididymis, where they play an important role in formation of the epididymal fluid. Evidence is accumulating that CFTR regulates other membrane transport proteins besides functioning as a cAMP-activated chloride channel. We have explored the possible interaction between epididymal CFTR and AQP-9 by cloning them from the rat epididymis and expressing them in Xenopus oocytes. The effects of the expressed proteins on oocyte water permeability were studied by immersing oocytes in a hypo-osmotic solution, and the ensuing water flow was measured using a gravimetric method. The results show that AQP-9 alone caused an increase in oocyte water permeability, which could be further potentiated by CFTR. This potentiation was markedly reduced by phloretin and lonidamine (inhibitors of AQP-9 and CFTR, respectively). The regulation of water permeability by CFTR was also demonstrated in intact rat epididymis luminally perfused with a hypo-osmotic solution. Osmotic water reabsorption across the epididymal tubule was reduced by phloretin and lonidamine. Elevation of intracellular cAMP with 3-isobutyl-1-methylxanthine increased osmotic water permeability, whereas inhibiting protein kinase A with H-89 (N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinoline sulfonamide hydrochloride) reduced it. These results are consistent with a role for CFTR in controlling water permeability in the epididymis in vivo. We conclude that this additional role of CFTR in controlling water permeability may have an impact on the genetic disease cystic fibrosis, in which men with a mutated CFTR gene have abnormal epididymis and infertility.
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44 Domain amplified Primer sequencea Corresponding size (kilobases) M595-C (C-terminal half of CFTR including R domain but no NBD1) E402X (N-terminal half of CFTR but no NBD1) W401M-D651X (NBD1 only) F: 5Ј-TTTGAAAAGTTGTGTCTGT-3Ј R: 5Ј-AGCCTCGAGCACTAGAGC-3Ј F: 5Ј-AGACATCATGCAGAAGTCGCCT-3Ј R: 5Ј-CTCCCAAAATGCTGTTACATT-3Ј F: 5Ј-ACAGCAACCTGGAGGAGGGATTTCAG-3Ј R: 5Ј-CTCGAGCATAAAAGTATCATACCCCAT-3Ј 2.7 1.2 0.8 a F, forward; R, reverse.
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ABCC7 p.Glu402* 12606488:44:129
status: NEW50 In brief, the truncated forms of CFTR, M595-C (C-terminal half of CFTR including R domain but lacking NBD1), E402X (N-terminal half of CFTR but lacking NBD1), and W401M-D651X (NBD1 only), were PCR amplified by specific primers (Table 1) and cloned into pGEM-T cloning vectors (Promega).
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ABCC7 p.Glu402* 12606488:50:109
status: NEW129 NBD1) nor E402X (C-terminal half of CFTR including R domain but lacking NBD1) potentiated AQP-9.
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ABCC7 p.Glu402* 12606488:129:10
status: NEW