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Pain Medicine  |   May 2000
Intravenous Anesthetics Differentially Modulate Ligand-gated Ion Channels
Article Information
Pain Medicine
Pain Medicine   |   May 2000
Intravenous Anesthetics Differentially Modulate Ligand-gated Ion Channels
Anesthesiology 5 2000, Vol.92, 1418-1425. doi:
Anesthesiology 5 2000, Vol.92, 1418-1425. doi:
SEVERAL members of the ligand-gated ion channel family have been identified as molecular targets of general anesthetics in the clinically relevant concentration range. Heteromeric neuronal nicotinic acetylcholine receptors (nAChRs) found in the central nervous system (CNS) are potential targets of volatile anesthetic drugs as they are inhibited at minimum alveolar concentration. 1,2 The intravenous anesthetic propofol does not modulate the central-type nAChRs in the clinically relevant concentration range. 1,2 
We hypothesize that the intravenous anesthetics ketamine, thiopental, and etomidate may have in common with volatile anesthetics modulation of nAChRs expressed in the CNS. We studied a central-type nAChR composed of α4 and β4 subunits. The α4 and β4 nAChR subunits are expressed in anatomic locations within the CNS, which make it a potential target for mediating anesthetic behavioral responses. Although RNA for the α4 subunit partner in the α4β4 nAChR is distributed diffusely throughout the CNS, 3 the β4 subunit has more specific CNS distribution. RNA message for β4 subunits is detected in the hippocampal formation, in layer I-IV of the cerebral cortex, in the cerebellum, in the medial habenula, in the interpeduncular and pontine and trigeminal nuclei, and in the locus coeruleus. The combination of α4 and β4 nAChR subunits is therefore anatomically located in an appropriate manner to subserve anesthetic behaviors such as sedation, amnesia, and central modification of autonomic reflexes. 4 The study of neuronal nAChR modulation by anesthetics is particularly pertinent because they have been implicated in memory, arousal, analgesia, and autonomic control. 5 
Although ketamine potentiates γ-aminobutyric acid type A (GABAA) receptor response at higher-than-clinical concentrations, concentration dependence has not been determined, and ketamine effect at clinical concentrations is unknown. 6 We hypothesize that ketamine is unusual among anesthetics in not potentiating the response to GABA at clinical concentrations.
Several traditional pharmacologic criteria have been used to select potentially relevant molecular targets of general anesthetics. 7,8 Among these criteria is the requirement that the target be modulated in the clinically relevant concentration range. The range of concentrations that is clinically relevant is controversial, and many drugs affect ligand-gated ion channels at high concentrations. However, if there is no effect on a molecular target at the clinical EC50for a drug, the target is unlikely to be responsible for drug-induced changes in behavior. The concentrations of the intravenous anesthetics that modulate α4β4 nAChRs may be compared with the clinical EC50s for those anesthetics, derived from the literature. The reported EC50s for anesthetics in humans vary considerably. One reason for the variability is that for many anesthetics, there is a great degree of protein binding, which has not always been taken into account. The concentration of ketamine measured in blood on awakening is reported to be between 2.7 and 4.7 μm. 9,10 Analgesia has been reported at 0.17 and 0.63 μm. 11 Ketamine is approximately 50% bound by protein in human plasma 12; therefore, the EC50concentration for free ketamine to produce anesthesia is between 1.3 and 2.4 μm. Analgesia is caused by 0.08–0.32 μm free ketamine. Fifty percent of patients fail to respond to a painful stimulus at 178 μm thiopental. When 85% protein binding is accounted for, the EC50for free thiopental is approximately 25 μm. 8 The EC50value for etomidate has been measured at 8.7 μm. Protein binding is 78%; thus, the EC50for free etomidate is approximately 2 μm. 13 
We have found that the intravenous anesthetics ketamine, thiopental, and etomidate differentially modulate both nAChRs and GABAAreceptors. Anesthetic drugs can perturb many systems; the crucial next step is in distinguishing targets that result in desirable anesthetic effects and undesirable side effects from those that are epiphenomena.
Materials and Methods
nAChRs and GABA Receptors in Xenopus  Oocytes
The chick α4 and β4 nAChR subunit cRNAs were prepared from appropriate cDNAs in a modified PGH19 vector using standard techniques. 14 The rat α1, β1, γ2s GABAAsubunit cRNAs were prepared from the appropriate cDNAs in a modified PGEM vector. The vector was linearized and used as a template for run-off transcription from the T7 (α4 nAChR and α1, β1, and γ2s GABAAreceptors) or SP6 (β4 nAChR) promoter. Xenopus laevis  oocytes were surgically removed from female frogs and defolliculated with collagenase. After incubation overnight in L-15 oocyte medium (Specialty Media, Phillipsburg, NJ), approximately 5 ng each subunit cRNA was injected per oocyte using a Nanoject Variable automatic injector (Drummond Scientific Co., Broomall, PA). The oocytes were incubated for 24–72 h in L-15 oocyte medium before physiologic assay.
Whole oocyte currents were recorded using a Gene-Clamp 500 two-microelectrode voltage-clamp amplifier with an active ground (Axon Instruments, Inc., Foster City, CA). The voltage, current, and active ground electrodes were filled with a 3-m KCl solution, such that voltage and current electrodes had a resistance of 1–5 MΩ. Extracellular recording solution consisted of 82.5 mm NaCl, 2 mm KCl, 1 mm MgCl2, 10 mm HEPES, 1 mm CaCl2, pH = 7.2. Calcium was omitted in experiments with GABAAreceptors in oocytes. Experiments were performed at room temperature (20–24°C). Ketamine (Parke-Davis, Morris Plains, NJ) was prepared as a 1-mm stock solution in external recording solution. Thiopental (Sigma, St.Louis, MO) was prepared as a 10-mm solution on the day the experiments were conducted. Etomidate (Abbott Laboratories, Chicago, IL) was prepared as a 1-mm solution. Test solutions were prepared by serial dilution. All anesthetic drugs were racemic mixtures.
Oocytes were held at a membrane potential of −60 mV (unless specified otherwise), and peak current was measured in response to ACh or GABA, with and without anesthetic. The drug solutions were placed in a closed syringe at the time of application and injected into a closed loop of tubing with a volume of 1 ml. When activated by a manual switch, the drug containing solution joined the column of solution that bathes a specially prepared recording dish consisting of a 125-μl cylindrical channel. Perfusate was run continually at approximately 4 ml/min. Drugs were thus applied for an approximately 15-s pulse of known volume and concentration. The cells were perfused with the test concentration of anesthetic for 5 min before agonist/anesthetic coapplication. Five minutes was allowed to elapse between repeated agonist application in all experiments to minimize the contribution of nAChR desensitization. This waiting time was adequate for recovery from desensitization in control experiments. A baseline control response to ACh or GABA was measured before each agonist–anesthetic coapplication. Response to agonist was measured after each anesthetic application. Responses that did not return to within 80% of baseline values were rejected for analysis. Each anesthetic response is expressed relative to the preceding control current amplitude. Unless otherwise indicated, test ACh concentrations were 1 mm, which is saturating for the α4β4 receptor (data not shown). Test GABA concentration was 0.2 μm, EC20derived in control experiments (data not shown). N ≥ 3 for each data point.
Statistics and Curve Fitting
Concentration–response curves were prepared by plotting the fraction of current remaining after the coapplication of ACh and varying concentrations of intravenous anesthetic relative to the current response elicited by ACh alone. These data were fit to a Hill equation,
y = 100/(1 + (x/IC50)n)
where y is the percentage of current remaining with antagonist application, and x is the concentration of antagonist. IC50is the dose at which 50% of available receptors are modulated, and n is the Hill coefficient. All fitting algorithms and graphs were produced with Microcal Origins 5.0 software (Microcal Software Inc., Northamton, MA). Responses were acquired on-line using Pclamp7 (Axon Instruments), low pass filtered at 5 kHz, and digitized (Digidata 1200 interface; Axon Instruments) using Plcamp7 software. Data are fit using a nonlinear regression method within Microcal Origin. Data are expressed as mean ± SE.
GABAAReceptors in Human Embryonic Kidney Cells
cDNAs for human GABA α1, γ2s and rat β2 subunits were expressed in human embryonic kidney (HEK) 293 cells (American Type Culture Collection, Rockville, MD) via  the pCIS2 vector, which contains the strong promotor from cytomegalovirus and a polyadenylation sequence from SV40. HEK cells were cultured in Eagle’s minimal essential media (Sigma) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), l-glutamine (0.292 μg/ml; Gibco BRL, Grand Island, NY), penicillin G sodium (100 U/ml; Gibco BRL), and streptomycin sulfate (100 μg/ml; Gibco BRL). For electrophysiology, cells were plated on glass coverslips coated with poly-d-lysine (Sigma). The cells were transfected using the calcium phosphate precipitation technique. 15 
Electrophysiologic recordings were performed at 22°C using whole-cell patch-clamp technique as previously described. 15 The coverslips were transferred 24–72 h after removal of the cDNA to a 70-ml chamber that was continuously perfused (2–3 ml/min) with extracellular medium containing 145 mm NaCl, 3 mm KCl, 1.5 mm CaCl2, 1 mm MgCl2, 5.5 mm d-glucose, and 10 mm HEPES, pH 7.4. The intracellular solution contained 145 m[scap[m N  -methyl-d-glucamine hydrocloride, 5 mm K2ATP, 5 mm HEPES/KOH, 2 mm MgCl2, 0.1 mm CaCl2, and 1.1 mm EGTA, pH 7.2. Pipette-to-bath resistance was 4–6 MΩ. Cells were voltage clamped at −60 mV. The test GABA concentration was always EC20. GABA and ketamine were applied with rapid solution changes (< 50 ms exchange time) to the cell by local perfusion using a motor-driven solution exchange device (Bio Logic Rapid Solution Changer RSC-100; Molecular Kinetics, Pullman, WA). 16 The solution changer was driven by protocols within the acquisition program Pclamp5 (Axon Instruments). Each data point represents five current measurements. Responses were digitized (TL-1-125 interface; Axon Instruments) using Pclamp5 and stored for off-line analysis. All concentration–response curves are plotted and analyzed using Microcal Origin 5.0 (Microcal Software Inc.). Data are expressed as mean ± SE. Data are fit using a nonlinear regression method within Microcal Origin.
Results
The three intravenous anesthetics studied—ketamine, thiopental, and etomidate—inhibit the ACh activation of the α4β4 nAChR; however, the effects of only ketamine and possibly thiopental occur in a clinically relevant concentration range (figs. 1A–F).
Fig. 1. Intravenous anesthetics, near clinical EC50, differentially modulate nAChRs expressed in Xenopus  oocytes. The modulation is concentration-dependent. (A  ) Ketamine 1 μm reversibly inhibits the maximal α4β4 nAChR response to 1 mm ACh by 80%. (B  ) Ketamine inhibition is concentration-responsive with an IC50 of 0.24 (± 0.3) μm and a Hill coefficient of 0.95 (± 0.1; n = 12). (C  ) Thiopental 25 μm reversibly inhibits the α4β4 nAChR response to ACh by 20%. (D  ) Thiopental at high clinical concentrations causes concentration-dependent inhibition with an IC50 of 84 (± 22) μm and a Hill coefficient of 0.75 (± 0.15; n = 12). (E  ) Etomidate 10 μm does not significantly inhibit the α4β4 nAChR response to ACh. (F  ) Etomidate at greater than clinical concentrations causes concentration-dependent inhibition with an IC50 of 33 (± 0.08) μm and a Hill coefficient of 2.1 (± 0.01; n = 12). Anesthetics were applied for 5 min before agonist–anesthetic coapplication.
Fig. 1. Intravenous anesthetics, near clinical EC50, differentially modulate nAChRs expressed in Xenopus 
	oocytes. The modulation is concentration-dependent. (A 
	) Ketamine 1 μm reversibly inhibits the maximal α4β4 nAChR response to 1 mm ACh by 80%. (B 
	) Ketamine inhibition is concentration-responsive with an IC50 of 0.24 (± 0.3) μm and a Hill coefficient of 0.95 (± 0.1; n = 12). (C 
	) Thiopental 25 μm reversibly inhibits the α4β4 nAChR response to ACh by 20%. (D 
	) Thiopental at high clinical concentrations causes concentration-dependent inhibition with an IC50 of 84 (± 22) μm and a Hill coefficient of 0.75 (± 0.15; n = 12). (E 
	) Etomidate 10 μm does not significantly inhibit the α4β4 nAChR response to ACh. (F 
	) Etomidate at greater than clinical concentrations causes concentration-dependent inhibition with an IC50 of 33 (± 0.08) μm and a Hill coefficient of 2.1 (± 0.01; n = 12). Anesthetics were applied for 5 min before agonist–anesthetic coapplication.
Fig. 1. Intravenous anesthetics, near clinical EC50, differentially modulate nAChRs expressed in Xenopus  oocytes. The modulation is concentration-dependent. (A  ) Ketamine 1 μm reversibly inhibits the maximal α4β4 nAChR response to 1 mm ACh by 80%. (B  ) Ketamine inhibition is concentration-responsive with an IC50 of 0.24 (± 0.3) μm and a Hill coefficient of 0.95 (± 0.1; n = 12). (C  ) Thiopental 25 μm reversibly inhibits the α4β4 nAChR response to ACh by 20%. (D  ) Thiopental at high clinical concentrations causes concentration-dependent inhibition with an IC50 of 84 (± 22) μm and a Hill coefficient of 0.75 (± 0.15; n = 12). (E  ) Etomidate 10 μm does not significantly inhibit the α4β4 nAChR response to ACh. (F  ) Etomidate at greater than clinical concentrations causes concentration-dependent inhibition with an IC50 of 33 (± 0.08) μm and a Hill coefficient of 2.1 (± 0.01; n = 12). Anesthetics were applied for 5 min before agonist–anesthetic coapplication.
×
Ketamine
Ketamine at its clinical EC50almost completely inhibits ACh-gated current from the α4β4 nAChR (fig. 1A). The inhibition by ketamine was readily reversible on washout of the drug. The application of ketamine alone at concentrations up to 1 mm had no direct effect on baseline membrane current. The inhibition by ketamine of the ACh response of the α4β4 nAChR is concentration-dependent (fig. 1B). When the concentration–response curve for ketamine inhibition of current response to 1 mm ACh is fit by the Hill equation, the IC50is determined to be 0.24 μm (± 0.03), and the Hill coefficient is 0.95 (± 0.01). After 5 min of ketamine preapplication, the first response to the application of agonist with ketamine is always larger than the response to subsequent applications (fig. 2A). Inhibition of the α4β4 nAChR response by any ketamine concentration is more potent when activated by a higher agonist concentration (fig. 2B). The inhibition of ACh response is not dependent on membrane holding potential from −30 to −60 mV (fig. 3).
Fig. 2. Evidence for use dependence of ketamine inhibition of the α4β4 nAChR. (A  ) After 5-min pretreatment with ketamine 0.1 μm, repeated applications of ketamine plus agonist result in increased inhibition. Inhibition with the first coapplication is 20%, with the second is 30%, and with the third is 38%. (B  ) Ketamine inhibition of the α4β4 nAChR response to 1 mm ACh is more potent than ketamine inhibition of the response to 100 μm ACh. When ACh 100 μm is the agonist, the IC50for ketamine is 1.63 (± 0.28) μm, and the Hill coefficient is 1.38 (± 0.27).
Fig. 2. Evidence for use dependence of ketamine inhibition of the α4β4 nAChR. (A 
	) After 5-min pretreatment with ketamine 0.1 μm, repeated applications of ketamine plus agonist result in increased inhibition. Inhibition with the first coapplication is 20%, with the second is 30%, and with the third is 38%. (B 
	) Ketamine inhibition of the α4β4 nAChR response to 1 mm ACh is more potent than ketamine inhibition of the response to 100 μm ACh. When ACh 100 μm is the agonist, the IC50for ketamine is 1.63 (± 0.28) μm, and the Hill coefficient is 1.38 (± 0.27).
Fig. 2. Evidence for use dependence of ketamine inhibition of the α4β4 nAChR. (A  ) After 5-min pretreatment with ketamine 0.1 μm, repeated applications of ketamine plus agonist result in increased inhibition. Inhibition with the first coapplication is 20%, with the second is 30%, and with the third is 38%. (B  ) Ketamine inhibition of the α4β4 nAChR response to 1 mm ACh is more potent than ketamine inhibition of the response to 100 μm ACh. When ACh 100 μm is the agonist, the IC50for ketamine is 1.63 (± 0.28) μm, and the Hill coefficient is 1.38 (± 0.27).
×
Fig. 3. Lack of voltage dependence of ketamine inhibition. Inhibition of α4β4 nAChR response to 1 mm ACh is not dependent on membrane holding potential between −30 and −60 mV (n = 16).
Fig. 3. Lack of voltage dependence of ketamine inhibition. Inhibition of α4β4 nAChR response to 1 mm ACh is not dependent on membrane holding potential between −30 and −60 mV (n = 16).
Fig. 3. Lack of voltage dependence of ketamine inhibition. Inhibition of α4β4 nAChR response to 1 mm ACh is not dependent on membrane holding potential between −30 and −60 mV (n = 16).
×
In marked contrast to the potent modulation by ketamine at nAChRs, ketamine does not modulate the function of the GABA α1β2γ2s receptor at clinically relevant concentrations. A clinically relevant concentration of 1 μm ketamine does not alter the response to GABA in this receptor when expressed in HEK cells (fig. 4) or Xenopus  oocytes (3 ± 6% potentiation, n = 3 oocytes). Ketamine did produce GABA potentiation in HEK cells but only at very high ketamine concentrations (> 500 μm), with an EC50of 1.2 mm (± 0.6 mm) and a Hill slope of 2.7 (± 0.3; n = 60).
Fig. 4. Ketamine does not modulate GABAAreceptor function in HEK 293 cells at clinically relevant concentrations. (A  ) Ketamine 10 μm does not modulate the α1β2γ2s GABAAreceptor. (B  ) Ketamine at higher-than-clinical concentrations causes concentration potentiation of GABA response with an EC50 of 1.2 mm (± 0.06 mm) and a Hill slope of 2.7 (± 0.3). Maximal efficacy of potentiation is 154% (± 4; n = 60).
Fig. 4. Ketamine does not modulate GABAAreceptor function in HEK 293 cells at clinically relevant concentrations. (A 
	) Ketamine 10 μm does not modulate the α1β2γ2s GABAAreceptor. (B 
	) Ketamine at higher-than-clinical concentrations causes concentration potentiation of GABA response with an EC50 of 1.2 mm (± 0.06 mm) and a Hill slope of 2.7 (± 0.3). Maximal efficacy of potentiation is 154% (± 4; n = 60).
Fig. 4. Ketamine does not modulate GABAAreceptor function in HEK 293 cells at clinically relevant concentrations. (A  ) Ketamine 10 μm does not modulate the α1β2γ2s GABAAreceptor. (B  ) Ketamine at higher-than-clinical concentrations causes concentration potentiation of GABA response with an EC50 of 1.2 mm (± 0.06 mm) and a Hill slope of 2.7 (± 0.3). Maximal efficacy of potentiation is 154% (± 4; n = 60).
×
Thiopental
Thiopental produces weak inhibition of central nAChRs within the clinically relevant range (fig. 1C). Thiopental inhibition of the α4β4 nAChR is also concentration-dependent (fig. 1D). A concentration–response curve with thiopental inhibition of the response to 1 mm ACh, fit by the Hill equation as above, yields an IC50of 84 μm (± 22) thiopental and a Hill coefficient of 0.75 (± 0.15; n = 12).
Etomidate
Etomidate does not alter the current response to ACh in a clinically relevant concentration range (fig. 1E). Etomidate inhibition of the α4β4 nAChR is concentration-dependent but occurs at well above a clinically relevant concentration range (fig. 1F). Etomidate inhibits the α4β4 nAChR response to ACh at high concentrations, with an IC50of approximately 33 μm (± 0.1) etomidate and a Hill number of 2.1 (± 0.0; n = 12).
Discussion
The α4β4 nAChR is differentially inhibited by intravenous general anesthetics. Ketamine, a dissociative anesthetic, is best known as a potent inhibitor of the N  -methyl-d-aspartate (NMDA) receptor. 17,18 Ketamine inhibits the NMDA receptor at concentrations between 2 and 50 μm. 17 These data demonstrate that ketamine is a more potent inhibitor of a central nAChR. Ketamine inhibition of the α4β4 nAChR response to 1 mm ACh is more potent than inhibition of the response to 100 μm ACh (fig. 2B). Thus, when a greater proportion of the nAChRs are in the open state, ketamine is a more potent inhibitor. These data suggest that the inhibition by ketamine of the neuronal nAChR is use-dependent, as it is in the NMDA receptor and muscle nAChR. 18,19 As further evidence for use dependence, the first response to the coapplication of ketamine and agonist is larger than subsequent responses, regardless of preincubation time with ketamine (fig. 2A). These data suggest that ketamine requires access to the nAChR in the open state for inhibition, although they do not prove that ketamine acts as an open channel blocker as at the NMDA receptor. 18 Inhibition by ketamine is insensitive to the membrane holding potential (fig. 3). The insensitivity of inhibition by this positively charged drug to static voltage changes makes a binding site near the surface of the channel likely. Further mechanistic information will come from experiments in small cells or excised patches that are more suited to mechanistic analysis.
Unlike many other anesthetics, ketamine does not affect GABAAor glycine receptors at clinically relevant concentrations (fig. 4). 20 This is the first complete concentration–response curve for ketamine potentiation at a recombinant GABAAreceptor, although potentiation at high concentrations has been shown previously. 6 The anesthetic effects of ketamine might be explained by inhibition of the nACh and NMDA receptors. Xenopus  oocytes also contain an endogenous calcium-gated chloride channel. Heteromeric nAChRs have a relatively small fractional calcium conductance compared with homomeric α7 nAChRs, 21 and if the general anesthetics were acting on this current, this would be expected to be a small effect. Effects at other undescribed targets may complement these effects.
Patients anesthetized with anesthetics that inhibit nAChRs (ketamine and volatile anesthetics) have analgesia at subanesthetic concentrations. 22,23 Analgesia with ketamine is present at blood ketamine concentrations of 0.08–0.32 μm. 11 The anesthetics, which do not inhibit nAChRs in the clinical range, such as etomidate and propofol, are not analgesic. 22 Inhibition of nAChRs is thus a candidate mechanism for general anesthetic analgesia. Nicotinic agonists are well known to have analgesic properties. Epibatidine, a potent, specific nicotinic agonist, has analgesic potency 200 times that of morphine. 24 Analgesia by agonist and antagonist need not be paradoxical. It is possible that agonist-induced analgesia occurs because of prolonged desensitization of nicotinic receptors.
Ketamine induces a state of consciousness that is different from that induced by other anesthetics. “Ketamine as a sole anesthetic produces a cataleptic state with nystagmus and intact corneal and light reflexes.”25 Ketamine is also the only anesthetic that does not potentiate GABAAreceptors in a clinically relevant range, with the exception of nitrous oxide and xenon, which, although less extensively studied, may also inhibit the NMDA receptor (fig. 4). 26–28 Although it is currently not possible to explain the mechanism of consciousness, it may be that anesthetics other than ketamine produce their particular state of unconsciousness via  potentiation of the GABAAresponse. Both the native transmitter GABA and the benzodiazepines that act specifically at GABAAreceptors cause unconsciousness.
The anesthetic state achieved with ketamine is better termed “inattentiveness to surroundings” than “unconsciousness.” With volatile anesthetics, that inattentiveness is perhaps overshadowed by unconsciousness during the anesthetic but can be appreciated on emergence from anesthesia. Patients typically awaken from a general anesthetic with, for example, 0.2% end-tidal isoflurane, measured by a gas analyzer (approximately 56 μm in solution). At this concentration, there remains approximately 20% central nicotinic inhibition, while GABAApotentiation is at threshold. 1,15 Drugs that do not inhibit nAChRs in a clinically relevant range, particularly propofol, are notable for the lack of inattentiveness and dysphoria on emergence from anesthesia. Prolonged inattentiveness, dysphoria, and perhaps other side effects of ketamine and volatile anesthetics may be the result of lingering nicotinic blockade. In particular, elderly patients and those suffering from Alzheimer’s or Parkinson’s disease, who have impaired central nicotinic systems, often emerge from anesthesia inattentive, disoriented, and dysphoric. 22,29 A common treatment for this “emergence delirium” is physostigmine, an acetylcholinesterase inhibitor that would increase the concentration of acetylcholine in the brain. These patients may represent a subgroup, which, because of preexisting abnormalities in their central cholinergic system, is particularly sensitive to nicotinic blockade by general anesthetics. It may be that this is a group of patients in which it is best to avoid general anesthetics that inhibit nAChRs. This area warrants further study and consideration.
Comparison of patterns of ligand-gated ion channel modulation with patterns of anesthetic behavior results in several hypotheses. The inhibition of nAChRs by general anesthetics may mediate analgesia, as well as inattentiveness and delirium. GABAAaugmentation may lead to a particular state of unconsciousness. Validation of these hypotheses and the definition of the central circuitry in which they occur will require further experiments. If these hypotheses prove valid, the designers of future anesthetic drugs will be able to test agents on heterologously expressed ion channels to design in  the desirable and out  the undesirable behavioral responses in humans.
The authors thank Drs. Neil Harrison and Carol Hirshman for their careful reading of the manuscript, and Dr. Lorna Role in The Center for Neurobiology at Columbia University for her guidance and the use of her facilities.
References
Flood P, Ramirez-Latorre J, Role L: Alpha 4 beta 2 neuronal nicotinic acetylcholine receptors in the central nervous system are inhibited by isoflurane and propofol, but alpha 7-type nicotinic acetylcholine receptors are unaffected. A nesthesiology 1997; 86:859–65Flood, P Ramirez-Latorre, J Role, L
Violet JM, Downie DL, Nakisa RC, Lieb WR, Franks NP: Differential sensitivities of mammalian neuronal and muscle nicotinic acetylcholine receptors to general anesthetics. A nesthesiology 1997; 86:866–74Violet, JM Downie, DL Nakisa, RC Lieb, WR Franks, NP
Wada E, Wada K, Boulter J, Deneris E, Heinemann S, Patrick J, Swanson LW: Distribution of alpha 2, alpha 3, alpha 4, and beta 2 neuronal nicotinic receptor subunit mRNAs in the central nervous system: A hybridization histochemical study in the rat. J Comp Neurol 1989; 284:314–35Wada, E Wada, K Boulter, J Deneris, E Heinemann, S Patrick, J Swanson, LW
Dineley-Miller K, Patrick J: Gene transcripts for the nicotinic acetylcholine receptor subunit, beta4, are distributed in multiple areas of the rat central nervous system. Brain Res Mol Brain Res 1992; 16:339–44Dineley-Miller, K Patrick, J
Flood P, Role LW: Neuronal nicotinic acetylcholine receptor modulation by general anesthetics. Toxicol Lett 1998; 100-101:149–53Flood, P Role, LW
Lin LH, Chen LL, Zirrolli JA, Harris RA: General anesthetics potentiate gamma-aminobutyric acid actions on gamma-aminobutyric acidAreceptors expressed by Xenopus oocytes: Lack of involvement of intracellular calcium. J Pharmacol Exp Ther 1992; 263:569–78Lin, LH Chen, LL Zirrolli, JA Harris, RA
Harrison N, Flood P: Molecular mechanisms of general anesthetic action. Sci Med 1998; 5:18–27Harrison, N Flood, P
Franks N, Lieb W: Molecular and cellular mechanisms of general anesthesia. Nature 1994; 367:607–14Franks, N Lieb, W
Little B, Chang T, Chucot L, Dill WA, Enrile LL, Glazko AJ, Jassani M, Kretchmer H, Sweet AY: Study of ketamine as an obstetric anesthetic agent. Am J Obstet Gynecol 1972; 113:247–60Little, B Chang, T Chucot, L Dill, WA Enrile, LL Glazko, AJ Jassani, M Kretchmer, H Sweet, AY
Idvall J, Ahlgren I, Aronsen KR, Stenberg P: Ketamine infusions: Pharmacokinetics and clinical effects. Br J Anaesth 1979; 51:1167–73Idvall, J Ahlgren, I Aronsen, KR Stenberg, P
Grant IS, Nimmo WS, Clements JA: Pharmacokinetics and analgesic effects of i. m. and oral ketamine. Br J Anaesth 1981; 53:805–10Grant, IS Nimmo, WS Clements, JA
Dayton PG, Stiller RL, Cook DR, Perel JM: The binding of ketamine to plasma proteins: Emphasis on human plasma. Eur J Clin Pharmacol 1983; 24:825–31Dayton, PG Stiller, RL Cook, DR Perel, JM
Giese JL, Stanley TH: Etomidate: A new intravenous anesthetic induction agent. Pharmacotherapy 1983; 3:251–8Giese, JL Stanley, TH
Sambrook J, Fritsch E, Maniatis T: Molecular Cloning. Plainview, Coldspring Harbor Press, 1989
Harrison NL, Kugler JL, Jones MV, Greenblatt EP, Pritchett DB: Positive modulation of human gamma-aminobutyric acid type A and glycine receptors by the inhalation anesthetic isoflurane. Mol Pharmacol 1993; 44:628–32Harrison, NL Kugler, JL Jones, MV Greenblatt, EP Pritchett, DB
Koltchine VV, Ye Q, Finn SE, Harrison NL: Chimeric GABAA/glycine receptors: Expression and barbiturate pharmacology. Neuropharmacology 1996; 35:1445–56Koltchine, VV Ye, Q Finn, SE Harrison, NL
MacDonald JF, Miljkovic Z, Pennefather P: Use-dependent block of excitatory amino acid currents in cultured neurons by ketamine. J Neurophysiol 1987; 58:251–66MacDonald, JF Miljkovic, Z Pennefather, P
Orser BA, Pennefather PS, MacDonald JF: Multiple mechanisms of ketamine blockade of N-methyl-D-aspartate receptors. A nesthesiology 1997; 86:903–17Orser, BA Pennefather, PS MacDonald, JF
Scheller M, Bufler J, Hertle I, Schneck HJ, Franke C, Kochs E: Ketamine blocks currents through mammalian nicotinic acetylcholine receptor channels by interaction with both the open and the closed state. Anesth Analg 1996; 83:830–6Scheller, M Bufler, J Hertle, I Schneck, HJ Franke, C Kochs, E
Mascia MP, Machu TK, Harris RA: Enhancement of homomeric glycine receptor function by long-chain alcohols and anaesthetics. Br J Pharmacol 1996; 119:1331–6Mascia, MP Machu, TK Harris, RA
McGehee DS, Role LW: Physiological diversity of nicotinic acetylcholine receptors expressed by vertebrate neurons. Annu Rev Physiol 1995; 57:521–46McGehee, DS Role, LW
Miller RD: Anesthesiology, 3rd Edition. New York, Churchill Livingstone, 1994
Levine LL, Winter PM, Nemoto EM, Uram M, Lin MR: Naloxone does not antagonize the analgesic effects of inhalation anesthetics. Anesth Analg 1986; 65:330–2Levine, LL Winter, PM Nemoto, EM Uram, M Lin, MR
Qian C, Li T, Shen TY, Libertine-Garahan L, Eckman J, Biftu T, Ip S: Epibatidine is a nicotinic analgesic. Eur J Pharmacol 1993; 250:R13–4Qian, C Li, T Shen, TY Libertine-Garahan, L Eckman, J Biftu, T Ip, S
Reich DL, Silvay G: Ketamine: An update on the first twenty-five years of clinical experience. Can J Anaesth 1989; 36:186–97Reich, DL Silvay, G
Jevtovic-Todorovic V, Todorovic SM, Mennerick S, Powell S, Dikranian K, Benshoff N, Zorumski CF, Olney JW: Nitrous oxide (laughing gas) is an NMDA antagonist, neuroprotectant and neurotoxin. Nat Med 1998; 4:460–3Jevtovic-Todorovic, V Todorovic, SM Mennerick, S Powell, S Dikranian, K Benshoff, N Zorumski, CF Olney, JW
Mennerick S, Jevtovic-Todorovic V, Todorovic SM, Shen W, Olney JW, Zorumski CF: Effect of nitrous oxide on excitatory and inhibitory synaptic transmission in hippocampal cultures. J Neurosci 1998; 18:9716–26Mennerick, S Jevtovic-Todorovic, V Todorovic, SM Shen, W Olney, JW Zorumski, CF
Franks NP, Dickinson R, de Sousa SL, Hall AC, Lieb WR: How does xenon produce anaesthesia (letter)? Nature 1998; 396:324Franks, NP Dickinson, R de Sousa, SL Hall, AC Lieb, WR
Golden W, Lavender R, Metzer W: Acute postoperative confusion and hallucinations in Parkinson’s disease. Ann Intern Med 1989; 111:218–22Golden, W Lavender, R Metzer, W
Fig. 1. Intravenous anesthetics, near clinical EC50, differentially modulate nAChRs expressed in Xenopus  oocytes. The modulation is concentration-dependent. (A  ) Ketamine 1 μm reversibly inhibits the maximal α4β4 nAChR response to 1 mm ACh by 80%. (B  ) Ketamine inhibition is concentration-responsive with an IC50 of 0.24 (± 0.3) μm and a Hill coefficient of 0.95 (± 0.1; n = 12). (C  ) Thiopental 25 μm reversibly inhibits the α4β4 nAChR response to ACh by 20%. (D  ) Thiopental at high clinical concentrations causes concentration-dependent inhibition with an IC50 of 84 (± 22) μm and a Hill coefficient of 0.75 (± 0.15; n = 12). (E  ) Etomidate 10 μm does not significantly inhibit the α4β4 nAChR response to ACh. (F  ) Etomidate at greater than clinical concentrations causes concentration-dependent inhibition with an IC50 of 33 (± 0.08) μm and a Hill coefficient of 2.1 (± 0.01; n = 12). Anesthetics were applied for 5 min before agonist–anesthetic coapplication.
Fig. 1. Intravenous anesthetics, near clinical EC50, differentially modulate nAChRs expressed in Xenopus 
	oocytes. The modulation is concentration-dependent. (A 
	) Ketamine 1 μm reversibly inhibits the maximal α4β4 nAChR response to 1 mm ACh by 80%. (B 
	) Ketamine inhibition is concentration-responsive with an IC50 of 0.24 (± 0.3) μm and a Hill coefficient of 0.95 (± 0.1; n = 12). (C 
	) Thiopental 25 μm reversibly inhibits the α4β4 nAChR response to ACh by 20%. (D 
	) Thiopental at high clinical concentrations causes concentration-dependent inhibition with an IC50 of 84 (± 22) μm and a Hill coefficient of 0.75 (± 0.15; n = 12). (E 
	) Etomidate 10 μm does not significantly inhibit the α4β4 nAChR response to ACh. (F 
	) Etomidate at greater than clinical concentrations causes concentration-dependent inhibition with an IC50 of 33 (± 0.08) μm and a Hill coefficient of 2.1 (± 0.01; n = 12). Anesthetics were applied for 5 min before agonist–anesthetic coapplication.
Fig. 1. Intravenous anesthetics, near clinical EC50, differentially modulate nAChRs expressed in Xenopus  oocytes. The modulation is concentration-dependent. (A  ) Ketamine 1 μm reversibly inhibits the maximal α4β4 nAChR response to 1 mm ACh by 80%. (B  ) Ketamine inhibition is concentration-responsive with an IC50 of 0.24 (± 0.3) μm and a Hill coefficient of 0.95 (± 0.1; n = 12). (C  ) Thiopental 25 μm reversibly inhibits the α4β4 nAChR response to ACh by 20%. (D  ) Thiopental at high clinical concentrations causes concentration-dependent inhibition with an IC50 of 84 (± 22) μm and a Hill coefficient of 0.75 (± 0.15; n = 12). (E  ) Etomidate 10 μm does not significantly inhibit the α4β4 nAChR response to ACh. (F  ) Etomidate at greater than clinical concentrations causes concentration-dependent inhibition with an IC50 of 33 (± 0.08) μm and a Hill coefficient of 2.1 (± 0.01; n = 12). Anesthetics were applied for 5 min before agonist–anesthetic coapplication.
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Fig. 2. Evidence for use dependence of ketamine inhibition of the α4β4 nAChR. (A  ) After 5-min pretreatment with ketamine 0.1 μm, repeated applications of ketamine plus agonist result in increased inhibition. Inhibition with the first coapplication is 20%, with the second is 30%, and with the third is 38%. (B  ) Ketamine inhibition of the α4β4 nAChR response to 1 mm ACh is more potent than ketamine inhibition of the response to 100 μm ACh. When ACh 100 μm is the agonist, the IC50for ketamine is 1.63 (± 0.28) μm, and the Hill coefficient is 1.38 (± 0.27).
Fig. 2. Evidence for use dependence of ketamine inhibition of the α4β4 nAChR. (A 
	) After 5-min pretreatment with ketamine 0.1 μm, repeated applications of ketamine plus agonist result in increased inhibition. Inhibition with the first coapplication is 20%, with the second is 30%, and with the third is 38%. (B 
	) Ketamine inhibition of the α4β4 nAChR response to 1 mm ACh is more potent than ketamine inhibition of the response to 100 μm ACh. When ACh 100 μm is the agonist, the IC50for ketamine is 1.63 (± 0.28) μm, and the Hill coefficient is 1.38 (± 0.27).
Fig. 2. Evidence for use dependence of ketamine inhibition of the α4β4 nAChR. (A  ) After 5-min pretreatment with ketamine 0.1 μm, repeated applications of ketamine plus agonist result in increased inhibition. Inhibition with the first coapplication is 20%, with the second is 30%, and with the third is 38%. (B  ) Ketamine inhibition of the α4β4 nAChR response to 1 mm ACh is more potent than ketamine inhibition of the response to 100 μm ACh. When ACh 100 μm is the agonist, the IC50for ketamine is 1.63 (± 0.28) μm, and the Hill coefficient is 1.38 (± 0.27).
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Fig. 3. Lack of voltage dependence of ketamine inhibition. Inhibition of α4β4 nAChR response to 1 mm ACh is not dependent on membrane holding potential between −30 and −60 mV (n = 16).
Fig. 3. Lack of voltage dependence of ketamine inhibition. Inhibition of α4β4 nAChR response to 1 mm ACh is not dependent on membrane holding potential between −30 and −60 mV (n = 16).
Fig. 3. Lack of voltage dependence of ketamine inhibition. Inhibition of α4β4 nAChR response to 1 mm ACh is not dependent on membrane holding potential between −30 and −60 mV (n = 16).
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Fig. 4. Ketamine does not modulate GABAAreceptor function in HEK 293 cells at clinically relevant concentrations. (A  ) Ketamine 10 μm does not modulate the α1β2γ2s GABAAreceptor. (B  ) Ketamine at higher-than-clinical concentrations causes concentration potentiation of GABA response with an EC50 of 1.2 mm (± 0.06 mm) and a Hill slope of 2.7 (± 0.3). Maximal efficacy of potentiation is 154% (± 4; n = 60).
Fig. 4. Ketamine does not modulate GABAAreceptor function in HEK 293 cells at clinically relevant concentrations. (A 
	) Ketamine 10 μm does not modulate the α1β2γ2s GABAAreceptor. (B 
	) Ketamine at higher-than-clinical concentrations causes concentration potentiation of GABA response with an EC50 of 1.2 mm (± 0.06 mm) and a Hill slope of 2.7 (± 0.3). Maximal efficacy of potentiation is 154% (± 4; n = 60).
Fig. 4. Ketamine does not modulate GABAAreceptor function in HEK 293 cells at clinically relevant concentrations. (A  ) Ketamine 10 μm does not modulate the α1β2γ2s GABAAreceptor. (B  ) Ketamine at higher-than-clinical concentrations causes concentration potentiation of GABA response with an EC50 of 1.2 mm (± 0.06 mm) and a Hill slope of 2.7 (± 0.3). Maximal efficacy of potentiation is 154% (± 4; n = 60).
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