Primary structure of an apical protein from Xenopus laevis that participates in amiloride-sensitive sodium channel activity.
High resistance epithelia express on their apical side an amiloride-sensitive sodium channel that controls sodium reabsorption. A cDNA was found to encode a 1,420-amino acid long polypeptide with no signal sequence, a putative transmembrane segment, and three predicted amphipathic alpha helices. A corresponding 5.2-kb mRNA was detected in Xenopus laevis kidney, intestine, and oocytes, with weak expression in stomach and eyes. An antibody directed against a fusion protein containing a COOH-terminus segment of the protein and an antiidiotypic antibody known to recognize the amiloride binding site of the epithelial sodium channel (Kleyman, T. R., J.-P. Kraehenbuhl, and S. A. Ernst. 1991. J. Biol. Chem. 266:3907-3915) immunoprecipitated a similar protein complex from [35S]methionine-labeled and from apically radioiodinated Xenopus laevis kidney-derived A6 cells. A single integral of 130-kD protein was recovered from samples reduced with DTT. The antibody also cross-reacted by ELISA with the putative amiloride-sensitive sodium channel isolated from A6 cells (Benos, D. J., G. Saccomani, and S. Sariban-Sohraby. 1987. J. Biol. Chem. 262:10613-10618). Although the protein is translated, cRNA injected into oocytes did not reconstitute amiloride-sensitive sodium transport, while antisense RNA or antisense oligodeoxynucleotides specific for two distinct sequences of the cloned cDNA inhibited amiloride-sensitive sodium current induced by injection of A6 cell mRNA. We propose that the cDNA encodes an apical plasma membrane protein that plays a role in the functional expression of the amiloride-sensitive epithelial sodium channel. It may represent a subunit of the Xenopus laevis sodium channel or a regulatory protein essential for sodium channel function.
Staub
O
O
Swiss Institute for Experimental Cancer Research, University of Lausanne, Epalinges.
Verrey
F
F
Kleyman
T R
TR
Benos
D J
DJ
Rossier
B C
BC
Kraehenbuhl
J P
JP
D000584:Amiloride / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002024:Bufo marinus; D016764:Cell Polarity; D003001:Cloning, Molecular; D004848:Epithelium; D015723:Gene Library; D007668:Kidney; D008565:Membrane Proteins / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012333:RNA, Messenger / Q000032:analysis; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000187:drug effects / Q000235:genetics*; D014018:Tissue Distribution; D029867:Xenopus Proteins; D014982:Xenopus laevis / Q000235:genetics*
D008565:Membrane Proteins; D012333:RNA, Messenger; C078824:Shroom1 protein, Xenopus; D015222:Sodium Channels; D029867:Xenopus Proteins; D000584:Amiloride; D012964:Sodium