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Previous experiments using genetic and pharmacological manipulations have provided strong evidence that etomidate impairs synaptic plasticity and memory by modulating α5-subunit containing GABAA receptors (α5-GABAARs). Because α5-GABAARs mediate tonic inhibition (TI) in hippocampal CA1 pyramidal cells and etomidate enhances TI, etomidate enhancement of TI in pyramidal cells has been proposed as the underlying mechanism (Martin et al., 2009). Here we tested this hypothesis by selectively removing α5-GABAARs from pyramidal neurons (CA1-pyr-α5-KO) and comparing the ability of etomidate to enhance TI and block LTP in fl-α5 (WT), global-α5-KO (gl-α5-KO), and CA1-pyr-α5-KO mice. Etomidate suppressed LTP in slices from WT and CA1-pyr-α5-KO but not gl-α5-KO mice. There was a trend toward reduced TI in both gl-α5-KO and CA1-pyr-α5-KO mice, but etomidate enhanced TI to similar levels in all genotypes. The dissociation between effects of etomidate on TI and LTP in gl-α5-KO mice indicates that increased TI in pyramidal neurons is not the mechanism by which etomidate impairs LTP and memory. Rather, the ability of etomidate to block LTP in WT and CA1-pyr-α5-KO mice, but not in gl-α5-KO mice, points toward α5-GABAARs on nonpyramidal cells as the essential effectors controlling plasticity in this in vitro model of learning and memory., (Copyright © 2015 the authors 0270-6474/15/359707-10$15.00/0.)

Enhancement of tonic inhibition mediated by extrasynaptic α5-subunit containing GABAA receptors (GABAARs) has been proposed as the mechanism by which a variety of anesthetics, including the general anesthetic etomidate, impair learning and memory. Since α5 subunits preferentially partner with β3 subunits, we tested the hypothesis that etomidate acts through β3-subunit containing GABAARs to enhance tonic inhibition, block LTP, and impair memory. We measured the effects of etomidate in wild type mice and in mice carrying a point mutation in the GABAAR β3-subunit (β3-N265M) that renders these receptors insensitive to etomidate. Etomidate enhanced tonic inhibition in CA1 pyramidal cells of the hippocampus in wild type but not in mutant mice, demonstrating that tonic inhibition is mediated by β3-subunit containing GABAARs. However, despite its inability to enhance tonic inhibition, etomidate did block LTP in brain slices from mutant mice as well as in those from wild type mice. Etomidate also impaired fear conditioning to context, with no differences between genotypes. In studies of recombinant receptors expressed in HEK293 cells, α5β1γ2L GABAARs were insensitive to amnestic concentrations of etomidate (1 μM and below), whereas α5β2γ2L and α5β3γ2L GABAARs were enhanced. We conclude that etomidate enhances tonic inhibition in pyramidal cells through its action on α5β3-containing GABAA receptors, but blocks LTP and impairs learning by other means - most likely by modulating α5β2-containing GABAA receptors. The critical anesthetic targets underlying amnesia might include other forms of inhibition imposed on pyramidal neurons (e.g. slow phasic inhibition), or inhibitory processes on non-pyramidal cells (e.g. interneurons)., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Electrochemical signaling in the brain depends on pentameric ligand-gated ion channels (pLGICs). Recently, crystal structures of prokaryotic pLGIC homologues from Erwinia chrysanthemi (ELIC) and Gloeobacter violaceus (GLIC) in presumed closed and open channel states have been solved, which provide insight into the structural mechanisms underlying channel activation. Although structural studies involving both ELIC and GLIC have become numerous, thorough functional characterizations of these channels are still needed to establish a reliable foundation for comparing kinetic properties. Here, we examined the kinetics of ELIC and GLIC current activation, desensitization, and deactivation and compared them to the GABAA receptor, a prototypic eukaryotic pLGIC. Outside-out patch-clamp recordings were performed with HEK-293T cells expressing ELIC, GLIC, or α1β2γ2L GABAA receptors, and ultra-fast ligand application was used. In response to saturating agonist concentrations, we found both ELIC and GLIC current activation were two to three orders of magnitude slower than GABAA receptor current activation. The prokaryotic channels also had slower current desensitization on a timescale of seconds. ELIC and GLIC current deactivation following 25 s pulses of agonist (cysteamine and pH 4.0 buffer, respectively) were relatively fast with time constants of 24.9 ± 5.1 ms and 1.2 ± 0.2 ms, respectively. Surprisingly, ELIC currents evoked by GABA activated very slowly with a time constant of 1.3 ± 0.3 s and deactivated even slower with a time constant of 4.6 ± 1.2 s. We conclude that the prokaryotic pLGICs undergo similar agonist-mediated gating transitions to open and desensitized states as eukaryotic pLGICs, supporting their use as experimental models. Their uncharacteristic slow activation, slow desensitization and rapid deactivation time courses are likely due to differences in specific structural elements, whose future identification may help uncover mechanisms underlying pLGIC gating transitions.

The prevalence of aromatic residues in the ligand binding site of the GABA(A) receptor, as with other cys-loop ligand-gated ion channels, is undoubtedly important for the ability of neurotransmitters to bind and trigger channel opening. Here, we have examined three conserved tyrosine residues at the GABA binding pocket (β(2) Tyr97, β(2) Tyr157, and β(2) Tyr205), making mutations to alanine and phenylalanine. We fully characterized the effects each mutation had on receptor function using heterologous expression in HEK-293 cells, which included examining surface expression, kinetics of macroscopic currents, microscopic binding and unbinding rates for an antagonist, and microscopic binding rates for an agonist. The assembly or trafficking of GABA(A) receptors was disrupted when tyrosine mutants were expressed as αβ receptors, but interestingly not when expressed as αβγ receptors. Mutation of each tyrosine accelerated deactivation and slowed GABA binding. This provides strong evidence that these residues influence the binding of GABA. Qualitatively, mutation of each tyrosine has a very similar effect on receptor function; however, mutations at β(2) Tyr157 and β(2) Tyr205 are more detrimental than β(2) Tyr97 mutations, particularly to the GABA binding rate. Overall, the results suggest that interactions involving multiple tyrosine residues are likely during the binding process., (© 2012 The Authors Journal of Neurochemistry © 2012 International Society for Neurochemistry.)

The GABA(A) receptor is an oligopentameric chloride channel that is activated via conformation changes induced upon the binding of the endogenous ligand, GABA, to the extracellular inter-subunit interfaces. Although dozens of amino acid residues at the α/β interface have been implicated in ligand binding, the structural elements that mediate ligand binding and receptor activation are not yet fully described. In this study, double-mutant cycle analysis was employed to test for possible interactions between several arginines (α₁R67, α₁R120, α₁R132, and β₂R207) and two aromatic residues (β₂Y97 and β₂F200) that are present in the ligand-binding pocket and are known to influence GABA affinity. Our results show that neither α₁R67 nor α₁R120 is functionally coupled to either of the aromatics, whereas a moderate coupling exists between α₁R132 and both aromatic residues. Significant functional coupling between β₂R207 and both β₂Y97 and β₂F200 was found. Furthermore, we identified an even stronger coupling between the two aromatics, β₂Y97 and β₂F200, and for the first time provided direct evidence for the involvement of β₂Y97 and β₂F200 in GABA binding. As these residues are tightly linked, and mutation of either has similar, severe effects on GABA binding and receptor kinetics, we believe they form a single functional unit that may directly coordinate GABA., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

The GABA(A) receptor is a multisubunit protein that transduces the binding of a neurotransmitter at an intersubunit interface into the opening of a central ion channel. The structural components that mediate the steps involved in this action are poorly defined. A large amount of work has focused on clarifying the specific functions and interactions of residues believed to surround the GABA binding pocket. Here, we explored two charged residues (β(2)Asp163 and α(1)Arg120), which have been suggested by homology models to participate in a salt-bridge interaction. When mutated to alanine, both single mutants, as well as the double mutant, increase EC(50-GABA), decrease the GABA binding rate, and accelerate deactivation and GABA unbinding rates. Double-mutant cycle analysis demonstrates that the effects of each alanine mutation on the GABA binding rate were additive and independent. In contrast, a significant coupling energy was found during an analysis of deactivation time constants. Using kinetic modeling, we further demonstrated that the GABA unbinding rates, in particular, are strongly coupled. These data suggest that β(2)Asp163 and α(1)Arg120 form a state-dependent salt bridge, interacting when GABA is bound to the receptor but not when the receptor is in the unbound state.