Rat brain dendrotoxin receptors associated with voltage-gated potassium channels: dendrotoxin binding and receptor solubilization

Abstract

Venom from the green mamba, Dendroaspis angusticeps, contains four polypeptides termed dendrotoxins (DaTXs) that block brain Ca-independent voltage-gated K channels. We compared the binding to rat brain receptors of two of these DTXs, alpha-DaTX and beta-DaTX, which preferentially block inactivating and noninactivating K channels, respectively. 125I-alpha-DaTX and 125I-beta-DaTX bind to single classes of receptor sites on synaptic membranes (KD = 0.7 and 36 nM for alpha-DaTX and beta-DaTX, respectively), with pH optima of about 6.5. The binding of both iodinated toxins was optimal in solutions containing 150 mM NaCl and decreased as Na was replaced with other alkali metal ions; the rank order for support of toxin binding was Na greater than K greater than Li greater than Rb. Cs (IC50 = 5-6 mM) prevented toxin binding, as did the divalent cations Ba and Ca (IC50 = 4-6 and 9-13 mM, respectively). The inhibition of 125I-alpha-DaTX binding by Cs and Ba was noncompetitive. The displacement of 125I-alpha-DaTX and 125I-beta-DaTX binding by the four unlabeled DaTXs was similar; the relative potency was alpha-DaTX greater than beta-DaTX greater than gamma-DaTX. The displacement curve for delta-DaTX did not parallel the others. When cross-linked with dimethylsuberimidate, both iodinated toxins covalently labeled membrane polypeptides of similar molecular weight (Mr = 65,000). The alpha-DaTX and beta-DaTX receptors were solubilized from rat synaptic membranes. Toxin binding to the soluble from rat synaptic membranes. Toxin binding to the soluble receptors was preserved in the presence of K and lecithin and decreased as K was replaced with Rb greater than Cs greater than Li greater than Na. The affinity of 125I-alpha-DaTX for the solubilized receptor was decreased 10-fold (KD = 7 nM); the affinity of 125I-beta-DaTX was decreased 3.5-fold (KD = 124 nM). However, the four unlabeled DaTXs retained their relative potencies for the inhibition of 125I-alpha-DaTX binding. The molecular weight of the solubilized receptor was estimated to be about 270,000 by sucrose density gradient centrifugation. These data raise the possibility that the inactivating ("A-type") and noninactivating voltage-gated K channels in rat brain may have similar subunits and that the channels may be composed of four Mr 65,000 polypeptides.