Purified Epithelial Na + Channel Complex Contains the Pertussis Toxin - sensitive Gai - 3 Protein

We have recently demonstrated that the amiloridesensitive Na+ channel in the apical membrane of the renal epithelial cell line, AB, is modulated by the ai-3 subunit of the Gi-3 protein. We also showed that a 700kDa protein complex can be purified from the membranes of A6 epithelia which (a) can reconstitute the amiloride-sensitive Na+ influx in liposomes and planar bilayer membranes and (b) consists of six major protein bands observed on reducing sodium dodecyl sulfatepolyacrylamide gels with molecular masses ranging from 36 to 320 kDa. The present study was undertaken to determine if the ai-3 subunit was a member of this Na+ channel complex. Gai structure and function were identified by Western blotting with specific Gai subunit antibodies and Na+ channel antibodies, through ADPribosylation with pertussis toxin, and by immunocytochemical localization of the Na+ channel and Gai proteins. We demonstrate that two protein substrates are ADP-ribosylated in the 700-kDa complex in the presence of pertussis toxin and are specifically immunoprecipitated with an anti-Na+ channel polyclonal antibody. One of these substrates, a 41-kDa protein, was identified as the ai.3 subunit of the G1-3 protein on Western blots with specific antibodies. Na+ channel antibodies do not recognize Gai.3 on Western blots of Golgi membranes which contain ai.3 but not Na+ channel proteins, nor do they immunoprecipitate from solubilized Golgi membranes; however, ai-3 is coprecipitated as part of the Na+ channel complex from A6 cell membranes by polyclonal Na+ channel antibodies. Both ai-3 and the Na+ channel have been localized in A6 cells by confocal imaging and immunofluoresence with specific antibodies and are found to be in distinct but adjacent domains of the apical cell surface. In functional studies, ai-3, but not w 2 , stimulates Na+ channel activity. These data are therefore consistent with the localization of Na+ channel activity and modulatory ai-3 protein at the apical plasma membrane, which together epresent a specific signal transduction pathway for ion channel regulation.