title: Functional characterization of human gamma-aminobutyric acidA receptors containing the alpha 4 subunit.
abstract: The alpha subunits are an important determinant of the pharmacology of gamma-aminobutyric acidA (GABAA) receptors with respect to agonists, antagonists, and modulatory compounds, particularly the benzodiazepines. The alpha 4 subunit is the least abundant subunit in the brain and the most similar in deduced primary amino acid sequence to the alpha 6 subunit. We demonstrate that the human alpha 4 subunit forms a functional receptor when expressed with beta gamma 2, demonstrating some properties similar to alpha 6 beta gamma 2 and some properties more akin to alpha 1 beta gamma 2. It also exhibited some properties that were unlike any other alpha subunit-containing receptor. GABA affinity seemed to be identical to that of the alpha 1 beta 1 gamma 2 receptor; however, the partial agonists 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol and piperidine-4-sulfonic acid showed lower efficacy than at either alpha 1 beta 1 gamma 2 or alpha 6 beta 1 gamma 2. Benzodiazepine pharmacology of alpha 4-containing receptors was similar to that of alpha 6-containing receptors with the exception of dimethoxy-4-ethyl-beta-carboline-3-carboxylate, which behaved as a partial inverse agonist. Pentobarbital potentiated alpha 4 beta 1 gamma 2 receptor GABA responses to a level comparable with alpha 6 beta 1 gamma 2 (approximately 700% of EC20); however, unlike alpha 6 beta 1 gamma 2 receptors, it did not elicit any direct activation of the receptor. Propofol also potentiated alpha 4 beta 1 gamma 2 GABA responses but to a level more comparable to that of alpha 1 beta 1 gamma 2, suggesting that these compounds act via different sites. Unlike other subunit combinations, propofol did not elicit a direct activation of the receptor. These results suggest that the mechanism for direct activation of the GABAA receptor by pentobarbital and propofol is absent on alpha 4-containing receptors. Furosemide, which non-competitively inhibits the GABAA receptor, showed 700-fold selectivity for alpha 6 beta 3 gamma 2 receptors over alpha 1-, alpha 2-, alpha 3-, and alpha 5-containing receptors and exhibited selectivity for alpha 4 beta 3 gamma 2 receptors (> 50-fold). These experiments reveal a unique pharmacology for alpha 4-containing receptors with some similarities to both alpha 6- and alpha 1-containing receptors.
authors.affiliation: Merck Sharp & Dohme Research Laboratories, Harlow, Essex, UK. keith_wafford@merck.com
authors.forename: K A
authors.initials: KA
authors.lastname: Wafford
authors.forename: S A
authors.initials: SA
authors.lastname: Thompson
authors.forename: D
authors.initials: D
authors.lastname: Thomas
authors.forename: J
authors.initials: J
authors.lastname: Sikela
authors.forename: A S
authors.initials: AS
authors.lastname: Wilcox
authors.forename: P J
authors.initials: PJ
authors.lastname: Whiting
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D002894:Chromosomes, Human, Pair 4; D017931:DNA Primers; D005260:Female; D018755:GABA Agonists / Q000494:pharmacology*; D018756:GABA Antagonists / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals / Q000737:chemistry; D011963:Receptors, GABA-A / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: D017931:DNA Primers; D018755:GABA Agonists; D018756:GABA Antagonists; D046911:Macromolecular Substances; D021382:Protein Sorting Signals; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D005680:gamma-Aminobutyric Acid