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Identifier 000449822
Title Allosteric regulation of CFTR by membrane phospholipid PIP2
Alternative Title Αλλοστερική ρύθμιση του CFTR από το μεμβρανικό φωσφολιπίδιο PIP2
Author Βυνιχάκη, Ιωάννα Μαρία
Thesis advisor Λογοθέτης, Διομήδης
Abstract Cystic Fibrosis is a lethal disease that arises from the dysfunction or even absence of the Cystic Fibrosis Transmembrane Conductance Regulator or CFTR. This protein is the single ion channel amongst the ABC superfamily, conducting Cl- ions across the apical plasma membrane of epithelial cells. Disease-causing mutations on CFTR can cause from protein-synthesis and maturation defects to activity dysfunctions. Τhe most common mutation is the deletion of Phe508 (ΔF508), that comprises 70% of the cases. Its structure contains two transmembrane domains (TMD1, TMD2) that form the channel pore, two nucleotide-binding domains (NBD1, NBD2) and a unique Regulatory Domain (RD). The opening and the closing of the channel (gating) is considered to be regulated by ATP binding to the conserved NBDs and the phosphorylation of the RD. On the other hand, phosphatidylinositol 4,5- biphosphate or PIP2 is a membrane phospholipid that is a major factor in ion-channel regulation, at times being an obligatory constituent for channel activity. In a 2004 scientific report, PIP2 was preliminarily shown to exert gating effects on both the unphosphorylated and the phosphorylated CFTR. However, since then, the relationship between CFTR and PIP2 has not been further clarified. Here we present data from inside-out, patch clamp electrophysiological experiments in Xenopus laevis oocytes that elaborate on this relationship at distinct stages of the CFTR activation cycle. Perfusion of CFTR with the water-soluble, short-chain dioctanoyl-PI(4,5)P2 (diC8-PIP2) in the presence of MgATP, was able to activate the unphosphorylated channel, as well as the phosphorylated CFTR, both during and after phosphorylation by Protein Kinase A (PKA). Intriguingly, the diC8-PIP2 effect appeared to be time-dependent during PKA phosphorylation, with early-on application of the phospholipid providing significantly higher current enhancement compared to later in the PKA phosphorylation stage. Additionally, PIP2 chelation with poly-L-Lysine (PL) provided interesting results when applied before and during phosphorylation. The unphosphorylated channel in ATP was activated by PL, while the polycation was able to significantly inhibit the current of the phosphorylated CFTR. Moreover, we established a CFTR activation and inhibition protocol in the whole-cell patch-clamp mode, that will allow us to study the CFTR-PIP2 relationship to a greater extent. Overall, we suggest that CFTR gating is modulated by PIP2 in a manner that is greatly dependent on the phosphorylation state of the channel.
Language English
Subject Fhosphorylation
Φωσφορυλίωση
Issue date 2022-07-29
Collection   School/Department--School of Medicine--Department of Medicine--Post-graduate theses
  Type of Work--Post-graduate theses
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