A topographical study of the electroplax sodium channel with site-directed antibodies

Three peptides corresponding to residues (13-23C), (Y1419-1431), and (1809-1820) of the electric eel sodium channel have been synthesized and used to raise antisera in rabbits. All the antibodies produced specifically recognized the corresponding peptides in an ELISA assay. However, their avidities to the channel protein were different. Two antibodies, against the sequence (1809-1820) and (13-23C) which are directed to the C-terminus region and to the adjacent portion of N-terminus, respectively, recognized the 250 kDa channel protein in the immunoblot and an ELISA assay. Binding of the two antibodies to the sodium channel in oriented electroplax membrane vesicles was increased 4-5-fold after permeabilizing the vesicles with 0.01% saponin, implying cytoplasmic orientation for the two peptides. The cytoplasmic orientation of the N- and C-terminal regions were further confirmed by immunogold electron microscopy. By contrast, antibody raised against the sequence (Y1419-1431) which has been proposed to be the transmembrane segment S4 of internal repeat IV, did not react with the channel protein not only after the saponin treatment but also even under the immunoblot conditions following SDS-PAGE. The antibody could recognize fragments of the channel protein after digestion with lysyl endoproteinase, suggesting that the region may apparently form a strictly well-ordered conformation in the transmembrane part of the channel molecule.