Vol. 298, Issue 3, 986-995, September 2001

Pentylenetetrazole-Induced Inhibition of Recombinant -Aminobutyric Acid Type A (GABA_A) Receptors: Mechanism and Site of Action

Ren-Qi Huang, Cathy L. Bell-Horner, Mohammed I. Dibas, Douglas F. Covey, John A. Drewe and Glenn H. Dillon

Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas (R.Q.H., C.B.H., M.I.D., G.H.D.); Department of Molecular Biology and Pharmacology, Washington University, St. Louis, Missouri (D.F.C.); and Cytovia, Inc., San Diego, California (J.A.D.)

Pentylenetetrazole (PTZ) is a central nervous system convulsant that is thought, based on binding studies, to act at the picrotoxin (PTX) site of the -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 122 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 122 receptors. Varying subunit configuration (change or lack of  subunit isoform or lack of 2 subunit) had modest effects on PTZ-induced inhibition, as evidenced by comparable IC₅₀ values (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  subunit. Using a one-site model for PTZ interaction with 122 receptors, the association rate (k₊₁) was found to be 1.14 × 10³ M¹ s¹ and the dissociation rate (k₁) was 0.476 s¹, 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. -Isopropyl--methyl--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_A receptor.