General Anesthetic Binding Sites in Human α4β3δ γ-Aminobutyric Acid Type A Receptors (GABAARs)

Extrasynaptic gamma-aminobutyric acid type A receptors (GABA(A)Rs), which contribute generalized inhibitory tone to the mammalian brain, are major targets for general anesthetics. To identify anesthetic binding sites in an extrasynaptic GABA(A)R, we photolabeled human alpha 4 beta 3 delta GABA(A)Rs purified in detergent with [H-3]azietomidate and a barbiturate, [H-3]R-mTFD-MPAB, photoreactive anesthetics that bind with high selectivity to distinct but homologous intersubunit binding sites in the transmembrane domain of synaptic alpha 1 beta 3 gamma 2 GABA(A)Rs. Based upon H-3 incorporation into receptor subunits resolved by SDS-PAGE, there was etomidate-inhibitable labeling by [H-3] azietomidate in the alpha 4 and beta 3 subunits and barbiturate-inhibitable labeling by [H-3] R-mTFD-MPAB in the beta 3 subunit. These sites did not bind the anesthetic steroid alphaxalone, which enhanced photolabeling, or DS-2, a delta subunit-selective positive allosteric modulator, which neither enhanced nor inhibited photolabeling. The amino acids labeled by [H-3] azietomidate or [H-3] R-mTFD-MPAB were identified by N-terminal sequencing of fragments isolated by HPLC fractionation of enzymatically digested subunits. No evidence was found for a delta subunit contribution to an anesthetic binding site. [H-3] azietomidate photolabeling of beta 3Met-286 in beta M3 and alpha 4Met-269 in alpha M1 that was inhibited by etomidate but not by R-mTFD-MPAB established that etomidate binds to a site at the alpha 3(+)-alpha 4(-) interface equivalent to its site in alpha 1 beta 3 gamma 2 GABA(A)Rs. [H-3] Azietomidate and [H-3] R-mTFD-MPAB photolabeling of beta 3Met-227 in beta M1 established that these anesthetics also bind to a homologous site, most likely at the beta 3(+)-beta 3(-) interface, which suggests a subunit arrangement of beta 3 alpha 4 beta 3 delta beta 3.