| 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.
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Allosteric regulation of CFTR by membrane phospholipid PIP2
