Characterization and cellular localization of the epithelial Na+ channel. Studies using an anti-Na+ channel antibody raised by an antiidiotypic route.
Amiloride-sensitive Na+ channels are expressed at the apical membrane of high resistance, Na+-transporting epithelial. The specific interaction of amiloride with this transport protein suggested the feasibility of raising anti-Na+ channel antibodies by an antiidiotypic approach designed to generate antibodies directed against the amiloride-binding domain on the channel. Antiidiotypic monoclonal antibody RA6.3 mimicked the effect of amiloride by inhibiting Na+ transport across A6 cell monolayers when applied to the apical cell surface. Inhibition of transport required pretreatment of the apical cell surface with trypsin in the presence of amiloride in order to enhance accessibility of the antibody to the amiloride-binding site. This antibody specifically immunoprecipitated a large 750,000-700,000 Da protein from [35S]methionine-labeled A6 cell cultures, which was resolved further under reducing conditions as a set of polypeptides with apparent molecular masses of 260,000-230,000, 180,000, 140,000-110,000, and 70,000 Da. The antibody recognized the 140,000-Da subunit, known to contain the amiloride-binding domain, on immunoblots of purified A6 cell Na+ channel. Immunoprecipitation of apical or basolateral plasma membrane proteins selectively labeled with 125I demonstrated that expression of the oligomeric Na+ channel was restricted to the apical plasma membrane. Immunocytochemical localization in A6 cultures revealed apical membrane as well as cytosolic immunoreactive sites. Immunostaining was also observed at or near the basolateral plasma membrane.
Kleyman
T R
TR
Department of Medicine, University of Pennsylvania, Philadelphia 19104.
Kraehenbuhl
J P
JP
Ernst
S A
SA
D000584:Amiloride / Q000494:pharmacology*; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology*; D001692:Biological Transport; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004848:Epithelium / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D007668:Kidney / Q000378:metabolism*; D011233:Precipitin Tests; D011817:Rabbits; D015222:Sodium Channels / Q000737:chemistry* / Q000187:drug effects / Q000276:immunology; D014982:Xenopus laevis
D000888:Antibodies, Anti-Idiotypic; D015222:Sodium Channels; D000584:Amiloride