The cellular pool of Na+ channels in the amphibian cell line A6 is not altered by mineralocorticoids. Analysis using a new photoactive amiloride analog in combination with anti-amiloride antibodies.

An amiloride-sensitive Na+ channel is found in the apical plasma membrane of high resistance, Na+ transporting epithelia. We have developed a method for the identification of this channel based on the use of a new high affinity photoreactive amiloride analog, 2'-methoxy-5'-nitrobenzamil (NMBA), and anti-amiloride antibodies to identify photolabeled polypeptides. NMBA specifically labels the putative Na+ channel in bovine kidney microsomes. A 130-kDa polypeptide is detected on immunoblots with anti-amiloride antibodies. NMBA is a potent inhibitor of Na+ transport in the established amphibian kidney epithelial cell line A6, and specifically labels a 130-kDa polypeptide. We utilized both NMBA photolabeling and [3H]benzamil binding in order to examine the cellular pool of putative channels following hormonal regulation of Na+ transport. This pool is not significantly altered by the mineralocorticoid agonist aldosterone or antagonist spironolactone, despite a 3.8-fold difference in transepithelial Na+ transport

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