Purified epithelial Na+ channel complex contains the pertussis toxin-sensitive Gαi-3 protein

Ausiello, Dennis A., Stow, Jennifer L., Cantiello, Horacio F., De Almeida, J. Bruno and Benos, Dale J. (1992) Purified epithelial Na+ channel complex contains the pertussis toxin-sensitive Gαi-3 protein. Journal of Biological Chemistry, 267 7: 4759-4765.

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Author Ausiello, Dennis A.
Stow, Jennifer L.
Cantiello, Horacio F.
De Almeida, J. Bruno
Benos, Dale J.
Title Purified epithelial Na+ channel complex contains the pertussis toxin-sensitive Gαi-3 protein
Formatted title
Purified epithelial Na+ channel complex contains the pertussis toxin-sensitive Gαi-3 protein
Journal name Journal of Biological Chemistry   Check publisher's open access policy
ISSN 0021-9258
1083-351X
Publication date 1992-03-05
Sub-type Article (original research)
Open Access Status File (Publisher version)
Volume 267
Issue 7
Start page 4759
End page 4765
Total pages 7
Place of publication Bethesda, MD, United States
Publisher American Society for Biochemistry and Molecular Biology
Language eng
Formatted abstract
We have recently demonstrated that the amiloride-sensitive Na+ channel in the apical membrane of the renal epithelial cell line, A6, is modulated by the αi-3 subunit of the Gi-3 protein. We also showed that a 700-kDa 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 sulfate-polyacrylamide gels with molecular masses ranging from 35 to 320 kDa. The present study was undertaken to determine if the αi-3 subunit was a member of this Na+ channel complex. Gαi structure and function were identified by Western blotting with specific Gαi subunit antibodies and Na+ channel antibodies, through ADP-ribosylation with pertussis toxin, and by immunocytochemical localization of the Na+ channel and Gαi 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 αi-3 subunit of the Gi-3 protein on Western blots with specific antibodies. Na+ channel antibodies do not recognize Gαi-3 on Western blots of Golgi membranes which contain αi-3 but not Na+ channel proteins, nor do they immunoprecipitate αi-3 from solubilized Golgi membranes; however, αi-3 is coprecipitated as part of the Na+ channel complex from A6 cell membranes by polyclonal Na+ channel antibodies. Both αi-3 and the Na+ channel have been localized in A6 cells by confocal imaging and immunofluorescence with specific antibodies and are found to be in distinct but adjacent domains of the apical cell surface. In functional studies, αi-3, but not αi-2, stimulates Na+ channel activity. These data are therefore consistent with the localization of Na+ channel activity and modulatory αi-3 protein at the apical plasma membrane, which together represent a specific signal transduction pathway for ion channel regulation.
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: Institute for Molecular Bioscience - Publications
 
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