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 (Gly 1-His 13), Leu 815-Gln 828 and the C-terminus (Ile 1002-Tyr 1006) 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 Asn 889-Gln 903 to cross-fractured basolateral plasma membranes suggesting that this epitope is located on the extracellular side of the membrane.