A Transmembrane Amino Acid in the GABA_A Receptor β₂ Subunit Critical for the Actions of Alcohols and Anesthetics

Abstract

Alcohols and inhaled anesthetics enhance the function of GABA A receptors containing ␣, ␤, and ␥ subunits. Molecular analysis has focused on the role of the ␣ subunits; however, there is evidence that the ␤ subunits may also be important. The goal of our study was to determine whether Asn265, which is homologous to the site implicated in the ␣ subunit (Ser270), contributes to an alcohol and volatile anesthetic binding site in the GABA A receptor ␤ 2 subunit. We substituted cysteine for Asn265 and exposed the mutant to the sulfhydryl-specific reagent octyl methanethiosulfonate (OMTS). We used two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes and found that, after OMTS application, GABA-induced currents were irreversibly potentiated in mutant ␣ 1 ␤ 2 (N265C)␥ 2S receptors [but not ␣ 1 ␤ 2 (I264C)␥ 2S ], presumably because of the covalent linking of octanethiol to the thiol group in the substituted cysteine. It is noteworthy that this effect was blocked when OMTS was applied in the presence of octanol. We found that potentiation by butanol, octanol, or isoflurane in the N265C mutant was nearly abolished after the application of OMTS, suggesting that an alcohol and volatile anesthetic binding site at position 265 of the ␤ 2 subunit was irreversibly occupied by octanethiol and consequently prevented butanol or isoflurane from binding and producing their effects. OMTS did not affect modulation or direct activation by pentobarbital, but there was a partial reduction of allosteric modulation by flunitrazepam and alphaxalone in mutant ␣ 1 ␤ 2 (N265C)␥ 2S receptors after OMTS was applied. Our findings provide evidence that Asn265 may contribute to an alcohol and anesthetic binding site.

The TM2 6′ Position of GABAA Receptors Mediates Alcohol Inhibition

“…Thus, in parallel to recent studies of GlyRs (Crawford et al, 2007) and nAChRs (Borghese et al, 2003), MTS reagents can be valuable tools for discriminating between multiple sites of alcohol interaction: substitution and labeling of our target residue revealed an inhibitory effect previously masked by the potentiating effects of alcohols at other sites in the same receptor. Furthermore, in parallel to recent work on the 15Ј potentiating site (McCracken et al, 2010), we showed that coapplication of methanol blocked the inhibitory effect of MMTS on the 6Ј cysteine mutant. This result is consistent with direct jpet.aspetjournals.org competition for a 6Ј-proximal inhibitory binding site, although we cannot exclude the possibility that allosteric modification of the receptor renders the 6Ј site inaccessible to MMTS.…”

“…Having tested a variety of ␣2␤2 mutants, we extended this work by showing that tryptophan substitution at ␣2(Thr6Ј) also reduces alcohol potentiation of ␥-containing receptors, which are the predominant population in brain (Möhler, 2006). Furthermore, although many structure/function studies on alcohol modulation have focused on GABA A R ␣ subunits (Mascia et al, 2000), McCracken et al (2010) recently demonstrated a critical role for the TM2 15Ј position in the GABA A R ␤2 subunit in receptor potentiation by alcohols and anesthetics. We also showed conservation of the inhibitory effect of MMTS labeling at the 6Ј position in ␣2 and ␤2 subunits, again in the context of ␥-containing receptors.…”

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

“…Bali and Akabas (50) introduced cysteine at two β2 positions, β2N265 and β2M286, where other mutations were known to influence propofol sensitivity in α1β2γ2 receptors (Figure 6 shows these residues in β3, which is highly homologous to β2 in this region). Both residues are predicted to contribute to the “β + ” transmembrane interface, although β2N265 may also be accessible from the “β − ” interfacial pocket (51). To achieve comparable mixes of receptor states during control and protection experiments, Bali and Akabas used a strategy different from ours, likely resulting in under 50% anesthetic site occupancy.…”

Functional Validation of Virtual Screening for Novel Agents with General Anesthetic Action at Ligand-Gated Ion Channels

“…Oocytes from female X. laevis frogs were extracted, isolated, and stored as described previously elsewhere (McCracken et al, 2010;Howard et al, 2011). For the expression of GLIC, 32 nl cDNA solution was injected into the oocyte nucleus via the animal pole using a microdispenser (Drummond Scientific, Broomall, PA) with a 20-30 mm outer diameter glass capillary tip pulled with a Flaming/Brown Micropipette Puller (Sutter Instrument Co., Novato, CA).…”