Intravenous Anesthetics Differentially Modulate Ligand-gated Ion Channels

The chick α4 and β4 nAChR subunit cRNAs were prepared from appropriate cDNAs in a modified PGH19 vector using standard techniques. 14The rat α1, β1, γ2s GABA^Asubunit 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 GABA^Areceptors) 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 GABA^Areceptors 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.

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.

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. 15The 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). 16The 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.

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).

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).

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).

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 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).

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. 17,18Ketamine inhibits the NMDA receptor at concentrations between 2 and 50 μm. 17These 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. 18,19As 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. 18Inhibition 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 GABA^Aor glycine receptors at clinically relevant concentrations (fig. 4). 20This is the first complete concentration–response curve for ketamine potentiation at a recombinant GABA^Areceptor, although potentiation at high concentrations has been shown previously. 6The 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, 21and 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. 22,23Analgesia with ketamine is present at blood ketamine concentrations of 0.08–0.32 μm. 11The anesthetics, which do not inhibit nAChRs in the clinical range, such as etomidate and propofol, are not analgesic. 22Inhibition 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. 24Analgesia 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.”25Ketamine is also the only anesthetic that does not potentiate GABA^Areceptors 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). 26–28Although 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 GABA^Aresponse. Both the native transmitter GABA and the benzodiazepines that act specifically at GABA^Areceptors 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 GABA^Apotentiation is at threshold. 1,15Drugs 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. 22,29A 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. GABA^Aaugmentation 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.