ABCMdb

PMID: 8663098

Becq F, Verrier B, Chang XB, Riordan JR, Hanrahan JW
cAMP- and Ca2+-independent activation of cystic fibrosis transmembrane conductance regulator channels by phenylimidazothiazole drugs.
J Biol Chem. 1996 Jul 5;271(27):16171-9., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC7 p.Gly551Asp CFTR channels bearing mutations at multiple phosphorylation sites, in the membrane domains, and in the first nucleotide binding domain (including the disease-causing mutations G551D and ⌬F508) all responded to phenylimidazothiazoles. Login to comment 4 ABCC7 p.Gly551Asp CFTR channels bearing mutations at multiple phosphorylation sites, in the membrane domains, and in the first nucleotide binding domain (including the disease-causing mutations G551D and DF508) all responded to phenylimidazothiazoles. Login to comment 34 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp 27, Issue of July 5, pp. 16171-16179, 1996 (c) 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. MATERIALS AND METHODS Cell Culture-CHO-K1 cells that had been stably transfected with pNUT vector alone (denoted CFTR(-)) or with wild-type CFTR (CFTR(ϩ)) or various mutated versions (G551D, R347D, R117H, and ⌬F508) in pNUT were used (8, 15). Login to comment 143 ABCC7 p.Gly551Asp ABCC7 p.Arg117His Activation of CFTR by Phenylimidazothiazole Drugs Functional Response of CFTR Channels with Disease-causing Mutations-Three CFTR mutations associated with severe (DF508 and G551D) or mild (R117H) forms of cystic fibrosis were studied after stable transfection into CHO cells. Login to comment 144 ABCC7 p.Gly551Asp ABCC7 p.Arg117His Functional Response of CFTR Channels with Disease-causing Mutations-Three CFTR mutations associated with severe (⌬F508 and G551D) or mild (R117H) forms of cystic fibrosis were studied after stable transfection into CHO cells. Login to comment 148 ABCC7 p.Arg117His The basic biophysical properties of DF508 and R117H versions of CFTR were similar to those reported previously (11, 28, 29). Login to comment 149 ABCC7 p.Arg117His ABCC7 p.Arg117His The basic biophysical properties of ⌬F508 and R117H versions of CFTR were similar to those reported previously (11, 28, 29). Login to comment 150 ABCC7 p.Arg117His R117H had a slightly lower unitary conductance and significantly reduced Po (Table I). Login to comment 152 ABCC7 p.Arg347Pro ABCC7 p.Arg347His ABCC7 p.Arg347Asp We also examined a CFTR mutation (R347D) that is at a residue where disease-causing mutations have been identified (R347P and R347H). Login to comment 153 ABCC7 p.Arg347Pro ABCC7 p.Arg347His ABCC7 p.Arg347Asp We also examined a CFTR mutation (R347D) that is at a residue where disease-causing mutations have been identified (R347P and R347H). Login to comment 154 ABCC7 p.Arg347Asp Spontaneous activity of R347D channels was never observed on resting cells (Table I) but was elicited by exposure to 15 mM forskolin (80% of patches; Fig. 6A) or l mM levamisole (60% of patches; Fig. 6, B and C). Login to comment 155 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp Spontaneous activity of R347D channels was never observed on resting cells (Table I) but was elicited by exposure to 15 ␮M forskolin (80% of patches; Fig. 6A) or l mM levamisole (60% of patches; Fig. 6, B and C). Login to comment 156 ABCC7 p.Arg347Asp The unitary conductance of R347D channels determined after stimulation by forskolin was 2.9 Ϯ 0.2 pS (n ϭ 8) and by levamisole was 2.8 Ϯ 0.3 pS (n ϭ 6; Fig. 6D). Login to comment 162 ABCC7 p.Gly551Asp G551D channel activity in the presence of PKA and ATP was lower than wild-type CFTR (Po 5 0.17 6 0.06, n 5 2.6 6 0.57, n 5 3 patches) but was nevertheless increased 2-fold by addition of bromotetramisole in the presence of PKA (Po 5 0.36 6 0.08, n 5 6.2 6 0.56, n 5 7 patches). Login to comment 163 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Arg347Asp G551D channel activity in the presence of PKA and ATP was lower than wild-type CFTR (Po ϭ 0.17 Ϯ 0.06, n ϭ 2.6 Ϯ 0.57, n ϭ 3 patches) but was nevertheless increased 2-fold by addition of bromotetramisole in the presence of PKA (Po ϭ 0.36 Ϯ 0.08, n ϭ 6.2 Ϯ 0.56, n ϭ 7 patches). Login to comment 164 ABCC7 p.Arg117His ABCC7 p.Arg347Asp Although these values for Po may be overestimated because they are based on the estimated number of channels without locking all the channels open using AMP-PNP, the increase in open probability was consistent and was also observed with the mutants R117H and R347D (data not shown). Login to comment 193 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Arg347Asp Our results can be summarized as follows: (i) levamisole and bromotetramisole promote the opening of cell-attached CFTR channels in the absence of forskolin, (ii) elevation of intracellular cAMP and Ca21 are not required for this stimulation, (iii) at least four mutant CFTRs (G551D, R117H, R347D, and DF508) can also be activated on-cell by these drugs in the absence of forskolin, (iv) both phenylimidazothiazoles further enhance CFTR channel activity when excised patches were exposed to high levels of PKA, indicating that their target is associated with the membrane, and (v) activation by both drugs requires some kinase activity. Login to comment 194 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Arg347Asp Our results can be summarized as follows: (i) levamisole and bromotetramisole promote the opening of cell-attached CFTR channels in the absence of forskolin, (ii) elevation of intracellular cAMP and Ca2ϩ are not required for this stimulation, (iii) at least four mutant CFTRs (G551D, R117H, R347D, and ⌬F508) can also be activated on-cell by these drugs in the absence of forskolin, (iv) both phenylimidazothiazoles further enhance CFTR channel activity when excised patches were exposed to high levels of PKA, indicating that their target is associated with the membrane, and (v) activation by both drugs requires some kinase activity. Login to comment 195 ABCC7 p.Arg347Asp Functional response of R347D stably expressed in CHO cells. Login to comment 196 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp Functional response of R347D stably expressed in CHO cells. Login to comment 197 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp Recordings of R347D channels at various potentials in the cell-attached configuration with 15 ␮M forskolin (A) or 1 mM levamisole (C). Login to comment 198 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp Note the different scales and reduced conductance of R347D mutant. Login to comment 199 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp B, histogram summarizing fraction of cell-attached patches that contained R347D channel activity in various conditions. Login to comment 200 ABCC7 p.Arg347Asp D, current-voltage relationship of R347D channels in cell-attached patches in the presence of 15 ␮M forskolin (circles) and 1 mM levamisole (squares). Login to comment 204 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Arg347Asp Frequency Control Levamisole Number of channels Po g pS CFTR 0/15 20/23 12 6 2.80 0.47 6 0.05 6.8 6 0.20 G551D 0/10 31/43 3 6 0.27 0.35 6 0.07 5.3 6 0.30 R117H 0/5 9/13 2 6 0.32 0.14 6 0.10 5.7 6 0.15 R347D 0/4 6/10 4.6 6 0.40 0.40 6 0.02 2.8 6 0.30 DF508 (37 &#b0;C) 0/10 0/15 DF508 (23 &#b0;C) 0/5 5/8 2.1 6 0.03 0.13 6 0.02 6.8 6 0.14 Activation of CFTR by Phenylimidazothiazole Drugs they are uncompetitive inhibitors of alkaline phosphatase (26, 31, 37). Login to comment 206 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Arg347Asp Frequency Control Levamisole Number of channels Po g pS CFTR 0/15 20/23 12 Ϯ 2.80 0.47 Ϯ 0.05 6.8 Ϯ 0.20 G551D 0/10 31/43 3 Ϯ 0.27 0.35 Ϯ 0.07 5.3 Ϯ 0.30 R117H 0/5 9/13 2 Ϯ 0.32 0.14 Ϯ 0.10 5.7 Ϯ 0.15 R347D 0/4 6/10 4.6 Ϯ 0.40 0.40 Ϯ 0.02 2.8 Ϯ 0.30 ⌬F508 (37 °C) 0/10 0/15 ⌬F508 (23 °C) 0/5 5/8 2.1 Ϯ 0.03 0.13 Ϯ 0.02 6.8 Ϯ 0.14 they are uncompetitive inhibitors of alkaline phosphatase (26, 31, 37). Login to comment 239 ABCC7 p.Gly551Asp Bromotetramisole (1 mM) and levamisole (1 mM) had no effect on short circuit current across trachea, jejunum, or caecum freshly isolated from normal and transgenic G551D mice.2 Interestingly, forskolin did cause significant stimulation of the short circuit current, probably due to cAMP-induced calcium mobilization and stimulation of Ca21 -activated chlorine conductance (45). Login to comment 240 ABCC7 p.Gly551Asp Bromotetramisole (1 mM) and levamisole (1 mM) had no effect on short circuit current across trachea, jejunum, or caecum freshly isolated from normal and transgenic G551D mice.2 Interestingly, forskolin did cause significant stimulation of the short circuit current, probably due to cAMP-induced calcium mobilization and stimulation of Ca2ϩ -activated chlorine conductance (45). Login to comment 242 ABCC7 p.Gly551Asp An intestinal phenotype does occur in the G551D mouse; however, it is unclear whether the same phosphatases regulate CFTR in airway and intestinal cells and the lack of effect might be explained by the known insensitivity of intestinal alkaline phosphatase to bromotetramisole. Login to comment 243 ABCC7 p.Gly551Asp An intestinal phenotype does occur in the G551D mouse; however, it is unclear whether the same phosphatases regulate CFTR in airway and intestinal cells and the lack of effect might be explained by the known insensitivity of intestinal alkaline phosphatase to bromotetramisole. Login to comment