Epithelial Na+ channels (ENaC) are important for regulation of Na+ and fluid homeostasis and blood pressure. It has been previously reported that phosphatidylinsositol 4,5-bisphosphate (PIP2) upregulates ENaC activity (Yue et al., 2002) and that PIP2 may mediate the regulation of ENaC by purinergic stimulation (Kunzelmann et al., 2005). Details of the mechanism by which PIP2 acts on ENaC are, however, currently unknown. In this project we used the whole-cell patch-clamp technique to measure ENaC activity in isolated mouse mandibular duct cells (Dinudom et al., 1998) in order to investigate the pathway through which PIP2 acts to increase ENaC activity. We found that the activity of ENaC in the duct cells is inhibited by inclusion in the pipette solution of a specific antibody directed against PIP2. Inclusion in the pipette solution of peptides corresponding to the N-termini of β- or γ-ENaC significantly decreased ENaC activity. These peptides contain consensus PIP2 binding motifs. We found that inclusion of 25 μM PIP2 in the pipette solution completely overcame the inhibitory effect of increased cytosolic Na+ on ENaC. Interestingly, PIP2 did not overcome the inhibitory effect of inclusion of recombinant Nedd4-2 protein in the pipette solution. Taken together, these data suggest that PIP2 is essential for maintaining ENaC activity in salivary duct cells and that it exerts its effect on ENaC by blocking the Na+ feedback pathway, possibly at a step upstream from Nedd4-2. The mechanism by which PIP2 inhibits Na+ feedback inhibition of ENaC is currently under investigation.
Yue G, Malik B, Yue G & Eaton DC (2002) Journal of Biological Chemistry, 277: 11965-11969.
Kunzelmann K, Bachhuber T, Regeer R, Markovich D, Sun J & Schreiber R (2005) FASEB Journal, 19: 142-143.
Dinudom A, Harvey KF, Komwatana P, Young JA, Kumar S, Cook DI (1998) Proceedings of the National Academy of Sciences USA, 95: 7169-7173.