Na-channels in membrane fragments from [formula omitted] electroplaques: Biochemical studies

Abstract

Na-channels are prepared in membrane fragments from the electric organ of Electrophorus electricus . To identify Na-channels in biochemical preparations and to study their binding properties, tetrodotoxin is tritiated to a high specific activity of 18 000 Ci/mol. This is achieved by a defined chemical procedure from tetrodotoxin via acetylanhydrotetrodotoxin to 3H-tetrodotoxin. 3H-tetrodotoxin blocks Na-current in Rana esculenta nodes at a concentration of 3 nM, whereas its apparent dissociation constant for binding to Electrophorus Na-channels is K D = (20 ± 10) nM. This difference may be due to a higher density of negative surface charges in nodal regions. Density gradient membrane fractions show specific binding of 3H-tetrodotoxin up to 5 pmol/mg. Using carrier-free column electrophoresis and lectin chromatography maximal specific binding between 25 and 30 pmol/mg have been obtained. In such membrane fractions receptor densities are close to values expected for extrasynaptic regions of the excitable face of the electroplaque. 22Na-efflux from vesicles is controlled by neurotoxins, when bulk Na- and K-concentrations are altered at the beginning of an efflux experiment. Efflux data and interaction of the vesicles with lectins suggest a high proportion of inside-out vesicles in density gradient fractions.