Abstract

The effects of synthetic alpha-agatoxins, corresponding to toxins found in the venom of the spider Agelenopsis aperta, on the N-methyl-D-aspartate (NMDA) receptor complex have been investigated by using ligand-binding assays with [3H]MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine] and electrophysiological studies of NMDA receptors expressed in Xenopus oocytes after injection of rat brain RNA. The alpha-agatoxins Agel-489 and Agel-505 (3-30 microM) enhanced the binding of [3H]MK-801 to NMDA receptors on membranes prepared from rat brain. The stimulatory effect of Agel-505 was attenuated by the polyamine antagonists diethylenetriamine and N-(3-aminopropyl)-1,10-diaminodecane. Higher concentrations of Agel-489 and Agel-505 inhibited the binding of [3H]MK-801. A related toxin, Argiotoxin-636, had inhibitory but not stimulatory effects on the binding of [3H]MK-801. In Xenopus oocytes voltage-clamped at -70 mV, Agel-505 inhibited responses to NMDA with an IC50 of 13 nM. This effect of Agel-505 occurred at concentrations approximately 10,000-fold lower than those that cause inhibition of [3H]MK-801 binding. Antagonism of NMDA-induced currents by Agel-505 in Xenopus oocytes was strongly voltage-dependent and may represent an open-channel blocking effect of the toxin, possibly due to an interaction at the Mg++ binding site. Although alpha-agatoxins can interact at the positive allosteric polyamine site on the NMDA receptor, stimulatory effects produced by this interaction may be masked in functional assays due to a separate action of the toxins as high-affinity, noncompetitive antagonists of the receptor.