The Journal of biological chemistry
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The cystic fibrosis transmembrane conductance regulator (CFTR) acts as a channel on the apical membrane of epithelia. Disease-causing mutations in the cystic fibrosis gene can lead to CFTR protein misfolding as in the case of the F508del mutation and/or channel dysfunction. Recently, a small molecule, VX-770 (ivacaftor), has shown efficacy in restoring lung function in patients bearing the G551D mutation, and this has been linked to repair of its channel gating defect. ⋯ Interestingly, we also found that VX-770 enhanced the channel activity of purified and mutant CFTR in the nominal absence of Mg-ATP. These findings suggest that VX-770 can cause CFTR channel opening through a nonconventional ATP-independent mechanism. This work sets the stage for future studies of the structural properties that mediate CFTR gating using VX-770 as a probe.
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Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel activated by multiple stimuli and is implicated in a variety of pain disorders. Dynamic sensitization of TRPV1 activity by A-kinase anchoring protein 150 demonstrates a critical role for scaffolding proteins in nociception, yet few studies have investigated scaffolding proteins capable of mediating receptor desensitization. In this study, we identify β-arrestin-2 as a scaffolding protein that regulates TRPV1 receptor activity. ⋯ In addition, we found that β-arrestin-2 scaffolding of phosphodiesterase PDE4D5 to the plasma membrane was required for TRPV1 desensitization. Importantly, inhibition of PDE4D5 activity reversed β-arrestin-2 desensitization of TRPV1. Together, these results identify a new endogenous scaffolding mechanism that regulates TRPV1 ligand binding and activation.
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N-type Ca(2+) channels (CaV2.2) are a nidus for neurotransmitter release and nociceptive transmission. However, the use of CaV2.2 blockers in pain therapeutics is limited by side effects resulting from inhibition of the physiological functions of CaV2.2 within the CNS. We identified an anti-nociceptive peptide (Brittain, J. ⋯ Similar results were obtained in DRGs from mice with a heterozygous mutation of Nf1 linked to neurofibromatosis type 1. Ct-dis peptide, administered intraperitoneally, exhibited antinociception in a zalcitabine (2'-3'-dideoxycytidine) model of AIDS therapy-induced and tibial nerve injury-related peripheral neuropathy. This study suggests that CaV peptides, by perturbing interactions with the neuromodulator CRMP2, contribute to suppression of neuronal hypersensitivity and nociception.
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G-protein coupled receptors (GPCRs) have long been known as receptors that activate G protein-dependent cellular signaling pathways. In addition to the G protein-dependent pathways, recent reports have revealed that several ligands called "biased ligands" elicit G protein-independent and β-arrestin-dependent signaling through GPCRs (biased agonism). Several β-blockers are known as biased ligands. ⋯ However, the metoprolol-induced fibrosis and cardiac dysfunction were not evoked in β-arrestin2- or GRK5-knock-out mice. Thus, metoprolol is a biased ligand that selectively activates a G protein-independent and GRK5/β-arrestin2-dependent pathway, and induces cardiac fibrosis. This study demonstrates the physiological importance of biased agonism, and suggests that G protein-independent and β-arrestin-dependent signaling is a reason for the diversity of the effectiveness of β-blockers.
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Triapine® (3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP)) is a drug in Phase II trials. One of its established cellular targets is the β(2) subunit of ribonucleotide reductase that requires a diferric-tyrosyl-radical [(Fe(III)(2)-Y·)(Fe(III)(2))] cofactor for de novo DNA biosynthesis. Several mechanisms for 3-AP inhibition of β(2) have been proposed; one involves direct iron chelation from β(2), whereas a second involves Y· destruction by reactive oxygen species formed in situ in the presence of O(2) and reductant by Fe(II)-(3-AP). ⋯ Three mammalian cell lines treated with 5 μm 3-AP reveal Y· loss and β(2) inactivation within 30-min of 3-AP-exposure, analyzed by whole-cell EPR and lysate assays, respectively. Selective degradation of apo- over [(Fe(III)(2)-Y·)(Fe(III)(2))]-β(2) in lysates, similar iron-content in β(2) immunoprecipitated from 3-AP-treated and untreated [(55)Fe]-prelabeled cells, and prolonged (12 h) stability of the inhibited β(2) are most consistent with Y· loss being the predominant mode of inhibition, with β(2) remaining iron-loaded and stable. A model consistent with in vitro and cell-based biochemical studies is presented in which Fe(II)-(3-AP), which can be cycled with reductant, directly reduces Y· of the [(Fe(III)(2)-Y·)(Fe(III)(2))] cofactor of β(2).