Purification and subunit structure of the (/sup 3/H)phenamil receptor associated with the renal apical Na/sup +/ channel

Journal Article · Wed Jul 01 00:00:00 EDT 1987 · Proc. Natl. Acad. Sci. U.S.A.; (United States)

DOI:https://doi.org/10.1073/pnas.84.14.4836· OSTI ID:5297815

Barbry, P; Chassande, O; Vigne, P; Frelin, C; Ellory, C; Cragoe, Jr, E J; Lazdunski, M

Sodium crosses the apical membrane of tight epithelia through a sodium channel, which is inhibited by the diuretic amiloride and by analogs such as phenamil. Target size analysis indicated that the functional size of the (/sup 3/H)phenamil binding sites associated with the epithelial Na/sup +/ channel from pig kidney is 90 +/- 10 kDa. The (/sup 3/H)phenamil receptor was solubilized by using 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate. The solubilized material displayed the same properties of interaction with amiloride and its derivatives as the membrane-bound receptor. A two-step purification of the epithelial Na/sup +/ channel was achieved by using QAE Sephadex chromatography and affinity chromatography on a Bandeiraea simplicifolia lectin column. It results in an 1100-fold purification of the Na/sup +/ channel as compared to pig kidney microsomes with a yield of 15% +/- 5%. The maximal specific activity was 3.7 nmol/mg of protein. NaDodSO/sub 4//polyacrylamide gel electrophoresis of the purified Na/sup +/ channel under nonreducing conditions showed the presence of a single major polypeptide chains of apparent molecular mass 185 kDa. Under disulfide-reducing conditions, the purified epithelial Na/sup +/ channel migrated as a single band of apparent molecular mass 105 kDa. It is suggested that the epithelial Na/sup +/ channel from pig kidney has a total molecular mass of 185 kDa and consists of two nearly identical 90- to 105-kDa polypeptide chains crosslinked by disulfide bridges.

• Research Organization: Centre National de la Recherche Scientifique, Nice (France)
• OSTI ID: 5297815
• Journal Information: Proc. Natl. Acad. Sci. U.S.A.; (United States), Vol. 84:14
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
DIURETICS
MEMBRANE TRANSPORT
RECEPTORS
MOLECULAR STRUCTURE
PURIFICATION
TRITIUM COMPOUNDS
CATIONS
ELECTROPHORESIS
KIDNEYS
POLYPEPTIDES
SODIUM COMPOUNDS
ALKALI METAL COMPOUNDS
BODY
CHARGED PARTICLES
DRUGS
IONS
LABELLED COMPOUNDS
MEMBRANE PROTEINS
ORGANIC COMPOUNDS
ORGANS
PEPTIDES
PROTEINS
550201* - Biochemistry- Tracer Techniques