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

Abstract Extra-synaptic γ-aminobutyric acid type A receptors (GABAAR), which contribute generalized inhibitory tone to the mammalian brain, are major targets for general anesthetics. To identify anesthetic binding sites in an extra-synaptic GABAAR, we photolabeled human α4β3δ GABAARs purified in detergent with [3H]azietomidate and a barbiturate, [3H]R-mTFD-MPAB, photoreactive anesthetics that bind with high selectivity to distinct but homologous intersubunit binding sites in the transmembrane domain of synaptic α1β3γ2 GABAARs. Based upon 3H incorporation into receptor subunits resolved by SDS-PAGE, there was etomidate-inhibitable labeling by [3H]azietomidate in the α4 and β3 subunits and barbiturate-inhibitable labeling by [3H]R-mTFD-MPAB in the β3 subunit. These sites did not bind the anesthetic steroid alphaxalone, which enhanced photolabeling, or DS-2, a δ subunit selective positive allosteric modulator, which neither enhanced nor inhibited photolabeling. The amino acids labeled by [3H]azietomidate or [3H]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 δ subunit contribution to an anesthetic binding site. [3H]azietomidate photolabeling of β3Met-286 in βM3 and α4Met-269 in α M1 that was inhibited by etomidate but not by R-mTFD-MPAB established that etomidate binds to a site at the β3+ - α4- interface equivalent to its site in α1 β3γ2 GABAARs. [3H]azietomidate and [3H]R-mTFD-MPAB photolabeling of β3Met-227 in βM1 established that these anesthetics also bind to an homologous site most likely at the β3+- β3- interface, which suggests a subunit arrangement of β3α4β3δβ3.