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

Xenopus laevis  female frogs were purchased from Xenopus I (Ann Arbor, MI) or Nasco (Fort Atkinson, WI). The use of experimental animals (frogs) was approved by the Animal Care and Use Committees of the Universities of Texas (Austin, Texas) and Colorado (Health Sciences Center, Denver, Colorado); all studies were performed following the appropriate guidelines. Acetylcholine chloride, atropine sulfate, collagenase type 1A, streptomycin/penicillin, gentamicin, and other reagents were purchased from Sigma Co. (St. Louis, MO). Isoflurane was obtained from Ohmeda Pharmaceutical Products (Liberty Corner, NJ). 1-chloro-1,2,2-trifluorocyclobutane (F3, 97%), 1,2-dicholorohexafluorocyclobutane (F6, 97%), and 2,3-chlorooctafluorobutane (F8, 97%) were from obtained from PCR Inc. (Gainesville, FL). XL-1 Blue cells were obtained from Stratagene (La Jolla, CA). The QIAFilter Maxi Kit was obtained from Qiagen (Chatworth, CA) and the mCAP mRNA capping kit was obtained from Stratagene (La Jolla, CA).

The hnAChR subunit cDNAs were provided by SIBIA Neurosciences (La Jolla, CA) in different expression vectors, α²and α³in pCMV-T7–3, α⁴and β⁴in pcDNA3 and β²in pSP64T. 20The cDNAs were transformed and amplified in XL-1 Blue cells and purified using the QIAFilter Maxi Kit. In vitro  transcripts were prepared using the mRNA capping kit.

Oocytes were obtained from Xenopus laevis  frogs kept in aquarium tanks at 19–21°C, on a 12-h light–dark cycle. Frogs were fed frog brittle (Nasco or Xenopus I) three times per week. Mature females were anesthetized by immersion for approximately 30 min in a 0.12% 3-aminobenzoic acid ethyl ester solution, a small incision was made in the abdominal wall, and a piece of ovary was removed and placed in Modified Barth's solution (88 mM NaCl, 1 mM KCl, 10 mM HEPES, 0.82 mM MgSO⁴, 2.4 mM NaHCO³, 0.91 mM CaCl², 0.33 mM Ca(NO³)², pH 7.5). Each frog underwent this procedure at most once a month.

To facilitate manual dissection of oocytes, a section of ovary was transferred from modified Barth's solution to a hypertonic buffer containing 108 mM NaCl, 2 mM KCl, 2 mM EDTA, 10 mM HEPES, pH 7.5, and theca and epithelial layers of mature oocytes (stages V and VI) were removed with surgical forceps. The follicular layer was removed by a 10-min immersion in 0.5 mg/ml collagenase in buffer containing 83 mM NaCl, 2 mM KCl, 1 mM MgCl², and 5 mM HEPES.

Oocytes were injected with 40 nl of diethyl pyrocarbonate-treated water containing 20–100 ng α^xβ^ysubunit combinations of cRNA in a 1:1 ratio. Injections were made with micropipettes (20 μm tip diameter) connected to a microdispenser (Drummond Scientific Co., Broomwall, PA) attached to a micromanipulator. After injection, oocytes were individually placed in wells of 96-well microtiter plates containing incubation medium (modified Barth's solution supplemented with 10 mg/l streptomycin, 10,000 U/l penicillin G, 50 mg/l gentamicin, 2 mM sodium pyruvate, and 0.5 mM theophylline) that was sterilized by passage through a 0.2-μm filter. Oocytes were incubated at 19°C for 3–5 days after injection.

Oocytes were placed in a rectangular chamber (approximately 100 μl) and perfused (2 ml/min) with buffer ND96 (96 mM NaCl, 2 mM KCl, 1 mM MgCl², 1.8 mM CaCl², 10 mM HEPES, pH 7.4) containing 1 μM atropine sulfate. In some experiments, the role of calcium influx through the nicotinic receptors was assessed by comparing responses obtained in ND96 with those obtained from calcium-free Ba²⁺–Ringer's (Ba-R) solution (115 mM NaCl, 2.5 mM KCl, 1.8 mM BaCl², and 10 mM HEPES, 1 μM atropine, pH 7.4). Oocytes were impaled with two glass electrodes (0.5–10 MΩ) filled with 3 M KCl and clamped at −70 mV using a Warner Instruments (Hamden, CT) oocyte clamp (model OC-725C).

Acetylcholine was applied for 20 s at 5-min intervals at an EC³⁰concentration in all experiments. Applications of 20 s were sufficient to allow currents to reach an equilibrium state in most oocytes (see Results). The EC³⁰for each subunit combination was calculated based on acetylcholine dose–response curves previously obtained in our laboratory (data not shown). Volatile compounds were preapplied for 2 min to allow complete equilibration in the bath and then immediately coapplied with acetylcholine for 20 s. Acetylcholine and anesthetic solutions were prepared on the day of the experiment. Saturated solutions of F3, F6, and F8 were obtained by 30-min bath sonication of 200 μl volatile anesthetic in 20 ml ND96 buffer. These solutions were equilibrated in a sealed vial protected from light for at least 24 h before use. The concentrations of the saturated solutions were 14.011 mM for F3, 0.225 mM for F6, and 0.035 mM for F8. Final solutions of those compounds were prepared from the respective saturated solution. The concentration in the figures represent bath concentrations that were calculated based on previously published values from our laboratory. 9All the experiments were performed at room temperature.

Results are expressed as percent of change from control responses, which were measured before and after each anesthetic application to account for possible shifts in the control current throughout the experiment. The “n” values refer to number of oocytes studied. Each experiment was performed with oocytes from at least two different frogs. Effects of anesthetics and nonimmobilizers were analyzed by one-sample Student t  test (against a theoretical mean of zero) and one-way analysis of variance using GraphPad Prizm software (San Diego, CA). Data are presented as mean ± SEM.

We first compared the time course of acetylcholine action and the concentration–response curves for acetylcholine using ND96 and calcium-free (Ba-R) solutions. The maximum effect of acetylcholine was achieved by 20 s (fig. 1), and this application time was used in all studies. The acetylcholine concentration–response curves were similar for ND96 and for Ba-R, indicating that calcium flux through the receptors did not influence acetylcholine potency (fig. 2).

We tested the effects of volatile anesthetic (isoflurane and F3) and nonimmobilizer (F6 and F8) compounds on the currents gated by acetylcholine in three different combinations of hnAChRs, α²β⁴, α³β⁴, and α⁴β², expressed in Xenopus  oocytes. Because working at high concentrations of agonist often causes problems, including desensitization of the receptors, an EC³⁰concentration of acetylcholine was used for all hnAChRs studied: 23 μM for α²β⁴, 127 μM for α³β⁴, and 0.5 μM for α⁴β².

For all subunit combinations tested, isoflurane inhibited acetylcholine-gated currents, and this inhibition was similar when experiments were performed using ND96 or Ba-R, indicating that calcium did not play a critical role in the actions of isoflurane (fig. 3). In Ba-R buffer, isoflurane markedly inhibited the maximal response to acetylcholine, with only small changes in the EC⁵⁰(fig. 4). Isoflurane, at a concentration corresponding to the minimum alveolar concentration (MAC, 320 μM), inhibited 83, 69, and 71% of acetylcholine-induced currents in the hnAChR subunit combinations α²β⁴, α³β⁴, and α⁴β², respectively, and the isoflurane IC⁵⁰values were 25, 56, and 82 μM, respectively (fig. 5). These receptors showed high sensitivity to isoflurane, with significant inhibition occurring at concentrations as low as 3.5 μM for α²β⁴and α³β⁴and 14 μM for α⁴β²nAChRs (Student t  test, P < 0.05).

F3 also inhibited all subunit combinations tested in a concentration-dependent manner (fig. 6; analysis of variance, P < 0.0001). 1 MAC F3 (0.8 mM) inhibited 64, 44, and 61% of currents gated in α²β⁴, α³β⁴, and α⁴β²hnAChRs, respectively. IC⁵⁰values for these receptors were 0.44, 1.17, and 0.33 mM, and all were significantly inhibited by the lowest concentration tested (100 μM, Student t  test, P < 0.05). Isoflurane (3.5–448 μM) and F3 (0.1–3.2 mM) did not activate any currents during the preapplication period in any of the hnAChRs tested.

F6, at concentrations corresponding to 0.5, 1.0, and 2.0 predicted MAC (8, 17, and 34 μM, respectively), did not induce any significant change in acetylcholine-gated currents in α²β⁴, α³β⁴and α⁴β²hnAChRs (fig. 7A; Student t  test, P < 0.05). F8, at concentrations ranging from 0.5 predicted MAC to 2.0 predicted MAC (4.4–18 μM), did not change the currents gated by α³β⁴and α⁴β²hnAChRs. F8 (4.4 μM and 18 μM) induced statistically significant potentiation of acetylcholine-gated currents in α²β⁴subunit combination (fig. 7B; 4.4 μM: 10 ± 4%; 18 μM: 17 ± 3%; Student t  test, P < 0.05). Despite the potentiation observed at 4.4 and 18 μM F8, 8.8 μM of this compound did not cause any changes in acetylcholine-gated currents in α²β⁴receptors (1.2 ± 2.7%, P > 0.05). Although statistically significant, the potentiation induced by F8 was very small and was not concentration-dependent.

Many proteins are affected by volatile anesthetics but are not necessarily important for anesthesia, e.g. , luciferase. 4One criterion for a role of a protein in the immobility component of anesthesia is that it should be sensitive to volatile anesthetics such as F3, but resistant to the structurally related nonimmobilizers F6 and F8. 8,9Previous studies asked whether non-neuronal nAChRs could differentiate among volatile anesthetics and nonimmobilizers, and conflicting results were found. Neuronal acetylcholine receptors from Torpedo nobiliana  were affected differentially by those compounds, 12but muscle nAChRs expressed in Xenopus  oocytes were inhibited by enflurane and by F6 and F8, although the inhibition by enflurane and the nonimmobilizers happened at distinct kinetic states of the receptor. 13In the current study, different combinations of neuronal hnAChR subunits were capable of differentiating among volatile anesthetics and nonimmobilizers. Therefore, there may be important differences in the intramolecular sites or mechanisms of anesthetic action between muscle and neuronal nAChRs, emphasizing the need for studies characterizing neuronal nicotinic receptors. Comparison of our results with other recent publications suggests that there are not marked species differences in the sensitivity of neuronal nAChRs to volatile anesthetics. For example, the IC⁵⁰for isoflurane inhibition of α⁴β²nAChRs was similar for receptors formed by subunits from rat, 18chicken, 19and human clones (current report). However, isoflurane inhibition of human nAChRs largely resulted from the noncompetitive mechanism, although acetylcholine dose–response curve was shifted slightly to the right, whereas chicken nAChRs were shown to be inhibited by the competitive antagonism. 19The human receptors have only recently been cloned, 20and our study provides the first data regarding anesthetic sensitivity of human nAChRs.

It is possible that the nonimmobilizers have a dual action on hnAChRs; that is, inhibition at the anesthetic site and enhancement at another site, and these effects could cancel each other, resulting in an apparent noneffect. However, it seems unlikely because a dual effect of those compounds has not been observed in other studies of ligand-gated ion channels. 3,10,22 

Neuronal acetylcholine receptors are part of the superfamily of ligand-gated ion channels and share with other members of the family differential responses to volatile anesthetics compared to nonimmobilizers. Conversely, one major distinction between nAChRs and the other members of this family is that GABA^A, glycine, and 5-HT³receptors are potentiated by anesthetics, 9,10,22but acetylcholine-induced currents in neuronal nAChRs are inhibited, as shown in the current study and other reports. 18,19It is important to emphasize that not all receptors differentiate among volatile anesthetics and nonimmobilizer compounds. Indeed, in a recent series of publications, Minami et al.  23–25demonstrated that metabotropic glutamate receptors (mGluR1, mGluR5), muscarinic m¹, and 5-HT^2Areceptors, which are all G-protein coupled, are inhibited by F3 and by F6 (albeit by distinct mechanisms), but not by F8. Considering that F6 suppresses learning in rodents, 26the authors suggested that G-protein–coupled receptors may be responsible for the amnesia caused by anesthetics.

Recently, it was shown that halothane and isoflurane both inhibit nicotine-induced release of dopamine by acting on presynaptic nAChRs in the rat striatum. 27Presynaptic neuronal nAChRs not only modulate release of dopamine, but also modulate the release of norepinephrine, glycine, GABA, 5-HT, acetylcholine, and glutamate. 17Therefore, inhibition of nAChRs by anesthetics may cause changes in the balance of neurotransmitter release at central levels, which could contribute to the mechanism of anesthesia.

In summary, heteromeric human neuronal nAChRs expressed in Xenopus laevis  oocytes were strongly inhibited by volatile anesthetics and were capable of differentiating between anesthetics and structurally related nonimmobilizers, suggesting the participation of these receptors in anesthesia.

The authors thank SIBIA Neurosciences for kindly providing the hnAChR subunit clones, Virginia Bleck and Kathryn J. Elliot for excellent technical assistance, and C. Fernando Valenzuela and Elizabeth L. Gonzales for many helpful discussions and suggestions.