A new variant of the subunit of renal Na,K-ATPase: Identification by mass spectrometry, antibody binding, and expression in cultured cells

The γ subunit is a specific regulator of Na,K-ATPase expressed mainly in kidney. On SDS-polyacryylamide gel electrophoresis, γ runs as a doublet, but the origin and significance of the doublet is obscure. Mass spectrometry of the γ chains of rat kidney Na,K-ATPase shows that γ(a) (upper) has a mass of 7184.0 ± 1 Da (carbamidomethyl cysteine), corresponding closely to that for the published sequence without the initiator methionine, while γ(b) (lower) has a mass of 7337.9 ± 1Da. Tryptic peptide mapping and sequencing by mass spectrometry reveals that the seven N-terminal residues of γ(a), TELSANH, are replaced by Ac-MDRWYL in γ(b), but otherwise the chains are identical. Antibodies raised against peptides TELSANHC and MDRWYLC recognize either γ(a) or γ(b) of the Na,K-ATPase, respectively. γ(a) or γ(b) cDNAs have been expressed in human embryonic kidney and HeLa cells. The major bands expressed correspond to γ(a) or γ(b) of renal Na,K-ATPase. Additional minor bands seen after transfection, namely γ(a) in human embryonic kidney and γ(b) in HeLa, are presumably cell-specific modifications. The present work clarifies earlier uncertainty regarding doublets seen in kidney and in transfected cells. In particular, the results show that renal Na,K-ATPase contains two variants of the γ subunit with different sequences but otherwise are unmodified. We discuss the possible functional significance of the two variants.