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

Abstract

Barbiturates act on GABA A 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 ␤ subunit, their agonist activity is substantially influenced by the ␣ subunit subtype. Pentobarbital is a more effective agonist than GABA only when receptors contain an ␣6 subunit. Results from chimeric ␣1/␣6 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 ␣6 subtype.Each of these sites was individually mutated in the ␣6 subunit to the corresponding residue of the ␣1 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, ␣6(T69K), altered pentobarbital efficacy. This single mutation reduced the response to pentobarbital to a level intermediate to the wild-type ␣1␤1␥2L and ␣6␤1␥2L 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 ␣1 subunit (K70T) did not alter the response to pentobarbital. This is the first identification of a structural difference in GABAR ␣ subtypes that regulates direct activation by barbiturates.The barbiturates are widely used sedative and anesthetic agents that act primarily by increasing the activity of GABA A receptors (GABARs) in the central nervous system. These drugs, like other i.v. anesthetics, have three distinct effects on GABAR activity. At low concentrations, they act at a positive allosteric site to increase the response to GABA, whereas at higher concentrations, they act as agonists, directly activating the receptor. At millimolar concentrations, an inhibitory activity appears. These three actions are affected differently by the subunit composition of the receptor , and several studies have shown that modulation and direct activation are structurally separable (Dalziel et al., 1999;Serafini et al., 2000;Chang et al., 2003). This suggests that distinct sites within the receptor regulate the binding and/or signal transduction pathways for the allosteric and agonist actions of the barbiturates.The GABARs are pentameric, ligand-gated chloride channels responsible for most fast inhibitory neurotransmission in the mammalian brain. These receptors can be constructed from a diverse array of GABAR subunits. In mammals, seven subunit families and 16 subunit subtypes have been reported . Formation of a functional receptor requires both ␣ and ␤ subunits, and there is considerable diversity ...

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