Contents

PIP₂ mediates sodium feedback inhibition in the epithelial sodium channel

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.