Andreas Buhr

The benzodiazepine binding site of GABAA receptors

The GABAA receptor belongs, along with the nicotinic acetylcholine receptor, the glycine receptor and the 5-HT3 receptor, to a family of homologous transmitter-gated ion channels mediating fast synaptic transmission. Many classes of drug interact with the GABAA receptor, which is the major inhibitory ion channel in the mammalian brain. Among these drugs are the allosteric modulators acting at the benzodiazepine binding site. In this article, Erwin Sigel and Andreas Buhr discuss recent studies that have identified amino acid residues that are thought to form the binding pocket for these compounds. These residues are probably located at subunit interfaces of the protein pentamer and at least some of them are homologous to residues implicated in channel agonist binding. This implies pseudosymmetry of channel agonist and channel modulatory sites, which may be, as recent data indicate, a general principle realized in other pseudosymmetric protein complexes.

Functional characterization of the new human GABAA receptor mutation β3(R192H)

Abstract. We screened 124 individuals for single nucleotide polymorphisms of the α1, β3 and γ2 genes of the GABAA receptor in the regions corresponding to the ligand-binding domains on the protein level. In a patient with chronic insomnia, a missense mutation was found in the gene of the β3 subunit. This mutation results in the substitution of the amino acid residue arginine for histidine in position 192 (β3(R192H)). The patient was found to be heterozygous for this mutation. Functional analysis of human α1β3(R192H)γ2S GABAA receptors using ultra fast perfusion techniques revealed a slower rate of the fast phase of desensitization compared with α1β3γ2S GABAA receptors. Additionally, current deactivation [a major determinant of inhibitory postsynaptic current (IPSC) duration] was faster in the mutated receptors. This raises the possibility of decreased GABAergic inhibition contributing to insomnia, as some members of the patients family also suffer from insomnia. The mutation β3(R192H) might, therefore, be linked to this condition. The intron/exon boundaries of the α1 subunit gene were also established and three additional variants were found in the α1 and β3 genes.

The glucose transporter of Escherichia coli: Purification and characterization by Ni+ chelate affinity chromatography of the IIBCGlc subunit

The IIBCGlc transmembrane subunit of the glucose transporter of E. coli containing a carboxy-terminal affinity tag consisting of six adjacent histidines was purified by nickel chelate affinity chromatography. The protein was constitutively overexpressed from a high copy number plasmid. 1.5 mg of 95% pure protein was obtained from 5 g (wet weight) cells. 70% of the phosphotransferase activity present in cell membranes was recovered. Adsorption to the nickel resin allows delipidation as well as rapid detergent exchange. The procedure was used to demonstrate exchange ofsubunits in the IIBCGlc dimer and it helds promise for the investigation of other protein-protein interactions.The IIBCGlc transmembrane subunit of the glucose transporter of E. coli containing a carboxy‐terminal affinity tag consisting of six adjacent histidines was purified by nickel chelate affinity chromatography. The protein was constitutively overexpressed from a high copy number plasmid. 1.5 mg of 95% pure protein was obtained from 5 g (wet weight) cells. 70% of the phosphotransferase activity present in cell membranes was recovered. Adsorption to the nickel resin allows delipidation as well as rapid detergent exchange. The procedure was used to demonstrate exchange ofsubunits in the IIBCGlc dimer and it helds promise for the investigation of other protein‐protein interactions.

Role of the Conserved Lysine Residue in the Middle of the Predicted Extracellular Loop Between M2 and M3 in the GABAA Receptor

Abstract : In α1, β2, and γ2 subunits of the γ‐aminobutyric acid A (GABAA) receptor, a conserved lysine residue occupies the position in the middle of the predicted extracellular loop between the transmembrane M2 and M3 regions. In all three subunits, this residue was mutated to alanine. Whereas the mutation in α1 and β2 subunits results each in about a sixfold shift of the concentration‐response curve for GABA to higher concentrations, no significant effect by mutation in the γ subunit was detected. The affinity for the competitive inhibitor bicuculline methiodide was not affected by the mutations in either the α1 subunit or the β2 subunit. Concentration‐response curves for channel activation by pentobarbital were also shifted to higher concentrations by the mutation in the α and β subunits. Binding of [3H]Ro 15‐1788 was unaffected by the mutation in the α subunit, whereas the binding of [3H]muscimol was shifted to lower affinity. Mutation of the residue in the α1 subunit to E, Q, or R resulted in an about eight‐, 10‐, or fivefold shift, respectively, to higher concentrations of the concentration‐response curve for GABA. From these observations, it is concluded that the corresponding residues on the α1 and β2 subunits are involved more likely in the gating of the channel by GABA than in the binding of GABA or benzodiazepines.

Amino acid residue 200 on the α1 subunit of GABAA receptors affects the interaction with selected benzodiazepine binding site ligands

Mutant alph1 subunits of the GABA(A) receptor were coexpressed in combination with the wild-type beta2 and gamma2 subunits in human embryonic kidney (HEK) 293 cells. The binding properties of various benzodiazepine site ligands were determined by displacement of ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a]-[1,4]benzodia zepine-3-carboxylate ([3H]Ro 15-1788). The mutation G200E led to a decrease in zolpidem and 3-methyl-6-[3-(trifluoromethyl)phenyl]-1,2,4-triazolo[4,3-b]pyridazine (CL 218872) affinity amounting to 16- and 8-fold. Receptors containing a conservative T206V substitution showed a 41- and 38-fold increase in methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) and CL 218872 affinity combined with a decrease in diazepam and zolpidem affinity, amounting to 7- and 10-fold. Two mutations, Q203A and Q203S showed almost no effects on the binding of benzodiazepine site ligands, indicating that this residue is not involved in the binding of benzodiazepines and related compounds.