ABSTRACT
BACKGROUND: The gamma-aminobutyric acid type A receptor (GABAA-R) beta subunits are critical targets for the actions for several intravenous general anesthetics, but the precise nature of the anesthetic binding sites are unknown. In addition, little is known about the role the fourth transmembrane (M4) segment of the receptor plays in receptor function. The aim of this study was to better define the propofol binding site on the GABAA-R by conducting a tryptophan scan in the M4 segment of the beta2 subunit. METHODS: Seven tryptophan mutations were introduced into the C-terminal end of the M4 segment of the GABAA-R beta2 subunit. GABAA-R subunit complementary DNAs were transfected into human embryonic kidney 293 cells grown on glass coverslips. After transfection (36-72 h), coverslips were transferred to a perfusion chamber to assay receptor function. Cells were whole cell patch clamped and exposed to GABA, propofol, etomidate, and pregnenolone. Chemicals were delivered to the cells using two 10-channel infusion pumps and a rapid solution exchanger. RESULTS: All tryptophan mutations were well tolerated, and with one exception, all resulted in minimal changes in receptor activation by GABA. One mutation, beta2(Y444W), selectively suppressed the ability of propofol to enhance receptor function while retaining normal sensitivity to etomidate and pregnenolone. CONCLUSIONS: This is the first report of a mutation that selectively reduces propofol sensitivity without altering the action of etomidate. The reduction in propofol sensitivity is consistent with the loss of a hydrogen bond within the propofol binding site. These results also suggest a possible orientation of the propofol molecule within its binding site.
Subject(s)
Anesthetics, Intravenous/pharmacology , Propofol/pharmacology , Receptors, GABA-A/drug effects , Receptors, GABA-A/genetics , Tyrosine/genetics , Amino Acid Sequence/genetics , Anesthetics, General/pharmacology , Cells, Cultured , Etomidate/pharmacology , Humans , Infusion Pumps , Molecular Sequence Data , Mutagenesis, Site-Directed/methods , Mutation/genetics , Patch-Clamp Techniques , Pregnenolone/pharmacology , Transfection , Tryptophan/geneticsABSTRACT
Fluoxetine has been reported to be a novel allosteric modulator of GABA(A) receptors with the notable exception of receptors that contain the alpha5-subunit isoform [Robinson, R.T., Drafts, B.C., Fisher, J.L., 2003. Fluoxetine increases GABA(A) receptor activity through a novel modulatory site. J. Pharmacol. Exp. Ther. 304, 978-984]. A mutagenic strategy has been used to investigate the structural basis for the insensitivity of this subunit. An alpha1/alpha5-subunit chimeragenesis approach first demonstrated the importance of the alpha1-subunit N-terminal sequence E165-D183 (corresponding to alpha5 E169-D187) in fluoxetine modulation. Specific amino acid substitutions in this domain subsequently revealed that a single mutation in the alpha5-subunit to the equivalent residue in alpha1 (T179A) was sufficient to confer fluoxetine sensitivity to the alpha5-containing receptor. However, the reciprocal mutation in the alpha1-subunit (A175T) did not result in a loss in sensitivity, suggesting the involvement of additional determinants for fluoxetine modulation. A comparative modeling approach was used to probe amino acids that may lie in close proximity to alpha1A175. This led serendipitously to the identification of a specific residue, alpha1F45, which, when mutated to an alanine, resulted in a significant decrease in potency for activation of the receptor by GABA and also reduced the efficacies of the partial agonists, THIP and P4S.
Subject(s)
Fluoxetine/pharmacology , Mutation/physiology , Receptors, GABA-A/drug effects , Selective Serotonin Reuptake Inhibitors/pharmacology , Amino Acid Substitution , Animals , Dose-Response Relationship, Drug , Drug Interactions , Electric Stimulation , GABA Agonists/pharmacology , Isoxazoles/pharmacology , Membrane Potentials/drug effects , Membrane Potentials/physiology , Membrane Potentials/radiation effects , Models, Molecular , Mutagenesis/physiology , Oocytes , Patch-Clamp Techniques/methods , Protein Subunits , Receptors, GABA-A/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/drug effects , Structure-Activity Relationship , Xenopus laevis , gamma-Aminobutyric Acid/pharmacologyABSTRACT
GABAA receptors that contain either the alpha4- or alpha6-subunit isoform do not recognize classical 1,4-benzodiazepines (BZDs). However, other classes of BZD site ligands, including beta-carbolines, bind to these diazepam-insensitive receptor subtypes. Some beta-carbolines [e.g. ethyl beta-carboline-3-carboxylate (beta-CCE) and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM)] display a higher affinity for alpha4- compared to alpha6-containing receptors. In order to identify the structural determinants that underlie these affinity differences, we constructed chimeric alpha6/alpha4 subunits and co-expressed these with wild-type rat beta2 and gamma2L subunits in tsA201 cells for radioligand binding analysis. After identification of candidate regions, site-directed mutagenesis was used to narrow the ligand selectivity to a single amino acid residue (alpha6N204/alpha4I203). Substitutions at alpha6N204 did not alter the affinity of the imidazobenzodiazepine Ro15-4513. A homologous mutation in the diazepam-sensitive alpha1 subunit (S205N) resulted in a 7-8-fold reduction in affinity for the beta-carbolines examined. Although the binding of the classical agonist flunitrazepam was relatively unaffected by this mutation in the alpha1 subunit, the affinity for Ro15-1788 and Ro15-4513 was decreased by approximately 19-fold and approximately 38-fold respectively. The importance of this residue, located in the Loop C region of the extracellular N-terminus of the subunit protein, emphasizes the differential interaction of ligands with the alpha subunit in diazepam-sensitive and -insensitive receptors.