Cystic Fibrosis is a disease that arises from the dysfunction or absence of the cystic fibrosis transmembrane conductance regulator or CFTR. This protein is the single ion channel member of the ABC superfamily and conducts Cl − and bicarbonate ions across the apical plasma membrane of epithelial cells. 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 closing of the channel (gating) is regulated by ATP binding to the NBDs and the phosphorylation of the RD. Phosphatidylinositol 4,5-biphosphate or PIP2 is a membrane phospholipid that regulates ion channel activity through electrostatic interactions. In 2004 PIP2 was reported to regulate gating of the unphosphorylated CFTR, while experiments using PIP2 on the phosphorylated channel remained inconclusive. No other reports on regulation of CFTR activity by PIP2 have been published. Here, we present data that elaborate on the CFTR-PIP2 interaction at distinct stages of the CFTR activation cycle. In inside-out patch clamp recordings from HEK293T cells, superfusion of CFTR with the water-soluble short-chain dioctanoyl-PI(4,5)P2 (diC8-PIP2) in the presence of MgATP activated the unphosphorylated channel, as well as the phosphorylated CFTR, both during and after phosphorylation by protein kinase A (PKA). Additionally, the PIP2 analog significantly accelerated CFTR activation kinetics by PKA. To examine the effect on CFTR of depleting membrane PIP2 levels, we utilized a powerful optogenetic tool in whole-cell recordings to specifically remove the P4 and P5 phosphates of the phospholipid. This intervention inhibited CFTR activity. Overall, our data suggest that CFTR gating is strongly modulated by PIP2 and hint at an interplay between channel, kinase, and PIP2.
Vynichaki et al. (Sun,) studied this question.