Pentylenetetrazole-induced inhibition of recombinant gamma-aminobutyric acid type A (GABA(A)) receptors: mechanism and site of action.

Pentylenetetrazole (PTZ) is a central nervous system convulsant that is thought, based on binding studies, to act at the picrotoxin (PTX) site of the gamma-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 alpha 1 beta 2 gamma 2 receptors, PTZ inhibited GABA-activated Cl(-) current in a concentration-dependent, voltage-independent manner, with an IC(50) of 0.62 +/- 0.13 mM. The mechanism of inhibition appeared competitive with respect to GABA in both rat and human alpha 1 beta 2 gamma 2 receptors. Varying subunit configuration (change or lack of alpha subunit isoform or lack of gamma 2 subunit) had modest effects on PTZ-induced inhibition, as evidenced by comparable IC(50) 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 alpha subunit. Using a one-site model for PTZ interaction with alpha 1 beta 2 gamma 2 receptors, the association rate (k(+1)) was found to be 1.14 x 10(3) 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. alpha-Isopropyl-alpha-methyl-gamma-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.

[1]  M. Dibas,et al.  The central nervous system convulsant pentylenetetrazole stimulates γ-aminobutyric acid (GABA)-activated current in picrotoxin-resistant GABAA receptors in HEK293 cells , 2000, Neuroscience Letters.

[2]  Donald B. Carter,et al.  Enhancement by GABA of the association rate of picrotoxin and tert‐butylbicyclophosphorothionate to the rat cloned α1β2γ2 GABAA receptor subtype , 1995 .

[3]  J. Bormann,et al.  The atypical M2 segment of the beta subunit confers picrotoxinin resistance to inhibitory glycine receptor channels. , 1992, The EMBO journal.

[4]  P. Bregestovski,et al.  Chloride channels of glycine and GABA receptors with blockers: Monte Carlo minimization and structure-activity relationships. , 2000, Biophysical journal.

[5]  D. Carpenter,et al.  Antagonists of gaba responses, studied using internally perfused frog dorsal root ganglion neurons , 1987, Neuroscience.

[6]  A. Constanti,et al.  Studies on the mechanism of action of picrotoxinin and other convulsants at the crustacean muscle GABA receptor , 1986, Proceedings of the Royal Society of London. Series B. Biological Sciences.

[7]  P. Schofield,et al.  Mutations Affecting the Glycine Receptor Agonist Transduction Mechanism Convert the Competitive Antagonist, Picrotoxin, into an Allosteric Potentiator (*) , 1995, The Journal of Biological Chemistry.

[8]  D. Vassilatis,et al.  Picrotoxin Blockade of Invertebrate Glutamate‐Gated Chloride Channels: Subunit Dependence and Evidence for Binding Within the Pore , 1999, Journal of neurochemistry.

[9]  J. Amin,et al.  Point mutations in the M2 region of the alpha, beta, or gamma subunit of the GABAA channel that abolish block by picrotoxin. , 1995, Receptors & channels.

[10]  S. Paul,et al.  Effects of tetrazole derivatives on [3H]diazepam binding in vitro: correlation with convulsant potency. , 1982, European journal of pharmacology.

[11]  J. Barker,et al.  Specific antagonism of GABA‐mediated postsynaptic inhibition in cultured mammalian spinal cord neurons , 1978, Neurology.

[12]  S. Moss,et al.  A Functional Comparison of the Antagonists Bicuculline and Picrotoxin at Recombinant GABAA Receptors , 1996, Neuropharmacology.

[13]  D. Covey,et al.  Multiple mechanisms of picrotoxin block of GABA‐induced currents in rat hippocampal neurons. , 1993, The Journal of physiology.

[14]  S. Lipton,et al.  Cloning of a gamma-aminobutyric acid type C receptor subunit in rat retina with a methionine residue critical for picrotoxinin channel block. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[15]  R. Mckernan,et al.  theta, a novel gamma-aminobutyric acid type A receptor subunit. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[16]  P. Seeburg,et al.  Stable expression of cloned rat GABAA receptor subunits in a human kidney cell line , 1993, Neuroscience Letters.

[17]  J P Changeux,et al.  Unconventional pharmacology of a neuronal nicotinic receptor mutated in the channel domain. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[18]  S. Paul,et al.  Convulsant potencies of tetrazoles are highly correlated with actions on GABA/benzodiazepine/picrotoxin receptor complexes in brain. , 1984, Life sciences.

[19]  D. J. Hinkle,et al.  Loreclezole inhibition of recombinant α1β1γ2L GABAA receptor single channel currents , 2000, Neuropharmacology.

[20]  J. Ferrendelli,et al.  Synthesis and structure-activity studies of alkyl-substituted gamma-butyrolactones and gamma-thiobutyrolactones: ligands for the picrotoxin receptor. , 1991, Journal of medicinal chemistry.

[21]  C. Newland,et al.  On the mechanism of action of picrotoxin on GABA receptor channels in dissociated sympathetic neurones of the rat. , 1992, The Journal of physiology.

[22]  M. Madeja,et al.  Effects of pentylenetetrazol on GABA receptors expressed in oocytes of Xenopus laevis: extra- and intracellular sites of action , 1996, Neuroscience Letters.

[23]  S. Moss,et al.  Subcellular localization of gamma-aminobutyric acid type A receptors is determined by receptor beta subunits. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[24]  R. ffrench-Constant,et al.  A point mutation in a Drosophila GABA receptor confers insecticide resistance , 1993, Nature.

[25]  A. Hackam,et al.  A single amino acid in gamma-aminobutyric acid rho 1 receptors affects competitive and noncompetitive components of picrotoxin inhibition. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[26]  M. Dibas,et al.  Influence of subunit configuration on the interaction of picrotoxin-site ligands with recombinant GABA(A) receptors. , 2000, Brain research. Molecular brain research.

[27]  K. Gee,et al.  3 alpha-Hydroxy-3 beta-trifluoromethyl-5 alpha-pregnan-20-one (Co 2-1970): a partial agonist at the neuroactive steroid site of the gamma-aminobutyric acidA receptor. , 1996, Molecular pharmacology.

[28]  G. Maksay,et al.  Convulsant/depressant site of action at the allosteric benzodiazepine-GABA receptor-ionophore complex. , 1983, Life sciences.

[29]  M. Akabas,et al.  Interaction of picrotoxin with GABAA receptor channel-lining residues probed in cysteine mutants. , 1995, Biophysical journal.

[30]  M. Ticku,et al.  Interactions of pentamethylenetetrazole and tetrazole analogues with the picrotoxinin site of the benzodiazepine-GABA receptor-ionophore complex. , 1984, European journal of pharmacology.

[31]  R. ffrench-Constant,et al.  A unique amino acid of the Drosophila GABA receptor with influence on drug sensitivity by two mechanisms. , 1994, The Journal of physiology.

[32]  J. Bloomquist.  Ion channels as targets for insecticides. , 1996, Annual review of entomology.

[33]  N. Akaike,et al.  Blockade of γ-aminobutyric acid-gated chloride current in frog sensory neurons by picrotoxin , 1988, Neuroscience Research.