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Pentameric ligand-gated ion channels (pLGICs), which mediate chemo-electric signal transduction in animals, have been recently found in bacteria. Despite clear sequence and 3D structure homology, the phylogenetic distance between prokaryotic and eukaryotic homologs suggests significant structural divergences, especially at the interface between the extracellular (ECD) and the transmembrane (TMD) domains. To challenge this possibility, we constructed a chimera in which the ECD of the bacterial protein GLIC is fused to the TMD of the human α1 glycine receptor (α1GlyR). Electrophysiology in Xenopus oocytes shows that it functions as a proton-gated ion channel, thereby locating the proton activation site(s) of GLIC in its ECD. Patch-clamp experiments in BHK cells show that the ion channel displays an anionic selectivity with a unitary conductance identical to that of the α1GlyR. In addition, pharmacological investigations result in transmembrane allosteric modulation similar to the one observed on α1GlyR. Indeed, the clinically active drugs propofol, four volatile general anesthetics, alcohols, and ivermectin all potentiate the chimera while they inhibit GLIC. Collectively, this work shows the compatibility between GLIC and α1GlyR domains and points to conservation of the ion channel and transmembrane allosteric regulatory sites in the chimera. This provides evidence that GLIC and α1GlyR share a highly homologous 3D structure. GLIC is thus a relevant model of eukaryotic pLGICs, at least from the anionic type. In addition, the chimera is a good candidate for mass production in Escherichia coli, opening the way for investigations of "druggable" eukaryotic allosteric sites by X-ray crystallography.

Introduction: Elevated concentrations of isovaleric (IVA), methylmalonic (MMA), and propionic acid are associated with impaired consciousness in genetic diseases (organic acidemias). We conjectured that part of the central nervous system depression observed in these disorders was due to anesthetic effects of these metabolites. We tested three hypotheses. First, that these metabolites would have anesthetic-sparing effects, possibly being anesthetics by themselves. Second, that these compounds would modulate glycine and gamma-aminobutyric acid (GABA(A)) receptor function, increasing chloride currents through these channels as potent clinical inhaled anesthetics do. Third, that these compounds would affect physical properties of lipids., Methods: Anesthetic EC(50)s were measured in Xenopus laevis tadpoles. Glycine and GABA(A) receptors were expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamping. Pressure-area isotherms of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayers were measured with and without added organic acids., Results: IVA acid was an anesthetic in tadpoles, whereas MMA and propionic acid decreased isoflurane's EC(50) by half. All three organic acids concentration-dependently increased current through alpha(1) glycine receptors. There were minimal effects on alpha(1)beta(2)gamma(2s) GABA(A) receptors. The organic acids increased total lateral pressure (surface pressure) of DPPC monolayers, including at mean molecular areas typical of bilayers., Conclusion: IVA, MMA, and propionic acid have anesthetic effects in tadpoles, positively modulate glycine receptor function and affect physical properties of DPPC monolayers.

Introduction: No theory of inhaled anesthetic action requires volatility of the anesthetic to accomplish the biophysical interaction of anesthetic with biological target. The identification of mutations that attenuate the effect of inhaled anesthetics on various receptors raises the possibility that nonvolatile compounds with anesthetic effects can be identified with the aid of these receptors. In previous studies, we identified compounds that were either charged or had an exceptionally low vapor pressure and which modulated anesthetic-sensitive receptors in a manner similar to inhaled anesthetics. We tested whether these, and another charged compound, shared a common mechanism with volatile anesthetics, by comparing their effect on wild-type gamma-aminobutyric acid type A (GABA(A)) or glycine receptors and mutant receptors that were engineered to be relatively resistant to inhaled anesthetics., Methods: The effect of beta-hydroxybutyric acid, ammonium chloride, diethylhexyl phthalate, and GABA were tested on homomeric alpha1 and mutant alpha1 (S267I) glycine receptors. The effect of sodium dodecyl sulfate and glycine were tested on alpha1 b2 gamma2s and mutant alpha1(S270I) beta2 gamma2s GABA(A) receptors. Receptors were expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamping. For both GABA(A) and glycine receptors, isoflurane and ethanol were used as positive controls and propofol as a negative control (i.e., unaffected by the mutation)., Results: Beta-hydroxybutyric acid, ammonium chloride, diethylhexyl phthalate, and GABA all enhanced glycine receptor function. This effect was reduced by the S267I mutations. Sodium dodecyl sulfate and glycine enhanced GABA(A) receptor function, and the S270I mutation attenuated this effect., Conclusion: These findings support the hypothesis that the compounds studied modulate GABA(A) or glycine receptors by a mechanism similar to that of isoflurane and ethanol. Comparing the effect of drugs on anesthetic-sensitive wild-type receptors with relatively less sensitive mutant receptors may help identify compounds with anesthetic effects.

Introduction: Intravenous (IV) fluid bags made of polyvinyl chloride (PVC) often contain the plasticizer di(2-ethylhexyl) phthalate (DEHP) to make the PVC flexible. Phthalate esters have been reported to inhibit neuronal nicotinic acetylcholine receptors, which are sensitive to many inhaled anesthetics. This raises the possibility that DEHP might modulate the function of other cys-loop receptors, such as gamma-amino butyric acid type A (GABA(A)) and glycine receptors, and that DEHP-plasticized PVC might interfere with electrophysiologic studies of anesthetic mechanisms on those receptors., Methods: alpha(1)beta(2) GABA(A) and alpha(1) glycine receptors were expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamping. We then measured the effect of buffers from IV bags containing DEHP-plasticized PVC, and of buffers saturated with DEHP, on agonist-induced currents., Results: Agonist-induced currents from glycine receptors were enhanced by buffers from IV bags containing DEHP-plasticized PVC by 291.9% +/- 84.5% (mean +/- se) and from saturated solutions of DEHP by 70.8% +/- 16.7%. Agonist-induced currents from alpha(1)beta(2) GABA(A) receptors were inhibited by buffers from IV bags containing DEHP-plasticized PVC by 19.3% +/- 3.2% and by 31.7% +/- 7.0% from buffers saturated with DEHP., Conclusions: The plasticizer DEHP modulates the function of both GABA(A) and glycine receptors. DEHP contamination can confound the results of electrophysiologic studies of anesthetic mechanisms on these receptors if DEHP-plasticized PVC is present in the experimental apparatus.

Introduction: A mechanism of anesthesia has recently been proposed which predicts that coreleased neurotransmitters may modulate neurotransmitter receptors for which they are not the native agonist in a manner similar to anesthetics., Methods: We tested this prediction by applying acetylcholine to a NR1/NR2A N-methyl-d-aspartate receptor, glycine to a wild-type alpha(1)beta(2) and anesthetic-resistant alpha(1)(S270I)beta(2) gamma-amino-butyric acid (GABA) type A receptor, and GABA to a homomeric alpha(1) wild type and anesthetic-resistant alpha(1) S267I glycine receptor. Receptors were expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamping., Results: We found inhibition of N-methyl-d-aspartate receptor function by acetylcholine, enhancement of glycine receptor function by GABA, and enhancement of GABA type A receptor function by glycine. As expected of compounds with anesthetic activity, GABA showed far less potentiation (enhancement) of the function of the anesthetic-resistant S267I glycine receptor than that of the wild-type receptor. Glycine potentiated the function of wild-type GABA type A receptors but inhibited the function of the anesthetic-resistant S270I GABA type A receptor., Conclusions: These results show that neurotransmitters that are coreleased onto anesthetic-sensitive receptors may modulate the function of receptors for which they are not the native agonist via an anesthetic-like mechanism. These findings lend support to a recent theory of anesthetic action.

Unlabelled: Many inhaled anesthetics potentiate the effect of glycine on inhibitory strychnine-sensitive glycine receptors in vitro, supporting the view that this receptor could mediate the immobility produced by inhaled anesthetics during noxious stimulation (i.e., would underlie minimum alveolar anesthetic concentration [MAC]). There are quantitative differences between anesthetics in their capacity to potentiate glycine's effect in receptor expression systems: halothane (most potentiation), isoflurane (intermediate), and cyclopropane (minimal). If glycine receptors mediate MAC, then their blockade in the spinal cord should increase the MAC of halothane more than that of isoflurane and isoflurane MAC more than cyclopropane MAC; the increases in MAC should be proportional to the receptor potentiation produced in vitro. Rats with chronically implanted intrathecal catheters were anesthetized with halothane, isoflurane, or cyclopropane. During intrathecal infusion of artificial cerebrospinal fluid, MAC was determined. Then MAC was re-determined during an infusion of 3, 12, 24, or 48 (isoflurane only) micro g/min of strychnine (strychnine blocks glycine receptors) in artificial cerebrospinal fluid. Strychnine infusion increased MAC in proportion to the enhancement of glycine receptors found in vitro. The maximum effect was with an infusion of 12 micro g/min. For the combined results at 12 and 24 micro g/min of strychnine, the increase in MAC correlated with the extent of in vitro potentiation (r(2) = 0.82). These results support the hypothesis that glycine receptors mediate part of the immobilization produced by inhaled anesthetics., Implications: In vitro, halothane potentiates glycine's effect on strychnine-sensitive glycine receptors more than isoflurane and isoflurane more than cyclopropane. The present in vivo work indicates that antagonism of the glycine receptor with strychnine increases minimum alveolar anesthetic concentration for halothane more than isoflurane and isoflurane more than cyclopropane. Such results support the notion that glycine receptors may mediate part of the immobility produced by inhaled anesthetics.

Unlabelled: Inhaled anesthetics produce immobility (a cardinal aspect of general anesthesia) by an action on the spinal cord, possibly by potentiating the responses of gamma-amino-n-butyric acid (GABA(A)) and glycine receptors to GABA and glycine. In this study, we antagonized GABA(A) and glycine responses by intrathecal administration of picrotoxin (a noncompetitive GABA(A) antagonist), strychnine (a competitive glycine antagonist), or combinations of these drugs. We measured the capacity of antagonist infusion to increase isoflurane MAC (the minimum alveolar concentration of anesthetic that prevents movement in response to noxious stimuli in 50% of subjects). We found that these potent GABA(A) and glycine receptor antagonists had a ceiling effect, either alone or in combination increasing the MAC of isoflurane by at most 47%., Implications: gamma-amino-n-butyric acid and glycine receptors may in part be responsible for the immobilizing action of isoflurane. They are not, however, the only receptors that contribute to isoflurane-induced immobility (i.e., that determine the MAC of isoflurane).