The spider toxin ω-Aga IIIA defines a high affinity site neuronal high voltage-activated calcium channels

Abstract

The spider toxin ω-agatoxin IIIA (ω-Aga-IIIA) is a potent inhibitor of high voltage-activated calcium currents in the mammalian brain. To establish the biochemical parameters governing its action, we radiolabeled the toxin and examined its binding to native and recombinant calcium channels. In experiments with purified rat synaptosomal membranes, both kinetic and equilibrium data demonstrate one-to-one binding of ω-Aga-IIIA to a single population of high affinity sites, with K(d) = ~9 pm and B(max) = ~1.4 pmol/mg protein. Partial inhibition of ω-Aga-IIIA binding by ω-conotoxins GVIA, MVMIA, and MVIIC identifies N and P/Q channels as components of this population, ω-Aga-IIIA binds to recombinant α(1B) and α(1E) calcium channels with a similar high affinity (K(d) = ~5-9 pM) in apparent one-to- one fashion. Results. From recombinant α(1B) binding experiments demonstrate virtually identical B(max) values for ω-Aga-IIIA and ω-conotoxIn MVUV, providing further evidence for a one-to-one stoichiometry of agatoxin binding to calcium channels. The combined evidence suggests that ω-Aga-IIIA defines a unique, high affinity binding site on N-, P/Q-, and R-type calcium channels.