The epithelial Na+ channel (ENaC) is located in the apical membrane of salt (re-) absorbing epithelia in kidney, colon, airways and glandular excretory ducts.
In this thesis it is demonstrated that both stimulation of purinergic receptors and activation of the cystic fibrosis transmembrane conductance regulator (CFTR) and other Cl- channels lead to inhibition of epithelial Na+ channels, resulting in reduced Na+ absorption. Activation of purinergic receptors is the starting point for a signalling cascade, leading to inhibition of ENaC via hydrolysis of the phospholipid PIP2. The Na+ channel is also inhibited by stimulation of CFTR- or other Cl- channels, which change the intracellular Cl- concentration upon activation in co-expression experiments. A correlation was found between the degree of ENaC inhibition and the increase in intracellular Cl- concentration. While binding of ENaC to PIP2 keeps the channel in an active state, interference of ENaC with intracelluar Cl- seems inhibitory on ENaC function.
To carry out CFTR studies under conditions like in native tissue, two new cell lines were created from MDCK type II cells, which stably express wt-CFTR or its mutant counterpart F508del-CFTR. The cell lines grow as tight monolayers and are therefore suitable for Ussing chamber experiments.
CK2 is a constitutively active protein kinase that influences the activity of the channel through its binding and phosphorylation of both beta- and gamma subunits of ENaC. Lack of these phosphorylation sites or dephosphorylation of one of these sites results in a significant attenuation of the activity of the epithelial Na+ channel. It suggests that phosphorylation of both beta- and gammaENaC by CK2 prevents binding of the ubiquitin ligase Nedd4-2 to ENaC. Lack of binding of Nedd4-2 to ENaC may result in both reduced endocytosis and accumulation of ENaC in the cell membrane as well as increase in ENaC activity
