ABSTRACT
The GABA(A) receptor system provides the major inhibitory control in the CNS, with the alpha 1 beta 2 gamma 2 subunit combination being the most abundant and widely distributed form of the receptor. The alpha1 subunit knock-out (alpha1 KO) mice had a surprisingly mild overt phenotype, despite having lost approximately 60% of all GABA(A) receptors. The alpha1 KO mice had normal spontaneous locomotor activity, but were more sensitive to the sedating/ataxic effects of diazepam than wildtype (WT) mice. Pharmacological modulation of dopamine and N-methyl-D-aspartate (NMDA) receptors also produced altered responses in alpha1 KO mice compared with WT mice. As expected, the NMDA receptor antagonist MK801, amphetamine and cocaine increased locomotor activity in WT mice. Although MK801 increased locomotor activity in alpha1 KO mice, amphetamine and cocaine induced stereotypy not hyperlocomotion. Binding studies showed no gross changes in the total number of D1, D2 or NMDA receptors. Furthermore, pre-pulse inhibition of acoustic startle and the effects of cocaine in conditioned place preference were similar in both alpha1 KO and WT mice, indicating selective rather that global changes in response to dopaminergic agents. These data demonstrate subtle changes in behaviours mediated by neurotransmitters other than GABA in alpha1 KO mice and suggest that compensation may have occurred beyond the GABAergic system.
Subject(s)
Amphetamine/pharmacology , Central Nervous System Stimulants/pharmacology , Cocaine/pharmacology , Dopamine Uptake Inhibitors/pharmacology , Motor Activity/drug effects , Protein Subunits/drug effects , Receptors, GABA-A/physiology , Animals , Benzazepines/pharmacokinetics , Binding, Competitive/drug effects , Diazepam/pharmacology , Dizocilpine Maleate/pharmacokinetics , Dopamine Antagonists/pharmacokinetics , Dose-Response Relationship, Drug , Electric Stimulation , Excitatory Amino Acid Antagonists/pharmacokinetics , GABA Modulators/pharmacology , Habituation, Psychophysiologic/drug effects , Habituation, Psychophysiologic/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Subunits/genetics , Protein Subunits/physiology , Receptors, Dopamine D1/drug effects , Receptors, Dopamine D1/metabolism , Receptors, Dopamine D2/drug effects , Receptors, Dopamine D2/metabolism , Receptors, GABA-A/genetics , Receptors, N-Methyl-D-Aspartate/drug effects , Receptors, N-Methyl-D-Aspartate/metabolism , Reflex, Startle/drug effects , Reflex, Startle/physiology , Spiperone/pharmacokineticsABSTRACT
L-655,708 is a ligand for the benzodiazepine site of the gamma-aminobutyric acid type A (GABA(A)) receptor that exhibits a 100-fold higher affinity for alpha5-containing receptors compared with alpha1-containing receptors. Molecular biology approaches have been used to determine which residues in the alpha5 subunit are responsible for this selectivity. Two amino acids have been identified, alpha5Thr208 and alpha5Ile215, each of which individually confer approximately 10-fold binding selectivity for the ligand and which together account for the 100-fold higher affinity of this ligand at alpha5-containing receptors. L-655,708 is a partial inverse agonist at the GABA(A) receptor which exhibited no functional selectivity between alpha1- and alpha5-containing receptors and showed no change in efficacy at receptors containing alpha1 subunits where amino acids at both of the sites had been altered to their alpha5 counterparts (alpha1Ser205-Thr,Val212-Ile). In addition to determining the binding selectivity of L-655,708, these amino acid residues also influence the binding affinities of a number of other benzodiazepine (BZ) site ligands. They are thus important elements of the BZ site of the GABA(A) receptor, and further delineate a region just N-terminal to the first transmembrane domain of the receptor alpha subunit that contributes to this binding site.
Subject(s)
Imidazoles/metabolism , Isoleucine/chemistry , Receptors, GABA-A/chemistry , Threonine/chemistry , Amino Acid Sequence , Amino Acid Substitution , Animals , Anti-Anxiety Agents/metabolism , Azides/metabolism , Benzodiazepines/metabolism , Binding Sites , Binding, Competitive , Carbolines/metabolism , DNA, Complementary , Female , Flumazenil/metabolism , Flunitrazepam/metabolism , GABA-A Receptor Agonists , Humans , Imidazoles/pharmacology , Ligands , Molecular Sequence Data , Mutagenesis, Site-Directed , Oocytes , Protein Binding , Protein Structure, Tertiary , Protein Subunits , Pyridazines/metabolism , Pyridines/metabolism , Receptors, GABA-A/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Structure-Activity Relationship , Transfection , Xenopus laevis , ZolpidemABSTRACT
A new class of N-(indol-3-ylglyoxylyl)piperidines are high affinity agonists at the benzodiazepine binding site of human GABA-A receptor ion-channels, with modest selectivity for receptors containing the alpha1 subunit over alpha2 and alpha3. All three receptor subtypes discriminate substantially between the two enantiomers of the chiral ligand 10.
