ABCMdb

PMID: 10220462

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 [PubMed]

Sentences

No. Mutations Sentence Comment

34 ABCC7 p.Glu402* (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). Login to comment 44 ABCC7 p.Cys590* 5310 terminal CFTR truncation including the N terminus, the first six transmembrane helices, and NBF-1 (C590X) was obtained as a SpeI͞KpnI CFTR cDNA fragment. Login to comment 45 ABCC7 p.Asp651* (v) The N-terminal CFTR truncation including the N terminus, the first six transmembrane helices, and an extended NBF-1, according to a remodeled NBF-1 (ref. 17; D651X), was generated with a SpeI͞BamHI CFTR fragment (M1-A457), and the PCR product was generated with 5Ј-GAGGAGGGATTTGGG- GAAT-3Ј (s) and 5Ј-TTAGAAAGAATCACATC- CCATGA-3Ј (as). Login to comment 46 ABCC7 p.Glu831* (vi) The N-terminal truncation including the N terminus, the first six transmembrane helices, NBF-1, and the R domain (E831X) was obtained by ligating a BamHI͞ KpnI fragment (G458͞E831X) with a SpeI͞BamHI fragment (M1-A457). Login to comment 97 ABCC7 p.Glu402* ABCC7 p.Cys590* ABCC7 p.Asp651* ABCC7 p.Glu831* 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). Login to comment 98 ABCC7 p.Glu402* 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). Login to comment 100 ABCC7 p.Glu831* When stimulated by IBMX and forskolin, only oocytes expressing the N-terminal half protein including the R domain (E831X) increased their whole-cell conductance, and no change could be observed for the other CFTR truncations FIG. 3. Summary of amiloride-sensitive whole-cell conductances measured in oocytes coexpressing ␣-, beta-, and ␥-ENaC and different ABC transporters. Login to comment 106 ABCC7 p.Glu831* When stimulated with IBMX and forskolin, only oocytes expressing the N-terminal half of CFTR including the R domain (E831X) responded with an increase of the whole-cell conductance. Login to comment 113 ABCC7 p.Glu831* A significant change in halide permeability and conductivity ratios could be observed only for full-length CFTR and E831X, indicating a shift from I- Ͼ Cl- toward Cl- Ͼ I- . Login to comment 114 ABCC7 p.Glu831* These results suggest that only E831X is able to form a cAMP-regulated Cl-conductance, and the other CFTR truncations are not. Login to comment 117 ABCC7 p.Cys590* As shown as an example in Fig. 6A, the N-terminal half of CFTR without the R domain (C590X) is able to inhibit ENaC. Login to comment 125 ABCC7 p.Glu831* When stimulated with IBMX and forskolin, only oocytes expressing either full-length CFTR or E831X had a change of the conductivity (GCl͞GI) and permeability (PCl͞PI) ratios toward Cl- Ͼ I-, indicating activation of CFTR typical Cl-conductance. Login to comment 128 ABCC7 p.Cys590* FIG. 6. Time course of the whole-cell conductance measured in oocytes coexpressing C590X and ␣-, beta-, and ␥-ENaC (A) or M595-C and ␣-, beta-, and ␥-ENaC (B). Login to comment 129 ABCC7 p.Cys590* Amiloride (Amil; 10 ␮mol͞liter) blocked most of the whole-cell conductance when applied under control conditions. In C590X-expressing oocytes, whole-cell conductances were inhibited, and the effect of amiloride was attenuated after stimulation with IBMX and forskolin. Login to comment 132 ABCC7 p.Glu402* 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. Login to comment 133 ABCC7 p.Glu402* 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. Login to comment 156 ABCC7 p.Arg117His ABCC7 p.Arg117His In fact, it has been shown that CFTR mutants producing only a little Cl-conductance such as R117H also inhibited in the range of 24% (19). Login to comment 160 ABCC7 p.Glu402* 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. Login to comment 169 ABCC7 p.Glu831* ABCC7 p.Glu831* The CFTR truncations presented here enhanced only those baseline Cl- currents that were not activated further by PKA, except for E831X. Login to comment