ABCMdb

PMID: 11597353

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., [PubMed]

Sentences

No. Mutations Sentence Comment

38 ABCC7 p.Gly551Asp ABCC7 p.Lys464Ala ABCC7 p.Asp572Asn ABCC7 p.Ser466Leu ABCC7 p.Arg487Gln ABCC7 p.Lys615Ala ABCC7 p.Arg516Ala Using similar PCR techniques, the NBF1 mutants of human CFTR vF508, G551D, S466L, K464A, D572N, KH483/484AA, R487Q, R516A, KR598/600GA, KK611/612AA and K615A were in vitro synthesized (Quickchange, Stratagene). Login to comment 63 ABCC7 p.Leu1480Val The C-terminal PDZ-BD of CFTR is not required for activation of CFTR or inhibition of ENaC in Xenopus oocytes The PDZ-BD at the C-terminal end of CFTR was mutated in vitro (L1480V-CFTR) and coexpressed together with the epithelial NaW channel in Xenopus oocytes. Login to comment 67 ABCC7 p.Leu1480Val A Cl3 conductance (GCFTR) of similar magnitude was activated in both wtCFTR and L1480V-CFTR expressing oocytes upon stimulation with IBMX and forskolin (I/F). Login to comment 69 ABCC7 p.Glu831* A truncated version of CFTR was expressed, comprising the 'rst transmembrane spanning domain, the 'rst nucleotide binding domain and the R domain (E831X-CFTR). Login to comment 71 ABCC7 p.Glu831* Nevertheless, a small but signi'cant portion of GAmil was inhibited during stimulation of E831X-CFTR (Fig. 2). Login to comment 72 ABCC7 p.Glu1474* In addition, we generated a CFTR mutant which only lacks the last six amino acids, encoding the PDZ-BD (E1474X-CFTR). Login to comment 74 ABCC7 p.Leu1480Val Original recordings of the whole cell currents measured in a Xenopus oocyte coexpressing L1480V-CFTR and ENaC. Login to comment 77 ABCC7 p.Glu1474* E1474X-CFTR generated a signi'cantly larger current than wtCFTR and largely downregulated GAmil (Fig. 2). Login to comment 78 ABCC7 p.Glu1474* 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 ABCC7 p.Glu831* ABCC7 p.Leu1480Val ABCC7 p.Glu1474* Coexpression of wtCFTR, L1480V-CFTR, E831X-CFTR and E1474X with ENaC. Login to comment 94 ABCC7 p.Glu831* ABCC7 p.Leu1480Val ABCC7 p.Glu1474* Stimulation of either wtCFTR, L1480V-CFTR, E831X-CFTR or E1474X signi'cantly enhanced CFTR whole cell Cl3 conductance. Login to comment 115 ABCC7 p.Gly551Asp It is shown that all mutations a&#a1;ected the CFTR Cl3 channel function, due to reduced expression (e.g. vF508) or defective function (e.g. G551D) of the mutant protein. Login to comment 116 ABCC7 p.Gly551Asp ABCC7 p.Asp572Asn ABCC7 p.Arg487Gln ABCC7 p.Lys615Ala The CFTR mutants K646A, R487Q, G551D, D572N and K615A did not generate signi'cant CFTR Cl3 conductances. Login to comment 118 ABCC7 p.Ser466Leu ABCC7 p.Arg516Ala The mutants S466L, KH483/484AA, R516A and KK611/612AA demonstrated a residual Cl3 channel function, which was paralleled by a limited ability to downregulate ENaC. Login to comment 150 ABCC7 p.Glu831* ABCC7 p.Leu1480Val ABCC7 p.Leu1480Val Although the L1480V-CFTR mutant allows only marginal binding of NHERF to CFTR [12], activation of L1480V-CFTR by IBMX and forskolin was comparable to that of wtCFTR and even the N-terminal half of CFTR (E831X) was still partially activated by increase in intracellular cAMP. Login to comment 151 ABCC7 p.Glu1474* 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 171 ABCC7 p.Gly551Asp ABCC7 p.Lys464Ala ABCC7 p.Asp572Asn ABCC7 p.Ser466Leu ABCC7 p.Arg487Ala We therefore introduced mutations into NBF1 sites which are essential for binding/hydrolysis of ATP and GTP and which have homology to GTP binding proteins such as Walker A (loop L1) (K464A, S466L), switch I motif (KH483/484AA, R487A), switch II motif (loop L4, G551D) and Walker B (D572N) [23]. Login to comment 172 ABCC7 p.Lys615Ala Furthermore, NBF1 sequences which could potentially serve as activator sequences for G proteins were mutated (KR598/600GA, KK611/ 612AA, K615A). Login to comment