Arginine residue 120 of the human GABAA receptor alpha 1, subunit is essential for GABA binding and chloride ion current gating.

The effect of mutating the conserved amino acid residue arginine 120 to lysine in the GABAA receptor alpha 1 subunit was studied. In electrophysiological experiments, the arginine 120 lysine (R120K) mutation in the alpha 1 subunit, when co-expressed with beta 2 and gamma 2 subunits in Sf-9 insect cells, induces a 180-fold rightward shift of the GABA dose-response curve compared with wild type alpha 1 beta 2 gamma 2s GABAA receptors. The diazepam potentiation of GABA-gated chloride ion currents was not affected.

Effects of 5-(4-piperidyl) isoxazol-3-ol (4-PIOL), a GABA(A) receptor partial agonist, on recombinant human GABA(A) receptors.

gamma-Aminobutyric acidA (GABA(A)) gated chloride ion channels were expressed from human recombinant cDNA using the baculovirus/Sf-9 insect cell expression system. The electrophysiological effects in whole-cell currents of 5-(4-piperidyl) isoxazol-3-ol (4-PIOL), a GABA(A) receptor partial agonist, were investigated on GABA(A) receptor complexes of alpha1beta2gamma2S subunits as well as a slightly modified construct of alpha1(valine 121)beta2gamma2S subunits. Here we report that (1)4-PIOL induces an inward whole-cell current in a concentration-dependent manner in both alpha1(val 121)beta2gamma2S and alpha1(ile 121)beta2gamma2S receptor subunit combinations.

Decreased agonist sensitivity of human GABA(A) receptors by an amino acid variant, isoleucine to valine, in the alpha1 subunit.

Recombinant human GABA(A) receptors were investigated in vitro by coexpression of cDNAs coding for alpha1, beta2, and gamma2 subunits in the baculovirus/Sf-9 insect cell system. We report that a single amino acid exchange (isoleucine 121 to valine 121) in the N-terminal, extracellular part of the alpha1 subunit induces a marked decrease in agonist GABA(A) receptor ligand sensitivity. The potency of muscimol and GABA to inhibit the binding of the GABA(A) receptor antagonist [3H]SR 95531 (2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl)pyridazinium bromide) was higher in receptor complexes of alpha1(ile 121) beta2gamma2 than in those of alpha1(val 121) beta2gamma2 (IC50 values were 32-fold and 26-fold lower for muscimol and GABA, respectively).

Unsaturated free fatty acids increase benzodiazepine receptor agonist binding depending on the subunit composition of the GABAA receptor complex.

It has been shown previously that unsaturated free fatty acids (FFAs) strongly enhance the binding of agonist benzodiazepine receptor ligands and GABAA receptor ligands in the CNS in vitro. To investigate the selectivity of this effect, recombinant human GABAA/benzodiazepine receptor complexes formed by different subunit compositions (alpha x beta y gamma 2, x = 1, 2, 3, and 5; y = 1, 2, and 3) were expressed using the baculovirus-transfected Sf9 insect cell system. At 10(-4) M, unsaturated FFAs, particularly arachidonic (20:4) and docosahexaenoic (22:6) acids, strongly stimulated (> 200% of control values) the binding of [3H]flunitrazepam ([3H]FNM) to the alpha 3 beta 2 gamma 2 receptor combination in whole cell preparations.