Topology of the C-Terminus of Sodium Hydrogen Exchanger Isoform-1: Presence of an Extracellular Epitope

In this study, the topology of the C-terminus of Na+/H+ exchanger-1 (NHE-1) was examined using red blood cell (RBC) membrane vesicles. Specific polyclonal antibodies were raised against the C-terminus of the rat NHE-1 using a glutathione S-transferase fusion protein antigen. Enzyme-linked immunosorbent assay (ELISA) and flow cytometer analyses were employed. NHE-1 antibodies recognized a 110-kDa protein in the rats and mice, but not in the humans. RBC vesicles were resealed using 1 mM MgCl2, and the right side out (RSOVs) and the inside out vesicles (ISOVs) were selected by concanavalin A. NHE-1 antibodies reacted strongly with the RSOVs and poorly with the ISOVs in an ELISA. The reaction with the unsealed vesicles was stronger than the resealed RSOVs. In contrast, the monoclonal α-actin antibodies reacted poorly with the RSOVs, but extremely strongly with the ISOVs. Flow cytometer analysis also showed a strong reaction of NHE-1 antibodies with the intact and permeabilized rat RBCs. The antibodies raised in this study also contain a population which reacts with the internal epitope in the NHE-1 C-terminus. These findings suggest that the NHE-1 C-terminus contains epitope(s) exposed extracellularly.