Single amino acid residue in the extracellular portion of transmembrane segment 2 in the nicotinic alpha7 acetylcholine receptor modulates sensitivity to ketamine

BACKGROUND: Ketamine inhibits the activation of both heteromeric and homomeric nicotinic acetylcholine receptors. The site of molecular interaction is unknown. METHODS: The inhibition of alpha7 nicotinic acetylcholine receptors by ketamine was compared to that of 5-hydroxytryptamine-3A (5HT3A) receptors that are resistant to ketamine inhibition in Xenopus laevis oocytes. To determine whether the region of transmembrane segments 2 and 3 is relevant for ketamine inhibition of nicotinic receptors, the authors identified single amino acid residues that differ in the sequence alignment of the two proteins. They created 22 mutant alpha7 nicotinic receptors that contain the single homologous amino acid residue in the 5HT3A sequence. RESULTS: Of the 22 mutant alpha7 nicotinic receptors tested, only one (alpha7 A258S) was significantly resistant to 20 microM ketamine. The ketamine concentration response relationship for the alpha7 A258S mutant was shifted to the right with the IC50 for ketamine increased from 17 +/- 2 for wild type to 30 +/- 3 microM in the mutant (P < 0.001). Agonist activation was unchanged by the mutation. The homologous amino acid residue in the 5HT3A receptor was mutated to the alanine that occurs in the wild-type nicotinic receptor. This mutation made the previously insensitive 5HT3A receptor sensitive to ketamine (P < 0.001). CONCLUSIONS: Conservative mutation of a single amino acid in the extracellular transmembrane segment 2 domain induces resistance to ketamine inhibition in the alpha7 nicotinic receptor and sensitivity to inhibition in the 5HT3A receptor. This region may represent a ketamine binding site in the alpha7 nicotinic receptor, or it may be an important transduction site for ketamine action.     

Subunit-dependent Inhibition of Human Neuronal Nicotinic Acetylcholine Receptors and Other Ligand-gated Ion Channels by Dissociative Anesthetics Ketamine and Dizocilpine

Background: The neuronal mechanisms responsible for dissociative anesthesia remain controversial. N-methyl-D-aspartate (NMDA) receptors are inhibited by ketamine and related drugs at concentrations lower than those required for anesthetic effects. Thus, the authors studied whether ligand-gated ion channels other than NMDA receptors might display a sensitivity to ketamine and dizocilpine that is consistent with concentrations required for anesthesia.

Methods: Heteromeric human neuronal nicotinic acetylcholine receptors (hnAChR channels [alpha]2[beta]2, [alpha]2[beta]4, [alpha]3[beta]2, [alpha]3[beta]4, [alpha]4[beta]2 and [alpha]4[beta]4), 5-hydroxytryptamine3 (5-HT3), [alpha]1[beta]2[gamma]2S[gamma]-aminobutyric acid type A (GABAA) and [alpha]1 glycine receptors were expressed in Xenopus oocytes, and effects of ketamine and dizocilpine were studied using the two-electrode voltage-clamp technique.

Results: Both ketamine and dizocilpine inhibited hnAChRs in a noncompetitive and voltage-dependent manner. Receptors containing [beta]4 subunits were more sensitive to ketamine and dizocilpine than those containing [beta]2 subunits. The inhibitor concentration for half-maximal response (IC50) values for ketamine of hnAChRs composed of [beta]4 subunits were 9.5-29 [mu]M, whereas those of [beta]2subunits were 50-92 [mu]M. Conversely, 5-HT3 receptors were inhibited only by concentrations of ketamine and dizocilpine higher than the anesthetic concentrations. This inhibition was mixed (competitive/noncompetitive). GABAA and glycine receptors were very resistant to dissociative anesthetics.     

Intravenous Anesthetics Differentially Modulate Ligand-gated Ion Channels

Background: Heteromeric neuronal nicotinic acetylcholine receptors (nAChRs) are potently inhibited by volatile anesthetics, but it is not known whether they are affected by intravenous anesthetics. Ketamine potentiates [gamma]-aminobutyric acid type A (GABAA) receptors at high concentrations, but it is unknown whether there is potentiation at clinically relevant concentrations. Information about the effects of intravenous anesthetics with different behavioral profiles on specific ligand-gated ion channels may lead to hypotheses as to which ion channel effect produces a specific anesthetic behavior.

Methods: A heteromeric nAChR composed of [alpha]4 and [beta]4 subunits was expressed heterologously in Xenopus laevis oocytes. Using the two-electrode voltage clamp technique, peak ACh-gated current was measured before and during application of ketamine, etomidate, or thiopental. The response to GABA of [alpha]1[beta]2[gamma]2s GABAA receptors expressed in human embryonic kidney cells and Xenopus oocytes was compared with and without coapplication of ketamine from 1 [mu]m to 10 mm.

Results: Ketamine caused potent, concentration-dependent inhibition of the [alpha]4[beta]4 nAChR current with an IC50 of 0.24 [mu]m. The inhibition by ketamine was use-dependent; the antagonist was more effective when the channel had been opened by agonist. Ketamine did not modulate the [alpha]1[beta]2[gamma]2s GABAA receptor response in the clinically relevant concentration range. Thiopental caused 27% inhibition of ACh response at its clinical EC50. Etomidate did not modulate the [alpha]4[beta]4 nAChR response in the clinically relevant concentration range, although there was inhibition at very high concentrations.     

Droperidol Inhibits GABAA and Neuronal Nicotinic Receptor Activation

Background: Droperidol is used in neuroleptanesthesia and as an antiemetic. Although its antiemetic effect is thought to be caused by dopaminergic inhibition, the mechanism of droperidol's anesthetic action is unknown. Because [gamma]-aminobutyric acid type A (GABAA) and neuronal nicotinic acetylcholine receptors (nAChRs) have been implicated as putative targets of other general anesthetic drugs, the authors tested the ability of droperidol to modulate these receptors.

Methods: [gamma]-Aminobutyric acid type A [alpha]1[beta]1[gamma]2 receptor, [alpha]7 and [alpha]4[beta]2 nAChRs were expressed in Xenopus oocytes and studied with two-electrode voltage clamp recording. The authors tested the ability of droperidol at concentrations from 1 nm to 100 [mu]m to modulate activation of these receptors by their native agonists.

Results: Droperidol inhibited the GABA response by a maximum of 24.7 +/- 3.0%. The IC50 for inhibition was 12.6 +/- 0.47 nm droperidol. At high concentrations, droperidol (100 [mu]m) activates the GABAA receptor in the absence of GABA. Inhibition of the GABA response is significantly greater at hyperpolarized membrane potentials. The activation of the [alpha]7 nAChR is also inhibited by droperidol, with an IC50 of 5.8 +/- 0.53 [mu]m. The Hill coefficient is 0.95 +/- 0.1. Inhibition is noncompetitive, and membrane voltage dependence is insignificant.     

Roles of Amino Acids and Subunits in Determining the Inhibition of Nicotinic Acetylcholine Receptors by Competitive Antagonists

Background: Binding sites for agonists and competitive antagonists (nondepolarizing neuromuscular blocking agents) are located at the [alpha]-[delta] and [alpha]-[varepsilon] subunit interfaces of adult nicotinic acetylcholine receptors. Most information about the amino acids that participate in antagonist binding comes from binding studies with (+)-tubocurarine and metocurine. These bind selectively to the [alpha]-[varepsilon] interface but are differentially sensitive to mutations. To test the generality of this observation, the authors measured current inhibition by five competitive antagonists on wild-type and mutant acetylcholine receptors.

Methods: HEK293 cells were transfected with wild-type or mutant ([alpha]Y198F, [varepsilon]D59A, [varepsilon]D59N, [varepsilon]D173A, [varepsilon]D173N, [delta]D180K) mouse muscle acetylcholine receptor complementary DNA. Outside-out patches were excised and perfused with acetylcholine in the absence and presence of antagonist. Concentration-response curves were constructed to determine antagonist IC50. An antagonist-removal protocol was used to determine dissociation and association rates.

Results: Effects of mutations were antagonist specific. [alpha]Y198F decreased the IC50 of (+)-tubocurarine 10-fold, increased the IC50 of vecuronium 5-fold, and had smaller effects on other antagonists. (+)-Tubocurarine was the most sensitive antagonist to [varepsilon]D173 mutations. [varepsilon]D59 mutations had large effects on metocurine and cisatracurium. [delta]D180K decreased inhibition by pancuronium, vecuronium, and cisatracurium. Inhibition by these antagonists was increased for receptors containing two [delta] subunits but no [varepsilon] subunit. Differences in IC50 arose from differences in both dissociation and association rates.     

Blockade and Activation of the Human Neuronal Nicotinic Acetylcholine Receptors by Atracurium and Laudanosine

Background: Curaremimetic nondepolarizing muscle relaxants are widely used in clinical practice to prevent muscle contraction either during surgery or during intensive care. Although primarily acting at the neuromuscular junction, these compounds can cause adverse effects, including modification of cardiac rhythm, arterial blood pressure, and in the worst cases, triggering of seizures. In this study, we assessed the interaction of atracurium and its metabolite, laudanosine, with neuronal nicotinic receptors.

Methods: The human neuronal nicotinic receptors [alpha]4[beta]2, [alpha]3[beta]4, [alpha]3[alpha]5[beta]4, and [alpha]7 are heterologously expressed in Xenopus laevis oocytes, and the effect of atracurium and its degradation product, laudanosine, were studied on these receptors.

Results: Atracurium and laudanosine inhibited in the micromolar range the major brain [alpha]4[beta]2 receptor and the ganglionic [alpha]3[beta]4 or [alpha]3[beta]4[alpha]5 and the homomeric [alpha]7 receptors. For all four receptors, inhibition was rapid and readily reversible within less than 1 min. Atracurium blockade was competitive at [alpha]4[beta]2 and [alpha]7 receptors but displayed a noncompetitive blockade at the [alpha]3[beta]4 receptors. Inhibition at this receptor subtype was not modified by [alpha]5. Laudanosine was found to have a dual mode of action; first, it competes with acetylcholine and, second, it blocks the ionic pore by steric hindrance. At low concentrations, these two drugs are able to activate both the [alpha]4[beta]2 and the [alpha]3[beta]4 receptors.     

Activation and Inhibition of Human Muscular and Neuronal Nicotinic Acetylcholine Receptors by Succinylcholine

Background: Succinylcholine is one of the most widely used muscle relaxants in clinical anesthesia and emergency medicine. Although the clinical advantages and cardiovascular side effects are well known, its mechanism of action within the human nicotinic cholinergic receptor system remains to be understood. The aim of this study was to investigate the effect of succinylcholine on human muscle and neuronal nicotinic acetylcholine receptor (nAChR) subtypes.

Methods: Xenopus laevis oocytes were injected with human messenger RNA for muscle and neuronal nAChR subunits. Receptor activation, desensitization, and inhibition induced by the natural ligand acetylcholine or by succinylcholine was studied using a multichannel two-electrode voltage clamp setup. Responses were measured as peak current and net charge.

Results: Succinylcholine concentration-dependently activated the muscle-type nAChR with an EC50 value of 10.8 [mu]m (95% confidence interval, 9.8-11.9 [mu]m), and after the initial activation, succinylcholine desensitized the muscle-type nAChR. Succinylcholine did not activate the neuronal nAChR subtypes [alpha]3[beta]2, [alpha]3[beta]4, [alpha]4[beta]2, or [alpha]7 at concentrations up to 1 mm and was a poor inhibitor at these receptor subtypes, with IC50 values above 100 [mu]m.     

Nonhalogenated Alkanes Cyclopropane and Butane Affect Neurotransmitter-gated Ion Channel and G-protein-coupled Receptors: Differential Actions on GABAA and Glycine Receptors

Background: Anesthetic mechanisms of nonhalogenated alkanes cyclopropane and butane are not understood. This study was designed to look at which neurotransmitter receptors are possible targets for these anesthetics.

Methods: Effects of cyclopropane and butane on eight recombinant receptors expressed in Xenopus oocytes were examined electrophysiologically. To address molecular mechanisms of interaction with glycine and [gamma]-aminobutyric acid type A (GABAA) receptors, cyclopropane was further tested on [alpha]1(S267C) glycine receptor and [alpha]2(S270X)[beta]1 GABAA receptors that were mutated to amino acids with larger side chains.

Results: Cyclopropane (1, 2, and 5 minimum alveolar concentration [MAC]) potentiated glycine responses by 39, 62, and 161%, respectively, and butane (1 MAC) potentiated by 64% with an increase in apparent affinity for glycine, but yielded barely detectable potentiation of GABAA receptors. The efficacy of cyclopropane for glycine receptors was less than isoflurane and halothane. The potentiation by cyclopropane was eliminated for the [alpha]1(S267C) glycine receptor. Mutant GABAA receptors in which the corresponding amino acid was substituted with larger amino acids did not produce significant potentiation. Cyclopropane and butane inhibited nicotinic acetylcholine and N-methyl-d-aspartate receptors, potentiated G-protein-coupled inwardly rectifying potassium channels, and did not change 5-hydroxytryptamine3A or muscarinic1 receptor function. Only cyclopropane markedly inhibited [alpha]-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors.     

Effects of Gaseous Anesthetics Nitrous Oxide and Xenon on Ligand-gated Ion Channels: Comparison with Isoflurane and Ethanol

Background: Ligand-gated ion channels are considered to be potential general anesthetic targets. Although most general anesthetics potentiate the function of [gamma]-aminobutyric acid receptor type A (GABAA), the gaseous anesthetics nitrous oxide and xenon are reported to have little effect on GABAA receptors but inhibit N-methyl-d-aspartate (NMDA) receptors. To define the spectrum of effects of nitrous oxide and xenon on receptors thought to be important in anesthesia, the authors tested these anesthetics on a variety of recombinant brain receptors.

Methods: The glycine, GABAA, GABA receptor type C (GABAC), NMDA, [alpha]-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainate, 5-hydroxytryptamine3 (5-HT3), and nicotinic acetylcholine (nACh) receptors were expressed in Xenopus oocytes and effects of nitrous oxide and xenon, and as equipotent concentrations of isoflurane and ethanol, were studied using the two-electrode voltage clamp.

Results: Nitrous oxide (0.58 atmosphere [atm]) and xenon (0.46 atm) exhibited similar effects on various receptors. Glycine and GABAA receptors were potentiated by gaseous anesthetics much less than by isoflurane, whereas nitrous oxide inhibited GABAC receptors. Glutamate receptors were inhibited by gaseous anesthetics more markedly than by isoflurane, but less than by ethanol. NMDA receptors were the most sensitive among glutamate receptors and were inhibited by nitrous oxide by 31%. 5-HT3 receptors were slightly inhibited by nitrous oxide. The nACh receptors were inhibited by gaseous and volatile anesthetics, but ethanol potentiated them. The sensitivity was different between [alpha]4[beta]2 and [alpha]4[beta]4 nACh receptors; [alpha]4[beta]2 receptors were inhibited by nitrous oxide by 39%, whereas [alpha]4[beta]4 receptors were inhibited by 7%. The inhibition of NMDA and nACh receptors by nitrous oxide was noncompetitive and was slightly different depending on membrane potentials for NMDA receptors, but not for nACh receptors.     

Ketamine Inhibits Presynaptic and Postsynaptic Nicotinic Excitation of Identified Cardiac Parasympathetic Neurons in Nucleus Ambiguus

Background: Ketamine increases both blood pressure and heart rate, effects commonly thought of as sympathoexcitatory. The authors investigated possible central nervous system actions of ketamine to inhibit cardiac parasympathetic neurons in the brainstem by inhibiting multiple nicotinic excitatory mechanisms.

Methods: The authors used a novel in vitro approach to study the effect of ketamine on identified cardiac parasympathetic preganglionic neurons in rat brainstem slices. The cardiac parasympathetic neurons in the nucleus ambiguus were retrogradely prelabeled with the fluorescent tracer by placing rhodamine into the pericardial sac. Dye-labeled neurons were visually identified for patch clamp recording. The effects of ketamine were tested on nicotine-evoked ligand-gated currents and spontaneous glutamatergic miniature synaptic currents (mini) in cardiac parasympathetic preganglionic neurons.

Results: Ketamine (10 [mu]m) inhibited (1) the nicotine (1 [mu]m)-evoked presynaptic facilitation of glutamate release (mini frequency, 18 +/- 7% of control; n = 9), and (2) the direct postsynaptic ligand-gated current (27 +/- 8% of control; n = 9), but ketamine did not alter the amplitude of postsynaptic miniature non-N-methyl-d-aspartate currents. [alpha] Bungarotoxin, an antagonist of [alpha]7 containing nicotinic presynaptic receptors, blocked ketamine actions on mini frequency (n = 10) but not mini amplitude.     

Stereospecific Interaction of Ketamine with Nicotinic Acetylcholine Receptors in Human Sympathetic Ganglion-like SH-SY5Y Cells

Background: Surprising clinical evidence suggests a block of sympathetic transmission by ketamine. The action of ketamine on nicotinic acetylcholine receptors (nAChRs) in human ganglions is unknown. Because ganglionic transmission depends on nAChRs, such information may help to clarify whether ketamine impairs ganglionic transmission in men. Because racemic ketamine as well as S (+)-ketamine are used clinically, the authors investigated stereospecific effects on human ganglionic nAChRs. Stereospecific psychomimetic effects have been attributed to voltage-dependent Kv channel inhibition; therefore the effects on nAChRs were compared with those on Kv channels present in the same cells.

Methods: Whole-cell currents through nAChRs and K channels were measured in SH-SY5Y cells with the patch-clamp technique by application of acetylcholine (1 mm, nAChRs) or by a step depolarization from a holding potential of -80 mV to +40 mV (K channels). Electrolyte conditions were identical for both currents.

Results: Racemic ketamine and the isomers inhibited nAChRs and K channels in a concentration-dependent and reversible manner. Racemic ketamine inhibited nAChRs and K channels, with the anesthetic concentration inducing the half-maximal effect being 1.4 and 300 [mu]m, respectively. Only inhibition of the nAChRs was stereoselective. The half-maximal concentrations were 0.8 and 3.6 [mu]m for S (+)- and R (-)-ketamine. The K channels were 350 and 70 times less sensitive to the effects of S (+)- and R (-)-ketamine.     

Effects of Thiopental and Its Optical Isomers on Nicotinic Acetylcholine Receptors

Background: With the exception of [gamma]-aminobutyric acidA (GABAA) receptors, the major molecular targets underlying the anesthetizing actions of thiopental have yet to be established. Neuronal nicotinic acetylcholine receptors (nAChRs) are closely related to GABAA receptors and hence might also be major targets. If so, they might be expected to be substantially inhibited by surgical concentrations (EC50 = 25 [mu]m) of thiopental and to display the same stereoselectivity as does general anesthesia.

Methods: Neuronal [alpha]4[beta]2, neuronal [alpha]7 and muscle [alpha][beta][gamma][delta] nAChRs were expressed in Xenopus oocytes. Peak acetylcholine-activated currents were measured at -70 mV using the two-electrode voltage clamp technique. Racemic thiopental and its two optical isomers were applied with and without preincubation and at high and low concentrations of acetylcholine.

Results: Inhibition of all three nAChRs was enhanced by preincubation with thiopental, a protocol that mimics the pharmacologic situation in vivo. Using this protocol, inhibition was further enhanced by high concentrations of acetylcholine, with IC50 = 18 +/- 2, 34 +/- 4, and 20 +/- 2 [mu]m (mean +/- SEM) thiopental for the neuronal [alpha]4[beta]2, neuronal [alpha]7 and muscle [alpha][beta][gamma][delta] nAChRs, respectively, with Hill coefficients near unity. Neither the neuronal [alpha]7 nor the muscle [alpha][beta][gamma][delta] nAChR differentiated between the optical isomers of thiopental. However, R (+)-thiopental was significantly more effective than the S (-) isomer at inhibiting the neuronal [alpha]4[beta]2 nAChR; interestingly, this is diametrically opposite to their stereoselectivity for general anesthesia.     

Spinal Muscarinic and Nicotinic Subtypes Activated by Clonidine in Postincisional Pain

Background: A recent model of acute incisional pain has been characterized that strongly parallels the postoperative period in patients experiencing evoked pain. In that setting, abundant literature has revealed antihypersensitive effects produced by intrathecally administered [alpha]2-adrenergic receptor agonists, such as clonidine, in both animals and humans. Recent reports have suggested an obligatory role of spinal acetylcholine receptors in the analgesic action of intrathecal clonidine. The authors sought to determine the involvement of spinal muscarinic and nicotinic receptor subpopulations in the antihypersensitivity effect of intrathecal clonidine in a rodent model for human postoperative pain.

Methods: After intrathecal catheterization, rats underwent superficial plantar incision. Clonidine or a combination of clonidine and muscarinic receptor subtype antagonists (M1, M2, M3, and M4) or nicotinic receptor subtype antagonists ([alpha]4[beta]2 and [alpha]7) were intrathecally administered, and withdrawal thresholds to mechanical stimuli were examined.

Results: Spinal clonidine maximally reduced hypersensitivity adjacent to the wound 30 min after its injection. When animals were intrathecally pretreated with the M1 muscarinic antagonist toxin MT-7, the M3 muscarinic antagonist 4-diphenylacetoxy-N-methylpiperidine, and the M4 muscarinic antagonist toxin MT-3, clonidine lost its antihypersensitive action. When animals were intrathecally pretreated with the [alpha]4[beta]2 nicotinic receptor antagonist dihydro-[beta]-erythroidine, but not with the [alpha]7 nicotinic receptor antagonist methyllycaconitine, the antihypersensitivity action of clonidine was abolished.     

Human Neuronal Nicotinic Acetylcholine Receptors Expressed in Xenopus Oocytes Predict Efficacy of Halogenated Compounds That Disobey the Meyer-Overton Rule

Background: According to the Meyer-Overton rule, anesthetic potency of a substance can be predicted by its lipid solubility, but a group of halogenated volatile compounds predicted to induce anesthesia does not obey this rule. Thus, these compounds are useful tools for studies of molecular targets of anesthetics. Human neuronal nicotinic acetylcholine receptor (hnAChR) subunits have been recently cloned, which allowed the authors to assess whether these receptors could differentiate among volatile anesthetic and nonimmobilizer compounds. This study provides the first data regarding anesthetic sensitivity of hnAChRs.

Methods: [alpha]2[beta]4, [alpha]3[beta]4, and [alpha]4[beta]2 hnAChRs were expressed in Xenopus oocytes, and effects of volatile anesthetics isoflurane and F3 (1-chloro-1,2,2-triflurocyclobutane, 1A) and nonimmobilizers F6 (1,2-dichlorohexafluorocyclobutane, 2N) and F8 (2,3-dichlorooctafluorobutane) on the peak acetylcholine-gated currents were studied using the two-electrode voltage-clamp technique.

Results: Isoflurane and F3 inhibited all the hnAChRs tested in a concentration-dependent manner. Isoflurane at a concentration corresponding to 1 minimum alveolar concentration (MAC) inhibited 83, 69, and 71% of ACh-induced currents in [alpha]2[beta]4, [alpha]3[beta]4, and [alpha]4[beta]2 hnAChRs, respectively, and 1 MAC of F3 inhibited 64, 44, and 61% of currents gated in those receptors. F6 (8-34[mu]M) did not cause any changes in currents gated by any of the receptors tested. F8 (4-18[mu]M) did not alter the currents gated in either [alpha]3[beta]4 or [alpha]4[beta]2 receptors, but caused a small potentiation of [alpha]2[beta]4 hnAChRs without a concentration-response relation.     

Blockade of Glutamate Receptors and Barbiturate Anesthesia: Increased Sensitivity to Pentobarbital-induced Anesthesia Despite Reduced Inhibition of AMPA Receptors in GluR2 Null Mutant Mice

Background: Barbiturates enhance [gamma]-aminobutyric acid type A (GABAA) receptor function and also inhibit the [alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of glutamate receptor. The relative contribution of these actions to the behavioral properties of barbiturates is not certain. Because AMPA receptor complexes that lack the GluR2 subunit are relatively insensitive to pentobarbital inhibition, GluR2 null mutant mice provide a novel tool to investigate the importance of AMPA receptor inhibition to the anesthetic effects of barbiturates.

Methods: GluR2 null allele (-/-), heterozygous (+/-), and wild-type (+/+) mice were injected with pentobarbital (30 and 35 mg/kg intraperitoneally). Sensitivity to anesthetics was assessed by measuring the latency to loss of righting reflex, sleep time, and the loss of corneal, pineal, and toe-pinch withdrawal reflexes. In addition, patch-clamp recordings of acutely dissociated CA1 hippocampal pyramidal neurons from (-/-) and (+/+) mice were undertaken to investigate the effects of barbiturates on kainate-activated AMPA receptors and GABA-activated GABAA receptors.

Results: Behavioral tests indicate that sensitivity to pentobarbital was increased in (-/-) mice. In contrast, AMPA receptors from (-/-) neurons were less sensitive to inhibition by pentobarbital (concentrations that produced 50% of the maximal inhibition [IC50], 301 vs. 51 [mu]M), thiopental (IC50, 153 vs. 34 [mu]M), and phenobarbital (IC50, 930 vs. 205 [mu]M) compared with wild-type controls, respectively. In addition, the potency of kainate was greater in (-/-) neurons, whereas no differences were observed for the potentiation of GABAA receptors by pentobarbital.     

Nonhalogenated alkanes cyclopropane and butane affect neurotransmitter-gated ion channel and G-protein-coupled receptors: differential actions on GABAA and glycine receptors

BACKGROUND: Anesthetic mechanisms of nonhalogenated alkanes cyclopropane and butane are not understood. This study was designed to look at which neurotransmitter receptors are possible targets for these anesthetics. METHODS: Effects of cyclopropane and butane on eight recombinant receptors expressed in Xenopus oocytes were examined electrophysiologically. To address molecular mechanisms of interaction with glycine and gamma-aminobutyric acid type A (GABA(A)) receptors, cyclopropane was further tested on alpha1(S267C) glycine receptor and alpha2(S270X)beta1 GABA(A) receptors that were mutated to amino acids with larger side chains. RESULTS: Cyclopropane (1, 2, and 5 minimum alveolar concentration [MAC]) potentiated glycine responses by 39, 62, and 161%, respectively, and butane (1 MAC) potentiated by 64% with an increase in apparent affinity for glycine, but yielded barely detectable potentiation of GABA(A) receptors. The efficacy of cyclopropane for glycine receptors was less than isoflurane and halothane. The potentiation by cyclopropane was eliminated for the alpha1(S267C) glycine receptor. Mutant GABA(A) receptors in which the corresponding amino acid was substituted with larger amino acids did not produce significant potentiation. Cyclopropane and butane inhibited nicotinic acetylcholine and N-methyl-D-aspartate receptors, potentiated G-protein-coupled inwardly rectifying potassium channels, and did not change 5-hydroxytryptamine(3A) or muscarinic(1) receptor function. Only cyclopropane markedly inhibited alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors. CONCLUSIONS: Glycine, nicotinic acetylcholine, and N-methyl-D-aspartate receptors are sensitive to nonhalogenated alkanes, and the authors propose that glycine and N-methyl-D-aspartate receptors are good candidates for anesthetic immobility. The authors also suggest that the distinct effects on glycine and GABA(A) receptors are not due to the small volumes of these anesthetics.     

Nonhalogenated Alkane Anesthetics Fail to Potentiate Agonist Actions on Two Ligand-gated Ion Channels

Background: Although ether, alcohol, and halogenated alkane anesthetics potentiate agonist actions or increase the apparent agonist affinity of ligand-gated ion channels at clinically relevant concentrations, the effects of nonhalogenated alkane anesthetics on ligand-gated ion channels have not been studied. The current study assessed the abilities of two representative nonhalogenated alkane anesthetics (cyclopropane and butane) to potentiate agonist actions or increase the apparent agonist affinity of two representative ligand-gated ion channels: the nicotinic acetylcholine receptor and [gamma]-aminobutyric acid type A (GABAA) receptor.

Methods: Nicotinic acetylcholine receptors were obtained from the electroplax organ of Torpedo nobiliana, and human GABAA receptors ([alpha]1[beta]2[gamma]2L) were expressed in human embryonic kidney 293 cells. The Torpedo nicotinic acetylcholine receptors apparent agonist affinity in the presence and absence of anesthetic was assessed by measuring the apparent rates of desensitization induced by a range of acetylcholine concentrations. The GABAA receptor's apparent agonist affinity in the presence and absence of anesthetic was assessed by measuring the peak currents induced by a range of GABA concentrations.

Results: Neither cyclopropane nor butane potentiated agonist actions or increased the apparent agonist affinity (reduced the apparent agonist dissociation constant) of the Torpedo nicotinic acetylcholine receptor or GABAA receptor. At clinically relevant concentrations, cyclopropane and butane reduced the apparent rate of Torpedo nicotinic acetylcholine receptor desensitization induced by low concentrations of agonist.     

Nonanesthetic Volatile Drugs Obey the Meyer-Overton Correlation in Two Molecular Protein Site Models

Background: Nonanesthetic volatile compounds fail to inhibit movement in response to noxious stimulation at concentrations predicted to induce anesthesia from their oil-water partitioning. Thus they represent tools to determine whether molecular models behave like the targets that mediate in vivo anesthetic actions. The effects of volatile anesthetics and non-anesthetics were examined in two experimental models in which anesthetics interact directly with proteins: the pore of the nicotinic acetylcholine receptor and human serum albumin.

Methods: Wild-type mouse muscle nicotinic receptors and receptors containing pore mutations ([Greek small leter alpha] S252I + [Greek small letter beta] T263I) were studied electrophysiologically in membrane patches from Xenopus oocytes. Patch currents evoked by brief pulses of acetylcholine were measured in the presence of enflurane and two nonanesthetics, 1,2-dichlorohexafluorocyclobutane and 2,3-dichlorooctafluorobutane. Nonanesthetic interactions with human serum album were assessed by quenching of intrinsic protein fluorescence.

Results: Both anesthetic and nonanesthetic volatile compounds inhibited wild-type and [Greek small letter alpha] S252I + [Greek small letter beta] T263I mutant nicotinic channels but displayed different selectivity for open versus resting receptor states. Median inhibitory concentrations (IC50 s) in wild-type nicotinic receptors were 870 +/- 20 [micro sign]M for enflurane, 37 +/- 3 [micro sign]M for 1,2-dichlorohexafluorocylcobutane, and 11.3 +/- 5.6 [micro sign]M for 2,3-dichlorooctafluorobutane. For all three drugs, ratios of wild-type IC50 s to mutant IC50mut ranged from 7-10, and ratios of wild-type IC50 s to predicted anesthetic median effective concentrations (EC50 s) ranged from 1.8-2.3. 1,2-Dichlorohexafluorocyclobutane quenched human serum albumin with an apparent dissociation constant (Kd) of 160 +/- 11 [micro sign]M. The ratios of dissociation constants to predicted EC50 s for the nonanesthetics were within a factor of two of the dissociation constant:EC (50) ratios calculated for halothane and chloroform from previous published results.     

Subunit-dependent inhibition of human neuronal nicotinic acetylcholine receptors and other ligand-gated ion channels by dissociative anesthetics ketamine and dizocilpine

Background The neuronal mechanisms responsible for dissociative anesthesia remain controversial. N-methyl-D-aspartate (NMDA) receptors are inhibited by ketamine and related drugs at concentrations lower than those required for anesthetic effects. Thus, the authors studied whether ligand-gated ion channels other than NMDA receptors might display a sensitivity to ketamine and dizocilpine that is consistent with concentrations required for anesthesia. METHODS: Heteromeric human neuronal nicotinic acetylcholine receptors (hnAChR channels alpha2beta2, alpha2beta4, alpha3beta2, alpha3beta4, alpha4beta2 and alpha4beta4), 5-hydroxytryptamine3 (5-HT3), alpha1beta2gamma2S gamma-aminobutyric acid type A (GABAA) and alpha1 glycine receptors were expressed in Xenopus oocytes, and effects of ketamine and dizocilpine were studied using the two-electrode voltage-clamp technique. RESULTS: Both ketamine and dizocilpine inhibited hnAChRs in a noncompetitive and voltage-dependent manner. Receptors containing beta1 subunits were more sensitive to ketamine and dizocilpine than those containing beta2 subunits. The inhibitor concentration for half-maximal response (IC50) values for ketamine of hnAChRs composed of beta4 subunits were 9.5-29 microM, whereas those of beta2 subunits were 50-92 microM. Conversely, 5-HT3 receptors were inhibited only by concentrations of ketamine and dizocilpine higher than the anesthetic concentrations. This inhibition was mixed (competitive/noncompetitive). GABAA and glycine receptors were very resistant to dissociative anesthetics. CONCLUSIONS: Human nAChRs are inhibited by ketamine and dizocilpine at concentrations possibly achieved in vivo during anesthesia in a subunit-dependent manner, with beta subunits being more critical than alpha subunits. Conversely, 5-HT3, GABAA, and glycine receptors were relatively insensitive to dissociative anesthetics.     

Blockade and activation of the human neuronal nicotinic acetylcholine receptors by atracurium and laudanosine

BACKGROUND: Curaremimetic nondepolarizing muscle relaxants are widely used in clinical practice to prevent muscle contraction either during surgery or during intensive care. Although primarily acting at the neuromuscular junction, these compounds can cause adverse effects, including modification of cardiac rhythm, arterial blood pressure, and in the worst cases, triggering of seizures. In this study, we assessed the interaction of atracurium and its metabolite, laudanosine, with neuronal nicotinic receptors. METHODS: The human neuronal nicotinic receptors alpha4beta2, alpha3beta4, alpha3alpha5beta4, and alpha7 are heterologously expressed in Xenopus laevis oocytes, and the effect of atracurium and its degradation product, laudanosine, were studied on these receptors. RESULTS: Atracurium and laudanosine inhibited in the micromolar range the major brain alpha4beta2 receptor and the ganglionic alpha3beta4 or alpha3beta4alpha5 and the homomeric alpha7 receptors. For all four receptors, inhibition was rapid and readily reversible within less than 1 min. Atracurium blockade was competitive at alpha4beta2 and alpha7 receptors but displayed a noncompetitive blockade at the alpha3beta4 receptors. Inhibition at this receptor subtype was not modified by alpha5. Laudanosine was found to have a dual mode of action; first, it competes with acetylcholine and, second, it blocks the ionic pore by steric hindrance. At low concentrations, these two drugs are able to activate both the alpha4beta2 and the alpha3beta4 receptors. CONCLUSION: Adverse effects observed during atracurium administration may be attributed, at least partly, to an interaction with neuronal nicotinic receptors.