Abstract
Extrasynaptic γ-aminobutyric acid type A receptors (GABAARs),which contribute generalized inhibitory tone to the mammalian brain, are major targets for general anesthetics. To identify anesthetic binding sites in an extrasynaptic 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 αM1 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 α1β3γ2 GABAARs. [3H]Azietomidate and [3H]R-mTFD-MPAB photolabeling of β3Met-227 in βM1 established that these anesthetics also bind to a homologous site, most likely at the β3+-β3- interface, which suggests a subunit arrangement of β3α4β3δβ3.
Highlights
Extrasynaptic ␥-aminobutyric acid type A receptors (GABAARs), which contribute generalized inhibitory tone to the mammalian brain, are major targets for general anesthetics
No evidence was found for a ␦ subunit contribution to an anesthetic binding site. [3H]azietomidate photolabeling of 3Met-286 in M3 and ␣4Met-269 in ␣M1 that was inhibited by etomidate but not by R-mTFD-MPAB established that etomidate binds to a site at the 3؉-␣4؊ interface equivalent to its site in ␣13␥2 GABAARs. [3H]Azietomidate and [3H]R-mTFDMPAB photolabeling of 3Met-227 in M1 established that these anesthetics bind to a homologous site, most likely at the 3؉-3؊ interface, which suggests a subunit arrangement of 3␣43␦3
Based upon the identification of photolabeled amino acids and the results of competition photolabeling assays carried out at the level of intact subunits, we demonstrate that etomidate, but not R-mTFD-MPAB, binds with high affinity to a site at the ϩ-␣Ϫ subunit interface in ␣43␦ GABAARs that is equivalent to its binding site in ␣13␥2 GABAARs
Summary
Azietomidate causes normal anesthesia in wild-type mice with the same potency as etomidate, and its action is attenuated in the knock-in mouse [14]. R-mTFD-MPAB causes general anesthesia in mice and is potent in wild-type and N256M knock-in mice [15], consistent with the location of its binding sites at the Ϫ subunit interfaces. We photolabeled detergent-solubilized, purified heterologous ␣43␦ GABAARs with [3H]azietomidate and [3H]R-mTFD-MPAB. Photolabeling of ␣43␦ GABAARs by [3H]azietomidate and [3H]R-mTFD-MPAB. Aliquots of ␣43␦ GABAAR (8 pmol of [3H]muscimol sites/aliquot) were photolabeled with 3 M [3H]azietomidate (lanes 2– 4) or 1 M [3H]R-mTFD-MPAB (lanes 5–7) in the absence (lanes 2 and 5) or presence of 1 mM etomidate (lanes 3 and 6) or 60 M R-mTFD-MPAB (lanes 4 and 7), and subunits were resolved by SDS-PAGE. Positive allosteric modulator selective for GABAARs containing a ␦ subunit [32], did not bind to these sites
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