ABCMdb

PMID: 15857825

Wang W, Li G, Clancy JP, Kirk KL
Activating cystic fibrosis transmembrane conductance regulator channels with pore blocker analogs.
J Biol Chem. 2005 Jun 24;280(25):23622-30. Epub 2005 Apr 27., 2005-06-24 [PubMed]

Sentences

No. Mutations Sentence Comment

27 ABCC7 p.Ser660Ala Cells expressing ⌬F508-CFTR or ⌬R-S660A-CFTR (where "R" is the regulatory domain) were grown for 1-2 days at 27 °C (low temperature-corrected) because these mutants are temperature-sensitive endoplasmic reticulum processing mutants that exhibit low surface expression when cells are cultured at 37 °C. Login to comment 68 ABCC7 p.Arg347Glu To determine whether NPPB activates CFTR by binding to the same (or different) site that causes a pore block, we tested its effects on a CFTR pore mutant (R347E) that is resistant to block by NPPB (19). Login to comment 69 ABCC7 p.Arg347Glu Fig. 1 (E and F) shows that NPPB stimulated the currents mediated by R347E-CFTR at positive potentials to a greater extent compared with wild-type CFTR at moderate levels of phosphorylation (high PKA concentration (110 units/ml), followed by PKI). Login to comment 70 ABCC7 p.Arg347Glu Unlike the wild-type channel, the R347E-CFTR currents were stimulated by NPPB even at negative potentials. Login to comment 71 ABCC7 p.Arg347Glu Thus, NPPB behaves more as a pure agonist for the R347E pore mutant, which implies that this compound stimulates channel opening by binding to a site that is distinct from the pore-blocking site. Login to comment 105 ABCC7 p.Ser660Ala Second, NPPB-AM markedly stimulated the currents mediated by a regulatory domain deletion mutant (⌬R-S660A-CFTR) (Fig. 3A) that exhibits low constitutive activity in the absence of PKA (23). Login to comment 109 ABCC7 p.Gly551Asp NPPB-AM Markedly Stimulates the Opening of ⌬F508-CFTR Channels under Conditions in Which Wild-type Channels Are Maximally Activated-We next tested the effects of NPPB and derivatives on the two most common CF mutants: G551D-CFTR and ⌬F508-CFTR. Login to comment 110 ABCC7 p.Gly551Asp G551D-CFTR is a gating mutant (24) that, unlike ⌬F508-CFTR, is trafficked to the cell surface with efficiency similar to that of wild-type CFTR. Login to comment 111 ABCC7 p.Gly551Asp The G551D mutation maps to a region in NBD1 that likely plays a role in MgATP binding or the conformational coupling between ATP binding and the opening of the pore within the transmembrane domains (ATP-binding cassette transporter signature sequence) (25). Login to comment 113 ABCC7 p.Gly551Asp However, G551D-CFTR activity was markedly stimulated by high doses of the charged parent compound (NPPB), doses that were impossible to achieve for the less soluble uncharged derivative (Fig. 3E). Login to comment 114 ABCC7 p.Gly551Asp In NPPB titration experiments, we observed an appreciable shift toward higher concentrations of NPPB for G551D-CFTR activation compared with wild-type channel activation (Fig. 3F). Login to comment 115 ABCC7 p.Gly551Asp This result implies that the G551D mutation in NBD1 reduces the apparent affinity of NPPB (and presumably of NPPB-AM) for its activation site. Login to comment 138 ABCC7 p.Arg347Glu F, at low doses, NPPB stimulates currents in both directions for moderately phosphorylated R347E-CFTR channels in an HEK-293T patch. Login to comment 157 ABCC7 p.Ser660Ala A, NPPB-AM and NPPB markedly stimulate ⌬R-S660A-CFTR currents minus PKA across an excised HEK-293T patch. Login to comment 168 ABCC7 p.Gly551Asp E, NPPB-AM weakly activates G551D-CFTR, whereas high doses of NPPB markedly stimulate this mutant at high PKA concentrations in excised HEK-293T patches. Login to comment 170 ABCC7 p.Gly551Asp See mean data in F. F, NPPB stimulates wild-type CFTR currents (WT; E) at lower doses than G551D-CFTR currents (●) in excised HEK-293T patches. Login to comment 172 ABCC7 p.Gly551Asp The conditions for G551D-CFTR were as described for E. Login to comment 173 ABCC7 p.Gly551Asp Shown are mean data at Ϯ80 mV obtained from four and seven experiments for the wild-type and G551D-CFTR channels, respectively. Data were normalized to the peak current induced by NPPB at each voltage. Login to comment 203 ABCC7 p.Ser660Ala NPPB-AM and NPPB are capable of stimulating CFTR opening without affecting channel phosphorylation by PKA, i.e. these compounds enhance wild-type or ⌬R-S660A-CFTR activity in the absence of active kinase. Login to comment 208 ABCC7 p.Ser660Ala In this regard, the ⌬R-S660A-CFTR construct used here (23) has much lower channel activity than highly phosphorylated wild-type channels (Po estimated at Ͻ0.05) (29), although this activity is not dependent on PKA. Login to comment 234 ABCC7 p.Ser660Ala Michael Welsh and John Wakefield for sharing the ⌬R-S660A-CFTR construct and the ⌬F508-CFTR-transfected CFBE41o-cells, respectively. Login to comment