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Identification of Structures within GABAA Receptor α Subunits That Regulate the Agonist Action of Pentobarbital

Barbiturates act on GABAA receptors (GABARs) through three distinct mechanisms, resulting in positive allosteric modulation, direct activation, and inhibition. These effects are observed at different concentrations and are differentially affected by some mutations and by the receptor's subunit composition. Mammalian GABARs can be formed from a combination of 16 different subunit subtypes. Although the effect of barbiturates depends largely on the β subunit, their agonist activity is substantially influenced by the α subunit subtype. Pentobarbital is a more effective agonist than GABA only when receptors contain an α6 subunit. Results from chimeric α1/α6 subunits suggested that structural differences within the extracellular N-terminal domain were responsible for this characteristic. Within this domain, we examined 15 amino acid residues unique to the α6 subtype. Each of these sites was individually mutated in the α6 subunit to the corresponding residue of the α1 subunit. The effect of the mutation on direct activation by pentobarbital was determined with whole-cell electrophysiological recordings. Our results indicate that only one of these mutations, α6(T69K), altered pentobarbital efficacy. This single mutation reduced the response to pentobarbital to a level intermediate to the wild-type α1β1γ2L and α6β1γ2L isoforms. The mutation did not affect the sensitivity of the receptor to GABA but did reduce the efficacy of etomidate, another i.v. anesthetic with activity similar to pentobarbital. The reverse mutation in the α1 subunit (K70T) did not alter the response to pentobarbital. This is the first identification of a structural difference in GABAR α subtypes that regulates direct activation by barbiturates.