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A scorpion toxin conjugated with biotin and avidin has been used to investigate the location of the voltage-dependent Na-channel in the electric organ of Electrophorus electricus. Distribution of the conjugated scorpion toxin was visualized under a light or an electron microscope by using biotinylated peroxidase and diaminobenzidine. A dense staining was observed on the innervated surface ofthe electroplax plasmalemma, but not on the non-innervated surface. Electron micrographs at a higher magnification showed that the caveolar invaginations of the innervated membrane surface were also stained. This specific staining was completely blocked by an excess amount ofunmodified scorpion toxin or by a high concentration of KC] in the incubation medium. The voltage-dependent Na-channel plays an important role in the production ofaction potential in various excitable cells (ll). Na-channel proteins have been purified from the eel electroplax (1), rat brain synaptosomes (10), and from the rat sarcolemma (3). It was shown that a major component of the Na-channel is a polypeptide with a molecular weight of about 250,000, and also that the purified Na-channel proteins can be incorporated into liposomes without loosing their physiological activity (16-18). Recently, the primary structure of the 250,000 component prepared from the eel electroplax was determined by the cDNA cloning technique (13). Distribution of the Na-channel in excitable cells was studied immunocytochemically by using polyclonal and monoclonal antibodies raised against the Na-channel from the eel electroplax (7, 8, 12), and from the rat sarcolemma (9). These studies revealed the characteristic distribution ofNa-channels in the electric eel electrocyte (7, 8), nodes of Ranvier (7, 9, 12), and skeletal muscles (9). However, these antibodies are highly species-specific and their cross-reactions are limited. This is due partially to differences in subunit composition of the Na-channel from different excitable membranes. The polypeptide toxin from the scorpion Leiurus quinquestriatus binds to the receptor site 3 of the Na-channel with a dissociation constant in nM range and affect the inactivation process in a highly specific manner (4, 14, 15). Although binding of scorpion toxin to the Na-channel is reversible and membrane potential-dependent, this interaction occurs in a wide variety of excitable cells. Therefore, scorpion toxin is potentially useful in a comparative study ofNa-channel distribution in different species. Fluorescently labeled or 1251-labeled scorpion toxin was used to stain the Na-channel in nodes of Ranvier (2) and in cultured neuronal cells (5). These approaches, however, were not successful in elucidating details of the Na-channel distribution. Recently, biotin-labeled scorpion toxin has been developed with its toxicity unimpaired (6). The biotin-avidin system combined with horseradish peroxidase visualizes the presence of biotinylated ligand at both the light and the electron microscopic level. In the present study, we employed the biotinylated scorpion toxin to demonstrate the location of Na-channels in the electric eel electrocyte. A crude venom of the scorpion Leiurus quinquestriatus was purchased from Sigma. The toxin V was purified from the venom according to Okamoto (14). Modification of the purified

biomedical research

&lt;b&gt;Localization of the voltage-dependent sodium channel in the electric eel electrocyte using biotin-labeled scorpion &lt;/b&gt;&lt;b&gt;toxin &lt;/b&gt;