Mutagenesis of the GABA(A) receptor alpha1 subunit reveals a domain that affects sensitivity to GABA and benzodiazepine-site ligands.

We have mutated several amino acids in the region of the GABA(A) receptor alpha1 subunit predicted to form a small extracellular loop between transmembrane domains two and three to investigate its possible role in ligand sensitivity. The mutations were S275T, L276A, P277A, V279A, A280S and Y281F. Mutant alpha1 subunits were co-expressed with beta2 and gamma2 subunits in tsA201 cells or Xenopus oocytes. Binding studies revealed that the only mutation that significantly affected [3H]Ro15-4513 binding was the V279A substitution which reduced the affinity for this ligand. Electrophysiological examination of mutant receptors revealed that L276A, P277A and V279A displayed rightward shifts of their GABA concentration-response curves, the largest occurring with the L276A mutant. The impact of these mutations on allosteric modulation by benzodiazepine-site ligands was examined. V279A reduced the potency of both flunitrazepam and Ro15-4513 but, in each case, their efficacy was enhanced. A280S resulted in a decrease in flunitrazepam efficacy without affecting its potency. Additionally, P277A and A280S resulted in Ro15-4513 losing its inverse agonist effect at these receptors. These results suggest that a domain within this small extracellular loop between TMII-TMIII plays a role in determining the sensitivity of GABA(A) receptors to both GABA and benzodiazepine-site ligands.

Characterization of the interaction of zopiclone with gamma-aminobutyric acid type A receptors.

Zopiclone is a cyclopyrrolone that is used clinically as a hypnotic. Although this drug is known to interact with neuronal gamma-aminobutyric acid type A receptors, its binding site(s) within the receptor oligomer has been reported to be distinct from that of the classical benzodiazepines. After photoaffinity labeling with flunitrazepam, receptors in rat cerebellar membranes showed differentially reduced affinity for flunitrazepam and zopiclone by 50- and 3-fold, respectively. Because histidine 101 of the alpha-subunit is a major site of photolabeling, we have made specific substitutions of this residue and studied the consequences on the binding properties of zopiclone and diazepam using recombinant alpha1beta2gamma2-receptors transiently expressed in tsA201 cells. Both compounds showed similar binding profiles with receptors containing mutated alpha-subunits, suggesting a similar interaction with the residue at position 101. At alpha1beta2gamma3-receptors, flunitrazepam affinity was dramatically decreased by approximately 36-fold, whereas the affinity for zopiclone was decreased only 3-fold, suggesting a differential contribution of the gamma-subunit to the binding pocket. Additionally, we used electrophysiological techniques to examine the contribution of the gamma-subunit isoform in the receptor oligomer to ligand recognition using recombinant receptors expressed in Xenopus oocytes. Both compounds are agonists at alpha1beta2gamma2- and alpha1beta2gamma3-receptors, with flunitrazepam being more potent but less efficacious. In summary, these data suggest that histidine 101 of the alpha1-subunit plays a similar role in ligand recognition for zopiclone, diazepam, and flunitrazepam.

The use of site-directed mutagenesis, transient transfection, and radioligand binding. A method for the characterization of receptor-ligand interactions.

Receptor-ligand interactions have traditionally been evaluated using a number of biochemical techniques including radioligand binding, photoaffinity labeling, crosslinking, and chemical modification. In modern biochemistry, these approaches have largely been superseded by site-directed mutagenesis in the study of protein function, owing in part to a better understanding of the chemical properties of oligonucleotides and to the ease with which mutant clones can now be generated. The Altered Sites II in vitro Mutagenesis System from the Promega Corporation employs oligonucleotides containing two mismatches to introduce specific nucleotide substitutions in the nucleic acid sequence of a target DNA. One of these mismatches will alter the primary sequence of a given protein, whereas the second will give rise to a silent restriction site that is used to screen for mutants. Transient transfection of tsA201 cells with mutant cDNA constructs using calcium phosphate as a carrier for plasmid DNA permits expression of recombinant receptors that can be characterized using radioligand binding assays. In this article, we focus on site-directed mutagenesis, heterologous expression in eukaryotic cells, and radioligand binding as a methodology to enable the characterization of receptor-ligand interactions.

Tyrosine 62 of the gamma-aminobutyric acid type A receptor beta 2 subunit is an important determinant of high affinity agonist binding.

The gamma-aminobutyric acid type A receptor (GABA(A)R) carries both high (K(D) = 10-30 nm) and low (K(D) = 0.1-1.0 microm) affinity binding sites for agonists. We have used site-directed mutagenesis to identify a specific residue in the rat beta2 subunit that is involved in high affinity agonist binding. Tyrosine residues at positions 62 and 74 were mutated to either phenylalanine or serine and the effects on ligand binding and ion channel activation were investigated after the expression of mutant subunits with wild-type alpha1 and gamma2 subunits in tsA201 cells or in Xenopus oocytes. None of the mutations affected [(3)H]Ro15-4513 binding or impaired allosteric interactions between the low affinity GABA and benzodiazepine sites. Although mutations at position 74 had little effect on [(3)H]muscimol binding, the Y62F mutation decreased the affinity of the high affinity [(3)H]muscimol binding sites by approximately 6-fold, and the Y62S mutation led to a loss of detectable high affinity binding sites. After expression in oocytes, the EC(50) values for both muscimol and GABA-induced activation of Y62F and Y62S receptors were increased by 2- and 6-fold compared with the wild-type. We conclude that Tyr-62 of the beta subunit is an important determinant for high affinity agonist binding to the GABA(A) receptor.