A functional membranous acceptor for dendrotoxin in rat brain: solubilization of the binding component

DTX, a single-chain polypeptide of M, 7000 from the venom of the eastern green mamba Dendroaspis angusticeps, has been shown to facilitate transmitter release at the neuromuscular junction (Harvey & Karlsson, 1982) and in the central nervous system (Docherty et al., 1983). It is highly toxic when administered centrally (Dolly et al., 19831, producing a convulsant action arising from its facilitation of the release of neurotransmitters. In the CAI region of the rat or guinea-pig hippocampus, this convulsive activity probably results from the inhibition of the A current, attributable to an outward K+ conductance (Dolly et al., 1984). By using ?sI-labelled DTX we recently demonstrated the occurrence of a high-affinity acceptor protein for DTX on synaptic membranes from rat cortex (KD = 0 . 4 3 n ~ , B,,,, = I . 1 pmol/mg of protein) which is located in discrete regions of the brain (Dolly et al., 1984). Cross-linking of IZSI-DTX to the acceptor and subsequent analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed a subunit of M, 65000 (Mehraban et al., 1984). Evidence for the involvement of the acceptor in the toxicity of DTX was obtained from structure-activity studies with toxins homologous to DTX. Accordingly, intraventricular injections into rat brain of DTX and four congeners (from related species of snake) revealed levels of toxicity commensurate with their potency in inhibiting the binding of 1?51-DTX to the membrane acceptor in ritro (Table I ) . Further characterization of the DTX-acceptor involved its successful solubilization in an active form by detergents such as Triton X-100 or deoxycholate. Extraction of rat cortex membranes with detergent in the presence of antiproteases in K+-containing buffers (50250mM) resulted in recovery of > 70% of activity relative to that in membranes; the Kd for DTX (Table I ) was not increased after solubilization. Omission of K + from the solubilization medium or its replacement with Na+ resulted in total loss of specific '?'I-DTX binding from the extract; however, Rb+ could effectively replace K + . As expected, heating the extract or digestion with trypsin (5mg/ml at 28°C for 40min) abolished all specific binding of 1?51-DTX. Sedimentation analysis of the extracted acceptor. followed by