Abstract

Pentylenetetrazole (PTZ) is a central nervous system convulsant that is thought, based on binding studies, to act at the picrotoxin (PTX) site of the γ-aminobutyric acid type A (GABA_A) receptor. In the present study, we have investigated the mechanism and site of action of PTZ in recombinant GABA_A receptors. In rat α1β2γ2 receptors, PTZ inhibited GABA-activated Cl⁻ current in a concentration-dependent, voltage-independent manner, with an IC₅₀ of 0.62 ± 0.13 mM. The mechanism of inhibition appeared competitive with respect to GABA in both rat and human α1β2γ2 receptors. Varying subunit configuration (change or lack of α subunit isoform or lack of γ2 subunit) had modest effects on PTZ-induced inhibition, as evidenced by comparable IC₅₀values (0.6–2.2 mM) in all receptor configurations tested. This contrasts with PTX and other PTX-site ligands, which have greater affinity in receptors lacking an α subunit. Using a one-site model for PTZ interaction with α1β2γ2 receptors, the association rate (k₊₁) was found to be 1.14 × 10³ M⁻¹ s⁻¹ and the dissociation rate (k₋₁) was 0.476 s⁻¹, producing a functional k_d of 0.418 mM. PTZ could only gain access to its binding site extracellularly. Single-channel recordings demonstrated that PTZ decreased open probability by increasing the duration of closed states but had no effect on single-channel conductance or open state duration. α-Isopropyl-α-methyl-γ-butyrolactone, a compound known to antagonize effects of PTX, also diminished the effects of PTZ. Taken together, our results indicate that pentylenetetrazole and picrotoxin interact with overlapping but distinct domains of the GABA_Areceptor.