Correction for Inhibition Leads to an Allosteric Co-Agonist Model for Pentobarbital Modulation and Activation of α1β3γ2L GABAA Receptors

Abstract

BackgroundPentobarbital, like propofol and etomidate, produces important general anesthetic effects through GABAA receptors. Photolabeling also indicates that pentobarbital binds to some of the same sites where propofol and etomidate act. Quantitative allosteric co-agonist models for propofol and etomidate account for modulatory and agonist effects in GABAA receptors and have proven valuable in establishing drug site characteristics and for functional analysis of mutants. We therefore sought to establish an allosteric co-agonist model for pentobarbital activation and modulation of α1β3γ2L receptors, using a novel approach to first correct pentobarbital activation data for inhibitory effects in the same concentration range.MethodsUsing oocyte-expressed α1β3γ2L GABAA receptors and two-microelectrode voltage-clamp, we quantified modulation of GABA responses by a low pentobarbital concentration and direct effects of high pentobarbital concentrations, the latter displaying mixed agonist and inhibitory effects. We then isolated and quantified pentobarbital inhibition in activated receptors using a novel single-sweep “notch” approach, and used these results to correct steady-state direct activation for inhibition.ResultsCombining results for GABA modulation and corrected direct activation, we estimated receptor open probability and optimized parameters for a Monod-Wyman-Changeux allosteric co-agonist model. Inhibition by pentobarbital was consistent with two sites with IC50s near 1 mM, while co-agonist model parameters suggest two allosteric pentobarbital agonist sites characterized by KPB ≈ 5 mM and high efficacy. The results also indicate that pentobarbital may be a more efficacious agonist than GABA.ConclusionsOur novel approach to quantifying both inhibitory and co-agonist effects of pentobarbital provides a basis for future structure-function analyses of GABAA receptor mutations in putative pentobarbital binding sites.

Competitive Antagonism of Anesthetic Action at the γ-Aminobutyric Acid Type A Receptor by a Novel Etomidate Analog with Low Intrinsic Efficacy

“…1 mM) and assuming a maximum P open value for GABA of 0.85 in this receptor subtype. 42 The curved lines in this figure are fits of this relationship to equation 3 with the number of anesthetic binding sites n constrained to 2 for etomidate (at the two β + –α − subunit interfacial sites) and 4 for naphthalene-etomidate (because our photoaffinity labeling studies suggest that it binds to the two β + –α − subunit interfacial sites and to the α + −β − and γ + −β − interfacial sites with similar affinities). 31,45,51 Based on this analysis, we determined that etomidate binds to the open state with an affinity that is 190-fold higher than to the closed state with microscopic dissociation constants of 0.23 μM (95% CI, 0.15 to 0.31 μM) and 44 μM (95% CI, 26 to 62 μM), respectively.…”

“…Etomidate and naphthalene-etomidate direct activation data were transformed to P open values by assuming that P open in the presence of a maximally activating GABA concentration (1 mM) is approximately 0.85. 41,42 The relationship between P open and the drug concentration was then fit to the allosteric equation (equation 3): 41 $P_{\mathit{open}}=\frac{1}{1+L_0{\left(\frac{1+\left[\mathit{drug}\right]/K{d}_{\mathit{closed}}}{1+\left[\mathit{drug}\right]/K{d}_{\mathit{open}}}\right)}^n}$where P open is the fraction of receptors that are open in the presence of etomidate or naphthalene-etomidate, [drug] is the concentration of either etomidate or naphthalene-etomidate, Kd closed and Kd open respectively are the microscopic dissociation constants of the drug in the closed and open states, and n is the number of drug binding sites. L 0 , the closed:open receptor ratio in the absence of drug, was fixed at a median literature value of 40,000.…”

Monod-Wyman-Changeux Allosteric Shift Analysis in Mutant α1β3γ2L GABAA Receptors Indicates Selectivity and Crosstalk among Intersubunit Transmembrane Anesthetic Sites

“…as described by Bevington and Robinson (2002). Wildtype L 0 has been reported over a wide range between 1100 and 70,000 (Chang and Weiss, 1999;Rüsch et al, 2004;Ziemba and Forman, 2016). Fitted wild-type log(d) values, and thus calculated Dlog(d) values for mutant receptors, were insensitive to fixed wild-type L 0 values between 5000 and 50,000.…”

“…Intravenous general anesthetics including etomidate, propofol, and barbiturates act as allosteric coagonists at GABA A receptors, positively modulating GABA activation at low concentrations and directly activating receptors at high concentrations (Brohan and Goudra, 2017). These actions, assessed electrophysiologically, are quantitatively described by two-state Monod-Wyman-Changeux (MWC) models (Rüsch et al, 2004;Ruesch et al, 2012;Ziemba and Forman, 2016;Steinbach and Akk, 2019).…”

A chimeric prokaryotic-eukaryotic pentameric ligand gated ion channel reveals interactions between the extracellular and transmembrane domains shape neurosteroid modulation

“…We envision that pentobarbital shifts the channel equilibrium towards an open state in GLIC-ρIII, which can explain both positive modulation and apparent direct activation. Consistent with this idea, the allosteric co-agonist model postulated by the Forman group posits that enhancement of GABA A R gating explains both positive modulation and direct activation by the allosteric activators etomidate (Rusch et al, 2004), propofol (Ruesch et al, 2012) and pentobarbital (Ziemba and Forman, 2016). Taken together, our data indicate that many of the functional and pharmacological properties of the GABAρ-TMD were preserved in the chimera.…”