Epithelial Na+ channels (ENaC) control sodium and fluid homeostasis and regulate blood pressure. In addition to being tightly regulated by well-known hormonal and homocellular factors, recent studies in A6 cells indicate that activity of ENaC may also be influenced by phosphatidylinositol 4,5-bisphosphate (PIP2), a membrane lipid1,2. Here we investigate whether PIP2 is involved in regulation of ENaC activity in mammalian epithelial cells by examining whether inhibition of PIP2 activity reduces ENaC activity in isolated mouse mandibular duct cells.
Amiloride-sensitive Na+ conductance was measured using whole-cell patch-clamp analysis in duct cells prepared from mandibular glands of euthanased Quackenbush mice3. Whole-cell current was measured three minutes after the whole-cell configuration was obtained. The amiloride-sensitive Na+ conductance was calculated from the difference between the currents observed with and without 100 μM amiloride.
Under control conditions, the amiloride-sensitive chord conductance of the duct cells was 275.2 ± 51.1 pS (n = 9). In the presence of an antibody directed against PIP2 (60 nM) in the pipette solution, the amiloride-sensitive conductance was reduced by 38.2% to 170.1 ± 12.5 pS (n = 8). Therefore, PIP2 activity may contribute to maintenance of ENaC function in duct cells. To confirm this finding, poly-L-lysine (300 μM) was added to the pipette solution to disrupt the electrostatic interaction between PIP2 and its target molecule. Poly-L-lysine reduced the amiloride-sensitive conductance by 32.8% from 304.0 ± 36.0 pS (n = 10) to 204.4 ± 44.8 pS (n = 7). The detailed mechanism of how PIP2 influences ENaC activity in duct cells is currently under investigation.
1. Dinudom, A., Harvey, K.F., Komwatana, P., Young, J.A., Kumar, S., Cook, D.I. (1998) Proceedings of the National Academy of Sciences of the United States of America 95, 7169-7173.
2. Ma, H.P., Saxena, S., Warnock, D.G. (2002) Journal of Biological Chemistry 277, 7641-7644.
3. Yue, G., Malik, B., Yue, G., Eaton, D.C. (2002) Journal of Biological Chemistry 277, 11965-11969.