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

Abstract

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.