Solubilization and physical characterization of acceptors for dendrotoxin and .beta.-bungarotoxin from synaptic membranes of rat brain

Abstract

Dendrotoxin (DTX), an Mr 7000 convulsant polypeptide from the venom of Dendroaspis angusticeps, or its facilitatory homologues act through blockade of certain voltage-sensitive K+ currents in a variety of neurons. High-affinity acceptors for DTX have been demonstrated in synaptic plasma membranes of rat or chick brain, and a fraction of these avidly bind beta-bungarotoxin (beta-BuTX), a presynaptically active protein whose lighter B polypeptide is homologous to this toxin. Extraction of rat synaptic plasma membranes using Triton X-100 in K+-containing buffer yielded binding sites with KD values of approximately 0.5 and 0.7 nM for 125I-labeled DTX and beta-BuTX, respectively. The content of high-affinity sites obtained for beta-BuTX, including the contribution of a lower affinity component, approximates to the Bmax (approximately 1.3 pmol/mg of protein) obtained for the apparent single set of DTX acceptors. On solubilization, the pharmacological specificity of the acceptor for neurotoxic DTX congeners was retained. 125I-beta-BuTX binding (2.1 nM) was blocked efficaciously by DTX (IC50 = 1.6 nM) while the binding of 2.1 nM 125I-DTX was inhibited completely by beta-BuTX (IC50 = 25 nM); the lower potency of the latter could relate to the noncompetitive nature of the mutual competition and to the presence of high- and low-affinity sites for beta-BuTX. On gel filtration, or sedimentation analysis in H2O/sucrose and 2H2O/sucrose gradients, one peak of DTX binding activity was observed, and this was inhibitable by beta-BuTX. From the hydrodynamic properties of the acceptor/detergent/lipid complex (s20,w = 13.2 S; Stokes radius = 8.6 nm), a molecular weight of 405,000-465,000 was estimated.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions between discrete neuronal membrane binding sites for the putative K+‐channel ligands β‐bungarotoxin, dendrotoxin and mast‐cell‐degranulating peptide

“…3 B), attenuation of specific binding attaining about 80% at 1 pM, with an ICsO of about 60 nM ('251-DTX concentration = 0.7 nM). This increased level of inhibition is in good agreement with that observed for solubilized DTX acceptors, again using imidazole buffer [33,34]; in both cases, the notably extended competition curves are consistent with the kinetic heterogeneity identified by direct binding of 1251-fl-BuTX under these conditions (Fig. 2).…”

Reduction of P/Q‐type calcium channels in the postmortem cerebellum of paraneoplastic cerebellar degeneration with Lambert‐Eaton myasthenic syndrome

“…To demonstrate that P/Q-type VGCCs were specifically reduced and that other channels or receptors were not reduced, we performed binding assays using 125 I--dendrotoxin (2,000Ci/mmol; Amersham, Buckinghamshire, UK) which is a voltage-gated potassium channel (VGKC) blocker, as previously described. 15 It has been demonstrated that VGKC was enriched in the molecular layer of the rat cerebellum. …”

Molecular aspects of neuronal voltage‐dependent K+ channels

“…Moreover, in the brain each toxin inhibits the binding of the other toxins (Bidard et al, 1987a;Rehm et al, 1988;Breeze and Dolly, 1989;Schweitz et al, 1989a;Brlu et al, 1990). Solubilization of the toxin binding sites with detergent does not prevent the mutual interaction between these sites and, furthermore, the toxin binding sites copurify (Rehm and Betz, 1984;Rehm et al, 1988;Black et al, 1988). Thus, the dendrotoxins, MCD, CTX and /?-BTX bind to the same (b) the apparent molecular mass of the glycosylated subunit determined by SDSjPAGE changes with the percentage of the gels (i.e.…”

“…The fact that both subpopulations have high-affinity sites for DTXl is in accord with the consistent finding that there are more high-affinity a-DTX and DTXI sites than high-affinity 8-BTX sites in brain membranes (Rehm and Lazdunski, 1988 b;Breeze and Dolly, 1989). It also explains why P-BTX is a less efficient inhibitor of a-DTX binding than a-DTX is for p-BTX binding Black et al, 1988;Brau et al, 1990).…”

“…The glycosylated 80-kDa subunits bind the several toxins, symbolized in Fig. 5 (Rehm and Betz, 1984;Black et al, 1988).…”