The epithelial sodium channel. Subunit number and location of the amiloride binding site

Sodium dodecyl sulfate gel electrophoresis of the radioiodinated native amiloride-sensitive epithelial sodium channel protein isolated from bovine renal papilla and cultured amphibian A6 cells under denatured and nonreduced conditions revealed an 125I-labeled protein band of Mr approximately 730,000...

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Bibliographic Details
Published in:The Journal of biological chemistry 1987-08, Vol.262 (22), p.10613-10618
Main Authors: Benos, D J, Saccomani, G, Sariban-Sohraby, S
Format: Article
Language:English
Subjects:
Affinity Labels
Amiloride - metabolism
Amphibians
Animals
Binding Sites
Biological and medical sciences
Cattle
Cell Line
Cell physiology
Epithelium - analysis
Fundamental and applied biological sciences. Psychology
Ion Channels - metabolism
kidney
Kidney - analysis
Membrane and intracellular transports
Molecular and cellular biology
Molecular Weight
Photochemistry
sodium
Sodium - metabolism
subunit structure
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Summary:Sodium dodecyl sulfate gel electrophoresis of the radioiodinated native amiloride-sensitive epithelial sodium channel protein isolated from bovine renal papilla and cultured amphibian A6 cells under denatured and nonreduced conditions revealed an 125I-labeled protein band of Mr approximately 730,000. Upon reduction, this protein was resolved into five major polypeptide bands with apparent average Mr values of 315,000, 149,000, 95,000, 71,000, and 55,000. The amiloride analog [3H]methylbromoamiloride has been used as a photoaffinity label to determine the location of the binding site for amiloride on the epithelial sodium channel protein. [3H]Methylbromoamiloride binds covalently to the sodium channel at high affinity binding sites with a half-maximal binding concentration of 0.2 microM. [3H]Methylbromoamiloride was specifically photoincorporated into the Mr approximately 150,000 polypeptide and this incorporation was blocked by addition of excess amiloride. These data suggest that the epithelial sodium channel protein is composed of at least five nonidentical polypeptide subunits, only one of which specifically binds amiloride.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(18)61007-5