Topology of Na,K-ATPase α subunit epitopes analyzed with oligopeptide-specific antibodies and double-labeling immunoelectron microscopy

Using four oligopeptide-specific polyclonal antibodies, we mapped the α subunit ofNa,K-ATPase by double-labeling immunoelectron microscopy combined with negative staining. The results show that the epitopes of the N-tenninus (Gly¹-His¹³), C-terminus (Ile¹⁰⁰²-Tyr¹⁰¹⁶) and Leu⁸¹⁵-Gln⁸²⁸ are located on the same face of crystallized Na,K-ATPase membranes from pig kidney, whereas the epitope Asn⁸⁸⁹-Gln⁹⁰³ is present on the opposite side. The present study demonstrates the cytoplasmic location of C-terminus and that Leu⁸²⁸-Gln⁸²⁸ is exposed on the eytoplasmic and Asn⁸⁸⁹-Gln⁹⁰³ on the extracellular side. The results are consistent with an eight- or ten-segment model, and support the existence of an M5/M6 loop and the presence of one transmembrane segment between Leu⁸¹⁵-Gln⁸²⁸ and Asn⁸⁸⁹-Gln⁹⁰³.