Cysteine Substitutions Define Etomidate Binding and Gating Linkages in the α-M1 Domain of γ-Aminobutyric Acid Type A (GABAA) Receptors

Abstract

Background: Etomidate induces anesthesia via intersubunit transmembrane sites in GABA A receptors. Results: In receptors engineered with ␣-M1 domain cysteines, GABA accelerates modification. Etomidate inhibits modification at three positions. Conclusion: Etomidate contacts a subdomain of ␣-M1 linked to channel gating, consistent with in silico model docking. Significance: We identify new structures that both bind anesthetic and modulate channel gating through rearrangement.

Mapping General Anesthetic Sites in Heteromeric γ-Aminobutyric Acid Type A Receptors Reveals a Potential For Targeting Receptor Subtypes

“…To further define the role of α-M1 in etomidate binding and modulation, we applied SCAMP to a series of 14 α1-M1 residues, from α1Q229 to α1Q242 spanning over three helical turns, in α1β2γ2L receptors (53). We found interesting functional phenotypes associated with these cysteine substitutions.…”

“…To test for binding effects of βN265 mutations, we exploited α1M236C, already established as protected by etomidate (53). In a wild-type background, α1M236C was readily protected by etomidate (as low as 3 µM) and propofol, but not by alphaxalone.…”

A Multifaceted GABAAReceptor Modulator: Functional Properties and Mechanism of Action of the Sedative-Hypnotic and Recreational Drug Methaqualone (Quaalude)

“…In contrast, there are compelling reasons to be cautious when speculating about the exact location of the binding site within this interface. Allosteric modulators bind quite differently to the transmembrane subunit interfaces in Cys-loop receptors (Hibbs and Gouaux, 2011;Trattnig et al, 2012;Sauguet et al, 2013;Stewart et al, 2013;Lansdell et al, 2015) and having only investigated two residues as putative binding partners, we can hardly claim to have obtained a detailed insight into the binding mode of methaqulone. Albeit the changed modulation displayed by methaqualone at a 1 M236W b 2 g 2S and a 1 b 2 M286W g 2S receptors could indicate involvement of these residues in modulator binding, it could also arise from allosteric effects of the introduced mutations (Siegwart et al, 2002;Chiara et al, 2012).…”

“…(1) /a (2) interface modulators Belelli et al, 1997;Hill-Venning et al, 1997;Halliwell et al, 1999;Khom et al, 2007). In the case of etomidate, the residue is believed not to participate in binding but rather to act as a transduction element between modulator binding and its effect on gating (Li et al, 2006;Desai et al, 2009;Chiara et al, 2012;Stewart et al, 2013;Stewart et al, 2014). In agreement with previous studies Siegwart et al, 2002;Desai et al, 2009;Stewart et al, 2014), the introduction of a N265M mutation in b 2 eliminated etomidate-mediated potentiation of a 1 b 2 g 2S receptor signaling completely, and interestingly the mutation had a similar detrimental effect on g 2S GABA A R signaling evoked by 100 mM etomidate and 300 mM methaqualone (means 6 S.E.M.…”

“…Elaborate photolabeling, substituted cysteine accessibility method, and mutagenesis studies have demonstrated the key importance of the a1-TM1 Met 236 and b2-TM3 Met 286 residues for the GABA A R modulation exerted by etomidate, and the two residues are believed to form direct interactions with the modulator (Siegwart et al, 2002;Li et al, 2006;Stewart et al, 2008;Chiara et al, 2012;Stewart et al, 2013). In concordance with previous studies (Siegwart et al, 2002;Stewart et al, 2008), etomidate (100 mM) displayed higher intrinsic agonist activity at the a 1 M236W b 2 g 2S GABA A R than at the WT a 1 b 2 g 2S receptor, whereas it was completely inactive at the a 1 b 2 M286W g 2S receptor (Fig.…”