[3H]phenamil binding protein of the renal epithelium Na+ channel. Purification, affinity labeling, and functional reconstitution.
This paper describes a large-scale purification procedure of the amiloride binding component of the epithelium Na+ channel. [3H]Phenamil was used as a labeled ligand to follow the purification. The first two steps are identical with those previously described [Barbry, P., Chassande, O., Vigne, P., Frelin, C., Ellory, C., Cragoe, E. J., Jr., & Lazdunski, M. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4836-4840]. A third step was a hydroxyapatite column. The purified material consisted of a homodimer of two 88-kDa proteins that migrated anomalously in SDS-PAGE to give an apparent Mr of 105,000. Deglycosylation by treatment with neuraminidase and endoglycosidase F or with neuraminidase and glycopeptidase F indicated that less than 5% of the mass of the native receptor was carbohydrate. Sedimentation analysis of the purified Na+ channel in H2O and D2O sucrose gradients and gel filtration experiments led to an estimated molecular weight of the [3H]phenamil receptor protein-detergent-phospholipid complex of 288,000 and of the native [3H]phenamil receptor protein of 158,000. [3H]Br-benzamil is another labeled derivative of amiloride that recognized binding sites that had the same pharmacological properties as [3H]phenamil binding sites and that copurified with them. Upon irradiation of kidney membranes, [3H]Br-benzamil incorporated specifically into a 185-kDa polypeptide chain under nonreducing electrophoretic conditions and a 105-kDa protein under reducing conditions. The same labeling pattern was observed at the different steps of the purification. Reconstitution of the purified phenamil receptor into large unilamellar vesicles was carried out. A low but significant phenamil- and amiloride-sensitive electrogenic Na+ transport was observed.
Barbry
P
P
Institut de Pharmacologie Moléculaire et Cellulaire, UPR 411 du CNRS, Valbonne, France.
Chassande
O
O
Marsault
R
R
Lazdunski
M
M
Frelin
C
C
D000345:Affinity Labels; D000584:Amiloride / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D004848:Epithelium / Q000648:ultrastructure; D006031:Glycosylation; D007668:Kidney / Q000648:ultrastructure*; D008055:Lipids; D008567:Membranes, Artificial; D011955:Receptors, Drug / Q000302:isolation & purification* / Q000378:metabolism; D015222:Sodium Channels / Q000378:metabolism*; D013552:Swine; D014316:Tritium
D000345:Affinity Labels; D008055:Lipids; D008567:Membranes, Artificial; D011955:Receptors, Drug; D015222:Sodium Channels; D014316:Tritium; D000584:Amiloride; C064150:6-bromobenzamil