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General Anesthetic Binding Sites in Human ?4?3? ?-Aminobutyric Acid Type A Receptors (GABAARs).


ABSTRACT: Extrasynaptic ?-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 ?4?3? 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 ?1?3?2 GABAARs. Based upon 3H incorporation into receptor subunits resolved by SDS-PAGE, there was etomidate-inhibitable labeling by [3H]azietomidate in the ?4 and ?3 subunits and barbiturate-inhibitable labeling by [3H]R-mTFD-MPAB in the ?3 subunit. These sites did not bind the anesthetic steroid alphaxalone, which enhanced photolabeling, or DS-2, a ? 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 ? 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 ?1?3?2 GABAARs. [3H]Azietomidate and [3H]R-mTFD-MPAB photolabeling of ?3Met-227 in ?M1 established that these anesthetics also bind to a homologous site, most likely at the ?3+-?3- interface, which suggests a subunit arrangement of ?3?4?3??3.

SUBMITTER: Chiara DC 

PROVIDER: S-EPMC5159512 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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

Chiara David C DC   Jounaidi Youssef Y   Zhou Xiaojuan X   Savechenkov Pavel Y PY   Bruzik Karol S KS   Miller Keith W KW   Cohen Jonathan B JB  

The Journal of biological chemistry 20161107 51


Extrasynaptic γ-aminobutyric acid type A receptors (GABA<sub>A</sub>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<sub>A</sub>R, we photolabeled human α4β3δ GABA<sub>A</sub>Rs purified in detergent with [<sup>3</sup>H]azietomidate and a barbiturate, [<sup>3</sup>H]R-mTFD-MPAB, photoreactive anesthetics that bind with high selectivity to distinct but homologous inters  ...[more]

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