ABSTRACT
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. The binding of the GABAA ligands [3H]muscimol and [3H]SR 95531 to alpha 1 (R120K) beta 2 gamma 2s GABAA receptors was abolished but the binding affinity of the benzodiazepine receptor ligand [3H]flunitrazepam was unchanged. These results suggest that the arginine residue 120 in the alpha 1 subtype of the GABAA receptor is essential for GABA binding.
Subject(s)
Arginine/genetics , Chloride Channels/physiology , Ion Channel Gating/physiology , Receptors, GABA-A/genetics , Receptors, GABA-A/metabolism , gamma-Aminobutyric Acid/metabolism , Amino Acid Sequence/genetics , Animals , Cell Line , Electric Conductivity , Humans , Insecta/cytology , Point Mutation/physiology , Protein Isoforms/genetics , Protein Isoforms/metabolismABSTRACT
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. (2) The 4-PIOL induced whole-cell currents were more pronounced in alpha1(val 121)beta2gamma2S than in alpha1(ile 121)beta2gamma2S receptor subunit combinations. (3) 4-PIOL inhibited GABA-induced responses on alpha1(ile 121)beta2gamma2S and alpha1(val 121)beta2gamma2S receptor combinations with similar potency.
Subject(s)
GABA Agonists/pharmacology , Isoxazoles/pharmacology , Piperidines/pharmacology , Receptors, GABA-A/physiology , gamma-Aminobutyric Acid/pharmacology , Animals , Cell Line , Humans , Kinetics , Macromolecular Substances , Membrane Potentials/drug effects , Mutagenesis, Site-Directed , Patch-Clamp Techniques , Receptors, GABA-A/biosynthesis , Receptors, GABA-A/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spodoptera , Transfection , ValineABSTRACT
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). The apparent affinity of the GABA(A) receptor antagonist bicuculline methiodide to inhibit the binding of [3H]SR 95531 did not differ between the two receptor complex variants. Electrophysiological measurements of GABA induced whole-cell Cl- currents showed a ten-fold decrease in the GABA(A) receptor sensitivity of alpha1 (val 121) beta2gamma2 as compared to alpha1(ile 121) beta2gamma2 receptor complexes. Thus, a relatively small change in the primary structure of the alpha1 subunit leads to a decrease selective for GABA(A) receptor sensitivity to agonist ligands, since no changes were observed in a GABA(A) receptor antagonist affinity and benzodiazepine receptor binding.
Subject(s)
GABA-A Receptor Agonists , Isoleucine/genetics , Valine/genetics , Baculoviridae/genetics , Cell Line , DNA Primers , GABA Agonists/pharmacology , GABA Antagonists/metabolism , GABA Antagonists/pharmacology , Humans , Patch-Clamp Techniques , Point Mutation , Pyridazines/metabolism , Receptors, GABA-A/genetics , Receptors, GABA-A/metabolism , Recombinant Proteins/agonists , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
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. No effect or small increases in levels of unsaturated FFAs on [3H]FNM binding to alpha 1 beta x gamma 2 and alpha 2 beta x gamma 2 receptor combinations were observed, and weak effects (130% of control values) were detected using the alpha 5 beta 2 gamma 2 receptor combination. The saturated FFAs, stearic and palmitic acids, were without effect on [3H]FNM binding to any combination of receptor complexes. The hydroxylated unsaturated FFAs, ricinoleic and ricinelaidic acids, were shown to decrease the binding of [3H]FNM only if an alpha 1 beta 2 gamma 2 receptor combination was used. Given the heterogeneity of the GABAA/ benzodiazepine receptor subunit distribution in the CNS, the effects of FFAs on the benzodiazepine receptor can be assumed to vary at both cellular and regional levels.
Subject(s)
Fatty Acids, Nonesterified/pharmacology , Flunitrazepam/metabolism , Receptors, GABA-A/metabolism , Animals , Baculoviridae , Cell Line , Cell Membrane/metabolism , Humans , Kinetics , Ligands , Macromolecular Substances , Receptors, GABA-A/chemistry , Receptors, GABA-A/drug effects , Recombinant Proteins/chemistry , Recombinant Proteins/drug effects , Recombinant Proteins/metabolism , Spodoptera , Structure-Activity Relationship , TransfectionABSTRACT
The nupG gene, encoding one of the two active nucleoside-transport systems in Escherichia coli K-12, has been cloned on the multicopy plasmid pBR322 and derivatives thereof. The recombinant plasmids complemented a chromosomal nupG mutation. A genetic map was determined by digestion with restriction endonucleases and the nucleotide sequence of a 3-kb stretch of DNA has been determined on fragments cloned into M13 phages. An open reading frame of 1254 bp, encoding a protein with a calculated molecular mass of 45.333 kDa, was deduced to be the coding region of nupG. Minicell-forming strains carrying plasmids containing this gene were shown to produce a hydrophobic, membrane-bound polypeptide with an apparent molecular mass of approximately 43 kDa.
