A genetically modified mouse model probing the selective action of ifenprodil at the N-methyl-D-aspartate type 2B receptor.

Selective antagonism of N-methyl-d-aspartate (NMDA) 2B subunit containing receptors has been suggested to have potential therapeutic application for multiple CNS disorders. The amino terminal NR2B residues 1 to 282 were found to be both necessary and sufficient for the binding and function of highly NR2B subunit specific antagonists like ifenprodil and CP-101,606. Using a genetic approach in mice, we successfully replaced the murine NR2B gene function by "knocking-in" (KI) a chimeric human NR2A/B cDNA containing the minimal domain abolishing ifenprodil binding into the endogenous NR2B locus. Patch-clamp recording from hippocampal cultures of the NR2B KI mice demonstrated that their NMDA receptors have reduced sensitivity to both ifenprodil and CP-101,606, as predicted, but also have a lower affinity for glycine. The NR2B KI mice exhibited normal locomotor activity making this ifenprodil-insensitive mouse model a valuable tool to test the specificity of NR2B selective antagonists in vivo.

Tracazolate reveals a novel type of allosteric interaction with recombinant gamma-aminobutyric acid(A) receptors.

Tracazolate, a pyrazolopyridine, is an anxiolytic known to interact with gamma-aminobutyric acid (GABA)(A) receptors, adenosine receptors, and phosphodiesterases. Its anxiolytic effect is thought to be via its interaction with GABA(A) receptors. We now report the first detailed pharmacological study examining the effects of tracazolate on a range of recombinant GABA(A) receptors expressed in Xenopus laevis oocytes. Replacement of the gamma2s subunit within the alpha1beta3gamma2s receptor with the epsilon subunit caused a dramatic change in the functional response to tracazolate from potentiation to inhibition. The gamma2s subunit was not critical for potentiation because alpha1beta3 receptors were also potentiated by tracazolate. gamma2/epsilon chimeras revealed a critical N-terminal domain between amino acids 206 and 230 of gamma2, governing the nature of this response. Replacement of the beta3 subunit with the beta1 subunit within alpha1beta3gamma2s and alpha1beta3epsilon receptors also revealed selectivity of tracazolate for beta3-containing receptors, determined by asparagine at position 265 within transmembrane 2. Replacement of gamma2s with gamma1 or gamma3 revealed a profile intermediate to that of alpha1beta1epsilon and alpha1beta1gamma2s. alpha1beta1delta receptors were also potentiated by tracazolate; however, the maximum potentiation of the EC(20) was much greater than on alpha1beta1gamma2. Concentration-response curves to GABA in the presence of tracazolate for alpha1beta1epsilon and alpha1beta1gamma2s revealed a concentration-related decrease in maximum current amplitude, but a leftward shift in the EC(50) only on alpha1beta1gamma2. Like alpha1beta1gamma2s, GABA concentration-response curves on alpha1beta1delta receptors were shifted to the left with increased maximum responses. Tracazolate has a unique pharmacological profile on recombinant GABA(A) receptors: its potency (EC(50)) is influenced by the nature of the beta subunit; but more importantly, its intrinsic efficacy, potentiation, or inhibition is determined by the nature of the third subunit (gamma1-3, delta, or epsilon) within the receptor complex.

Mechanism of alpha-subunit selectivity of benzodiazepine pharmacology at gamma-aminobutyric acid type A receptors.

Benzodiazepine pharmacology at the GABA(A) receptor is dependent on the alpha and gamma subunit isoforms present. Ligands with higher affinity for certain isoforms--selective compounds--have been classified into benzodiazepine type I and II and into diazepam-sensitive and diazepam-insensitive receptors. A single amino acid position (alpha1G201/alpha3E225) has been identified which discriminates BZI and BZII receptors. The role of this residue has been explored by mutagenesis of alpha1 position 201 and the pharmacology of recombinant receptors examined using BZI receptor agonists. Ligand affinity is reduced by increasing side chain volume at alpha1G201 suggesting that steric inhibition underlies alpha-subunit selectivity. A second amino acid (alpha1H102/alpha6R100) determines diazepam sensitivity. The nature of the amino acid at this position was also examined by mutagenesis. Flumazenil and Ro15-4513 (ethyl 8-azido-6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a]-[1,4]benzodiazepine-3-carboxylate) binding affinity correlated weakly with the amino acid hydrophobicity suggesting a weak hydrophobic interaction between the ligand and alpha1H102.