Epitope topology of Na,K‐ATPase α subunit analyzed in basolateral cell membranes of rat kidney tubules

Abstract

For topological analysis of integral membrane protein in situ, we used a novel immunoelectron microscopic technique, SDS-digested freeze-fracture replica labeling (SDS-FRL), and oligopeptide-specific antibodies to clarify the sidedness of Na,K-ATPase α subunit epitopes in basolateral cell membranes of kidney tubules. Unfixed tissue slices from rat kidney outer medulla were frozen with liquid helium, freeze-fractured, and replicated. After digestion with SDS to solubilize unfractured membranes and cytoplasm, the platinum/carbon replicas, along with attached cytoplasmic and exoplasmic membrane halves, were processed for immunocytochemistry. Immunogold labeling using antibodies against the N-terminus (Gly¹-His¹³), Leu⁸¹⁵-Gln⁸²⁸ and the C-terminus (Ile¹⁰⁰²-Tyr¹⁰⁰⁶) was superimposed on the images of the electron microscope protoplasmic fracture face of the basolateral plasma membranes, thus demonstrating cytoplasmic locations of these epitopes. On the contrary, SDS-FRL showed specific binding of Asn⁸⁸⁹-Gln⁹⁰³ to cross-fractured basolateral plasma membranes suggesting that this epitope is located on the extracellular side of the membrane.