Binding of the proline-rich region of the epithelial Na+ channel to SH3 domains and its association with specific cellular proteins

Abstract

The amiloride-sensitive epithelial Na+ channel (ENaC) is an important component of the Na+-reabsorption pathway in many epithelia. The identification of three subunits of ENaC (α, β and γ), as well as results from a number of functional and biochemical studies, suggests that functional Na+ channels are composed of a complex of proteins. To learn about possible interactions of the channel with other proteins, we studied the α-subunit of rat and human ENaC. We found that the proline-rich C-terminal domains of both rat and human α-ENaC, expressed as glutathione S-transferase fusion proteins, bound to SH3 domains in vitro. A 116 kDa protein from a human lung adenocarcinoma cell line (H441) was specifically bound by the human α-ENaC C-terminal fusion protein and by a shorter 18-amino acid proline-rich peptide derived from the larger fusion protein. The 116 kDa protein was not glycosylated and was not phosphorylated on tyrosine or by cyclic AMP-dependent protein kinase (PKA). A 134 kDa protein which was also bound by the human α-ENaC C-terminal fusion protein was a substrate for phosphorylation by PKA. These data suggest that the proline-rich C-terminal tail of α-ENaC may interact with other proteins that control its function, regulation or localization.