Tetrodotoxin receptors in membrane fragments: purification from Electrophorus electricus electroplax and binding...

A tetrodotoxin receptor-rich preparation of membrane fragments from the electric organ of Electrophorus electricus is described. The specific binding of neurotoxins and freeze-fracture electron microscopy are used as tools to identify and to characterize membrane fractions. Freeze-fracture electron micrographs of the electric organ demonstrate a high density of membrane particles in the extrasynaptic regions. Density gradient fractions show a broad distribution of [3H]tetrodotoxin, [3H]saxitoxin and 125I-labelled bungarotoxin binding in the range of 1.04--1.15 g/ml sucrose densities, with specific neurotoxin binding up to approx. 5 pmol/mg protein. Carrier-free column electrophoresis of density gradient fractions yields a subfraction with tetrodotoxin and alpha-neurotoxin binding up to 30 pmol/mg protein. The major part of the membrane fragments forms vesicles, which are separated by lectin chromatography into an outside-out and inside-out population. The latter represents at least 50% of the material of a density gradient fraction. For the association of tetrodotoxin, a bimolecular kinetic constant kf greater than or equal to 3.10(5) M-1.s-1 is determined. The dissociation constant is k'b = 2.5.10(-2)s-1. These data are in agreement with a thermodynamic dissociation constant of Kd = 20 nM as determined earlier for E. electricus membrane fragments by equilibrium methods (Grünhagen, H.H., Rack, M., Stämpfli, R., Fasold, H. and Reiter, P. (1981) Arch. Biochem. Biophys. 206, in the press). However, these association kinetics of tetrodotoxin binding in vitro are significantly different from kinetics determined electrophysiologically in Rana (Wagner, H.H. and Ulbricht, W. (1975) Pflügers Arch. 359, 297--315) or Xenopus (Schwarz, J.R., Ulbricht, W. and Wagner, H.H. (1973) J. Physiol. 233, 167--194).