ABCMdb

PMID: 9252549

Wilkinson DJ, Strong TV, Mansoura MK, Wood DL, Smith SS, Collins FS, Dawson DC
CFTR activation: additive effects of stimulatory and inhibitory phosphorylation sites in the R domain.
Am J Physiol. 1997 Jul;273(1 Pt 1):L127-33., [PubMed]

Sentences

No. Mutations Sentence Comment

15 ABCC7 p.Ser737Ala In contrast, alanine substitution at serine-737 or -768 actually decreased the KA for activation, suggesting that phosphorylation at either of these sites is inhibitory. Login to comment 64 ABCC7 p.Lys1250Ala Interpretation of Dose Response and Rate Analyses In a previous study (20), we used a CFTR (K1250A) mutant that is hypersensitive to activation by IBMX [half-maximal activation constant (&) = 0.07 mM] to obtain evidence that IBMX blocks CFTR channels with a half-maximal inhibition constant (&) of -9.5 mM. Login to comment 85 ABCC7 p.Ser660Ala ABCC7 p.Ser670Ala Activation constants for wild-type and single serine-to-alanine mutant CFTRs PICA Phosphorylation CFTR K4, n-m n Class In vivo In vitro Wild type 0.65 t 0.08 26 S64lA 0.6520.12 14 N No concensus S660A 1.21+ 0.19* 11 S ++ ++ S670A 0.99t0.12* 12 s ? Login to comment 87 ABCC7 p.Ser737Ala ABCC7 p.Ser795Ala ABCC7 p.Ser813Ala ABCC7 p.Ser686Ala ABCC7 p.Ser768Ala ABCC7 p.Ser712Ala ABCC7 p.Ser700Ala S686A 0.75 ?I 0.07 9 N PKC site S700A 0.86 t 0.07* 12 S ++ +++ S712A 0.67 t 0.12 9 N - ++ S737A 0.35 5 0.05* 8 I +++ +++ S768A 0.09 IT 0.03* 8 I - ++++ S795A 1.24 + 0.22* 9 S +++ ++++ S813A 3.18-+0.36* 6 S ++++ ++ Values of half-maximal inhibition constant for activation (KA) are means + SE by 3-isobutyl-1-methylxanthine (IBMX) obtained for wild-type cystic fibrosis transmembrane conductance regulator (CFTR) and variants with single serine-to-alanine substitutions. Login to comment 107 ABCC7 p.Ser660Ala ABCC7 p.Ser737Ala ABCC7 p.Ser795Ala ABCC7 p.Ser813Ala ABCC7 p.Ser768Ala Symbols show averaged IBMX dose-response data for wild-type CFTR and several single-site serine-to-alanine mutants (S660A, S737A, S768A, S795A, and S813A). Login to comment 119 ABCC7 p.Ser813Ala 100 i - wild type m m - S813A 0 S768,813A A S737,795,813A v S660,795,813A S660,737,795, E Fig. 2. Login to comment 125 ABCC7 p.Ser813Ala For reference, the activation profiles of wild-type CFTR and single-site mutant S813A are shown by the thick continuous and dashed curves, respectively. Login to comment 129 ABCC7 p.Ser813Ala One triple mutant (S737, 795, 813A) eliminated one inhibitory site (S737) and two stimulatory sites (S795 and S813), and the sensitivity to activation was not significantly different from that of the single-site mutant, S813A, as if the effects of eliminating S737 and S795 roughly canceled. Login to comment 130 ABCC7 p.Ser813Ala In contrast, the other triple mutant (S660, 795, 813A), which lacked the three strongest stimulatory sites, exhibited a KA that was substantially greater than that of S813A, indicating that the effects produced by eliminating each of these sites summed such that the activation sensitivity of CFTR was considerably impaired. Login to comment 132 ABCC7 p.Ser813Ala The value of KA was significantly reduced compared with that observed with the triple mutant (S660, 795,813A) but was significantly greater than that of S813A, again indicating a summation of effects such that the elimination of S737 partially ameliorated the reduced activation sensitivity produced by eliminating S660 and S795. Login to comment 135 ABCC7 p.Ser768Ala CFTR 1O-3 min-l n min 1O-3 mix1 n Wild type 664+51 20 6.0 + 0.3 8826 16 S768A 1,055 5 66* 9 12.7 5 1.7* 112? Login to comment 136 ABCC7 p.Ser813Ala 26 7 S813A 533t75* 8 2.7 + 0.3* 173515* 9 Summary of activation and deactivation parameters (means 5 SE) for wild-type CFTR and the alanine substitution mutants of the strongest inhibitory (S768) and stimulatory (S813) phosphorylation sites. Login to comment 145 ABCC7 p.Ser813Ala The data displayed in Table 3 indicate that the rate of approach to the activated steady state was decreased by -20% in the S813A construct, whereas the deactivation parameters, the latency, and the subsequent rate of exponential decline (*&E) indicated a destabilization of the active state. Login to comment 148 ABCC7 p.Ser813Ala Deactivation parameters were consistent with stabilization of the active state; the latency was double that of wild-type CFTR, Y 80 zl 2 60 0 wt 0 S813A B 5 minutes z 80 iii n 60 0 10 20 30 40 50 minutes Fig. 3. Login to comment 149 ABCC7 p.Ser813Ala ABCC7 p.Ser768Ala Representative time courses for the activation (A) and deactivation (B) of wild-type (wt) CFTR and single-site mutants S768A and S813A after, respectively, exposure to 10 PM forskolin and 5 mM IBMX and the removal of these drugs from the perfusate. Login to comment 174 ABCC7 p.Ser768Ala It is particularly interesting that S768A, the most important inhibitory site, according to the dose-response assay, has not been clearly identified as an in vivo site. Login to comment 189 ABCC7 p.Ser660Ala Gadsby and Nairn (10) called attention to the fact that the open probability of constructs like CFTRAR-S660A is well below that of wild-type channels. Login to comment