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 y-aminobutyric acid type A (GABA(A)) receptor.
Nicotinic acetylcholine receptors were obtained from the electroplax organ of Torpedo nobiliana, and human GABA(A) receptors (alpha1beta2gamma2L) 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 GABA(A) 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.
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 GABA(A) receptor. At clinically relevant concentrations, cyclopropane and butane reduced the apparent rate of Torpedo nicotinic acetylcholine receptor desensitization induced by low concentrations of agonist.
Our results suggest that the in vivo central nervous system depressant effects of nonhalogenated alkane anesthetics do not result from their abilities to potentiate agonist actions on ligand-gated ion channels. Other targets or mechanisms more likely account for the anesthetic activities of nonhalogenated alkane anesthetics.
THE Torpedo nicotinic acetylcholine receptor (nAchR), γ-aminobutyric acid type A receptor (GABAAR), the glycine receptor, and 5-hydroxytryptamine3receptor together form an anesthetic-sensitive superfamily of ligand-gated ion channels (LGICs) that are critical for synaptic function. 1,2Members of this superfamily are widely considered to be the most important targets of general anesthetics and are frequently used in studies of anesthetic molecular mechanisms. 3–11Such studies have shown that, at clinically relevant concentrations (≤ 3 minimum alveolar concentration [MAC]), general anesthetics representing a wide range of chemical classes (including halogenated alkanes, halogenated and nonhalogenated ethers, and alcohols) increase ion flux or apparent desensitization rates induced by low agonist concentrations. When experiments have been performed using a range of agonist concentrations, this action has been shown to reflect a reduction in the receptor’s apparent agonist dissociation constant (Kd) (an increase in apparent agonist affinity). 3,8,12–15
Despite a large body of literature documenting the effects of anesthetics on a variety of LGICs, there have been no studies to assess the effects of nonhalogenated alkane anesthetics on any LGIC. Nonhalogenated alkanes, in particular cyclopropane, were commonly used in the past as clinical inhalational anesthetics before they were supplanted by nonflammable halogenated analogs. The goal of the current study was to test the hypothesis that agonist potentiation of LGICs might also play an important role in the clinical actions of nonhalogenated alkane anesthetics. This was achieved by assessing the abilities of cyclopropane and butane to increase the apparent agonist affinities of the two best characterized LGICs: the nAchR and the GABAAR. Our results reveal that, in contrast to nearly all other previously studied anesthetics, cyclopropane and butane do not potentiate agonist actions on LGICs at clinically relevant concentrations. This suggests that other molecular mechanisms account for the in vivo anesthetic activities of nonhalogenated alkane anesthetics.
Materials and Methods
Torpedo nobiliana was obtained from Biofish Associates (Georgetown, MA). Diisopropylfluorophosphate and acetylcholine were purchased from Sigma Chemical Co. (St. Louis, MO). Alkane anesthetics were purchased from Aldrich (Milwaukee, WI). The fluorescent partial agonist, [1-(5-dimethylaminonaphthalene)sulfonamido] n-hexanoic acid β-(N -trimethylammonium bromide) ethyl ester (Dns-C6-Cho), was synthesized according to the procedure of Waksman et al . 16
Preparation and Characterization of Nicotinic Acetylcholine Receptor–rich Membranes
Receptor membranes were obtained from freshly dissected T. nobiliana electric organs and prepared using sucrose density gradient centrifugation as described by Braswell et al. 17and in accordance with guidelines established by the Massachusetts General Hospital Animal Care Committee (Boston, MA). Membranes were stored at −80°C in Torpedo physiologic solution (250 mm NaCl, 5 mm KCl, 3 mm CaCl, 2 mm MgCl2, 5 mm NaH2PO4, and 0.02% NaN3, pH 7.0) and thawed on the day of use. Acetylcholinesterase activity was inhibited by exposing membranes to 1.0 mm diisopropylfluorophosphate for 60 min before dilution with Torpedo physiologic solution to the desired receptor concentration. The number of agonist binding sites was determined from Dns-C6-Cho titrations as previously described. 18
Determination of the Apparent Rate of Acetylcholine-induced Desensitization
The apparent rate of agonist-induced desensitization was determined with a double-agonist pulse assay using a sequential mixing stopped-flow spectrofluorometer (Applied Photophysics, Leatherhead, United Kingdom). 8In brief, receptor-rich membranes were first preincubated with acetylcholine for periods ranging from 15 ms to several minutes. The number of receptors able to be activated (nondesensitized) that remained after this preincubation period was then quantified from the amplitude of the rapid fluorescence signal observed when the membrane–acetylcholine solution was rapidly mixed with an assay solution containing the fluorescent partial agonist Dns-C6-Cho and a high, channel-activating concentration of acetylcholine (5 mm). In each experiment, receptor membranes (0.8-μm agonist binding sites) were loaded into one of the spectrofluorometer’s gas-tight premix syringes, and acetylcholine was loaded into the other gas-tight premix syringe. The solutions were rapidly mixed (1 ms mixing time; 1:1 vol:vol) and allowed to preincubate for the desired time. The nAchR–acetylcholine solution was then mixed (1 ms mixing time; 1:1 vol:vol) with an assay solution containing 10 mm acetylcholine and 20 μm Dns-C6-Cho, and the fluorescence emission was recorded. Where appropriate, solutions also contained anesthetic at the desired concentration. Solutions remained within a closed system made of inert materials (glass and Teflon), and anesthetic loss during experiments was negligible as determined by gas chromatographic analysis. An excitation wavelength of 290 nm was provided by a 150-W xenon arc lamp, and the monochromator bandpass was 5 nm. Fluorescence emission greater than 500 nm was measured through a high-pass filter. Fluorescence intensity was recorded for 500 ms after the second mixing step. All experiments were performed at 20 ± 0.3°C.
Cell Culture and Transfection with Human γ-Aminobutyric Acid Type A Receptor cDNAs
Human embryonic kidney cells (HEK 293; American Type Culture Collection, Rockville, MD) were cultured in minimum essential medium Eagle (ATCC), supplemented with 10% horse serum, 1% penicillin–streptomycin (all from Gibco BRL, Life Technologies, Grand Island, NY), and maintained at 37°C in a 5% CO2incubator. For transient transfection, the calcium phosphate precipitation technique was used as described by Chen and Okayama. 19Cells were plated on protamine-coated glass coverslips and transfected with cDNAs for the α1, β2, and γ2Lsubunits of human GABAAR in pCDM8 vectors with a wt/wt ratio of 1α:2β:5γ. Cells were cotransfected with an expression plasmid (πH3-CD8) for the lymphocyte surface antigen CD8-α, which was supplied by Dr. Gary Yellen (Harvard Medical School, Boston, MA). After transfection, cells were incubated for 36–72 h. Shortly before experiments, cells were incubated briefly with polystyrene microspheres, precoated with anti-CD8 antibody (Dynabeads M-450 CD8, Dynal, Great Neck, NY). Thus, cells expressing CD8 were decorated with beads and could be distinguished from untransfected ones. 20
γ-Aminobutyric Acid Type A Receptor Electrophysiology
For electrophysiology, the medium was replaced by an extracellular solution with 162 mm NaCl, 5.3 mm KCl, 0.67 mm Na2HPO4, 0.22 mm KH2PO4, 15 mm HEPES, 5.6 mm glucose, and 2 mm CaCl2, adjusted to a pH of 7.30 with NaOH. Patch pipettes were fire-polished, resulting in open tip resistances of 2–5 MΩ. Pipettes were filled with intracellular solution containing 140 mm KCl, 2 mm MgCl2, 11 mm EGTA, 10 mm HEPES, and 10 mm glucose, adjusted to a pH of 7.30 with KOH. GABA solutions were prepared shortly before the experiments. Recordings were performed at room temperature (20–22°C) using standard outside-out or whole cell patch clamp techniques. Patches or cells were clamped to −50 mV during recordings; series resistance was compensated. Currents through the patch clamp amplifier (Axopatch 200A; Axon Instruments, Foster City, CA) were filtered (eight-pole bessel, 2 kHz) and digitized at 5–10 kHz using commercial software (pClamp 8.0, Axon Instruments). Rapid superfusate switching (0.2–1.2 ms, measured with open pipette junction currents) was performed using a piezo-driven quad tube as previously described. 21Standard protocols repetitively applied different concentrations of GABA alone or in combination with cyclopropane, butane, isoflurane, or octanol at the desired concentrations as pulses lasting 1.5–2.5 s. When anesthetics were applied, they were applied simultaneously with GABA and without preincubation. A 20-s interval between pulses was used for recovery of channel activity from desensitization. Control currents elicited with GABA alone were checked before and after application of anesthetic. Data were analyzed only if precontrol and postcontrol peak currents differed less than 10%. Cyclopropane and butane were stored in airtight syringes during the experiments. All parts of the application system consist of glass or Teflon tubing material.
Neuronal Nicotinic and N -methyl-d-aspartate Receptor Studies
Xenopus frogs were maintained and treated in accordance with regulations specified by the Massachusetts General Hospital Animal Care Committee (Boston, MA). Methods for oocyte expression of ion channels have been previously described. 22Capped mRNAs were synthesized in vitro from linearized cDNA templates using SP6 or T7 RNA polymerase kits (Ambion, Austin, TX) and purified with affinity beads (BIO-101, Vista, CA). Oocytes were injected with mixtures of mRNAs (20–100 ng) encoding neuronal nAchR subunits α4and β2(wt/wt ratio = 1) or N -methyl-d-aspartate (NMDA) receptor subunits NR1b and NR2a (wt/wt ratio = 1) and incubated at 18°C for 48–96 h. For two-microelectrode electrophysiology, oocytes were placed in a 0.1-ml perfusion chamber at room temperature (20–22°C) and voltage clamped at −50 mV via glass electrodes filled with 3 m KCl (0.2–1 MΩ). Oocytes expressing neuronal nAchRs were superfused with calcium-free ND-96 (96 mm NaCl, 2 mm KCl, 1 mm BaCl2, 0.8 mm MgCl2, 10 mm HEPES, pH 7.6) at 2–3 ml/min, and currents were activated with buffer containing 200 μm acetylcholine. Oocytes expressing NMDA receptors were superfused in calcium- and magnesium-free ND-96 (MgCl2replaced with BaCl2) containing 10 μm glycine, and currents were activated with 100 μm NMDA. Near the middle of a 60-s agonist pulse, anesthetic–agonist solutions (delivered from closed glass syringe reservoirs via Telfon tubing and valves) were introduced for 12–16 s. Currents were digitized at 50–200 Hz and recorded on a personal computer.
Preparation of Anesthetic Solutions
Saturated solutions of cyclopropane or butane were made by bubbling the gases though the desired buffer solution (25–50 ml) within a 250-ml glass bottle sealed with a Teflon septum. A gas inlet and outlet line passed into the bottle though the septum. During the 1–2-h equilibration period at room temperature, solutions were continuously stirred to achieve a saturated aqueous solution. After equilibration, the outlet line was cleared by aspiration, and the saturated solutions were drawn into a gas-tight syringe that was then connected directly to the spectrofluorometer (for nAchR studies) or application system (for GABAAR, neuronal nAchR, and NMDA receptor studies) via a Teflon stopcock. In parallel experiments using the same technique, the concentrations of cyclopropane and butane in saturated solutions (10.6 and 1.1 mm, respectively) were determined by gas chromatography to be within 10% of that predicted by their aqueous:gas partition coefficients. 23Saturated solutions of isoflurane (15 mm) and octanol (4.5 mm) were prepared by mixing excess anesthetic with buffer overnight. To achieve the desired anesthetic concentration, saturated solutions were diluted with buffer within a gas-tight syringe.
Conversion of Minimum Alveolar Concentration to Aqueous EC50
The approach for converting MAC values into aqueous EC50values (and vice versa ) has been described by Franks and Lieb. 24All MAC values for inhalational anesthetics are for humans. 23For butane, MAC was approximated as the EC50value reported by Firestone et al. 23For ethanol and octanol, MAC values are for rats. 25
Each data point represents the mean of at least three measurements, and the error bars indicate the SD. Statistical analysis was performed using an unpaired Student t test and analysis of variance (Prism version 3.0, Graphpad Software, San Diego, CA). P < 0.05 was considered significant. Data points on acetylcholine concentration-apparent desensitization rate curves were fit to a Hill equation in the form: where kappis the experimentally determined apparent rate of desensitization at each acetylcholine concentration, kmaxis the maximum apparent rate of desensitization induced by high acetylcholine concentrations, Kdappis the nAchR’s apparent Kdfor acetylcholine, and n is the Hill coefficient. The reported errors for all fitted parameters are the SDs derived from the curve fit.
For electrophysiolgic experiments using the GABAAR, an analogous Hill equation was used: where I is the experimentally determined peak current at each GABA concentration, Imaxis the maximal peak current induced by high GABA concentrations, Kdappis the GABAAR’s apparent Kdfor GABA, and n is the Hill coefficient. Again, the reported errors for all fitted parameters are the SDs derived from the curve fit.
Anesthetic Action on the Nicotinic Acetylcholine Receptor’s Apparent Agonist Affinity
In the presence or absence of anesthetic, the apparent rate of Torpedo nAchR desensitization increased with acetylcholine concentration before reaching a maximum at high concentrations (fig. 1A). From the acetylcholine concentration dependence of the apparent rate of acetylcholine-induced desensitization, we calculated the receptor’s apparent Kdfor this agonist (equation 1). In the absence of anesthetic, Torpedo nAchRs in this membrane preparation had an apparent Kdfor acetylcholine of 8 ± 2 μm and a maximum rate of desensitization of 5.7 ± 0.3 s−1. Preequilibration with 5.3 mm cyclopropane (a one-half saturated aqueous solution and equivalent to 6.5 MAC) had no effect on either the apparent Kdfor acetylcholine or the maximal rate of acetylcholine-induced desensitization (7.9 ± 0.5 μm and 5.8 ± 0.1 s−1). Figure 1Bshows the acetylcholine concentration dependence of the apparent rate of Torpedo nAchR desensitization in the absence of anesthetic and in the presence of 9.3 mm cyclopropane (equivalent to 11.4 MAC) using a different receptor preparation. In the absence of anesthetic, Torpedo nAchRs in this membrane preparation had an apparent Kdfor acetylcholine of 9 ± 1 μm and a maximum rate of desensitization of 6.6 ± 0.3 s−1. Preequilibration with 9.3 mm cyclopropane reduced the apparent Kdfor acetylcholine by just 30% to 6 ± 1 μm (not statistically significant) and had no detectable effect on the maximal rate of desensitization.
Figure 2shows the effect of butane on the acetylcholine concentration–response curve for Torpedo nAchR desensitization. At a concentration of 0.55 mm (equivalent to 3 MAC), butane failed to reduce the Torpedo nAchR’s apparent Kdfor acetylcholine (10 ± 1 μm vs. a control value of 8 ± 2 μm) or to alter the maximal rate of desensitization (5.6 ± 0.2 s−1vs. a control value of 5.7 ± 0.3 s−1).
To further assess the agonist-potentiating abilities of cyclopropane and butane and to compare them with other anesthetics, we measured the abilities of anesthetics to increase the apparent rate of Torpedo nAchR desensitization induced by 1 μm acetylcholine, an EC10agonist concentration (fig. 3). We used a single-receptor preparation to more easily compare the effects of different anesthetics on the kinetics of agonist-induced desensitization without the potentially confounding effects of preparation-to-preparation variability on anesthetic sensitivity. At a concentration of 0.22 mm (0.8 MAC), isoflurane increased the apparent rate of acetylcholine-induced desensitization by 56% from a control value of 0.36 ± 0.05 s−1to 0.56 ± 0.01 s−1. The apparent rate of desensitization increased with isoflurane concentration, with no evidence of saturation over the concentration range studied. By 0.88 mm, the highest concentration studied, isoflurane increased the apparent desensitization rate to 1.4 ± 0.2 s−1, a fourfold increase over the control. Ethanol and octanol also increased the apparent rate of acetylcholine-induced desensitization at pharmacologically relevant concentrations and in a concentration-dependent manner. However, at relevant concentrations, cyclopropane and butane actually reduced the apparent rate of acetylcholine-induced desensitization rate. At higher, supraphysiologic (toxic) concentrations, the apparent desensitization rate was not significantly different from control.
Anesthetic Action on the γ-Aminobutyric Acid Type A Receptor’s Apparent Agonist Affinity
The inset in figure 4Ashows typical current traces induced by 3 μm GABA (an EC5agonist concentration) in the absence of anesthetic (control) and in the presence of 1.6 MAC cyclopropane. Cyclopropane did not potentiate currents induced by this low concentration of GABA. For comparison, the inset also shows the significant potentiating effect of 1.6 MAC isoflurane on currents using the same patch. The effect of 1.6 MAC cyclopropane on the GABAAR’s apparent agonist Kdwas determined by fitting plots of the normalized peak current as a function of GABA concentration in the presence or absence of cyclopropane to equation 2(fig. 4A). Using this analysis, it was determined that cyclopropane did not reduce the apparent agonist Kdas the apparent Kdvalues for GABA in the absence and presence of cyclopropane were 23 ± 3 and 33 ± 5 μm, respectively. The inset in figure 4Bshows typical current traces induced by 3 μm in the absence of anesthetic and in the presence of 1.6 MAC butane and 1.6 MAC octanol. Although octanol significantly potentiated currents induced by 3 μm GABA, butane had no effect. The plot in figure 4Bshows the effect of 1.6 MAC butane on the GABA concentration–response relation. In common with cyclopropane, 1.6 MAC butane failed to significantly potentiate currents induced by this low concentration of agonist or to significantly reduce the GABAAR’s apparent Kdfor GABA (18 ± 2 μm as compared with a control value of 23 ± 3 μm).
Inhibition of Neuronal Nicotinic Acetylcholine Receptors and N -methyl-d-Aspartate Receptors
Because our results indicated that nonhalogenated alkane anesthetics do not potentiate LGICs, we considered the possibility that they might act, instead, by inhibiting receptor function. To test this hypothesis, we determined whether cyclopropane and butane inhibit ion flux through neuronal nAchRs and NMDA receptors. Figures 5A and Bdemonstrate that clinically relevant concentrations of cyclopropane inhibit ion flux through neuronal nAchRs and NMDA receptors. Similarly, butane inhibits both neuronal nAchRs and NMDA receptors at clinically relevant concentrations (data not shown).
The nAchR and GABAAR are the two best characterized members of the superfamily of LGICs. Functional studies indicate that the binding of two agonist molecules induces resting state receptors to undergo a rapid conformational transition to an ion-permeable open-channel state. 26–28Prolonged agonist exposure induces nAchRs and GABAARs to undergo a slower conformational transition to one or more nonconducting desensitized state(s). 26,29,30For both receptor families, the processes of agonist binding, channel gating, and desensitization may be most simply represented by the following kinetic scheme: where A is the agonist, R is the closed resting state, A2R is the closed preopen state, A2O is the open channel state, and A2D is the closed desensitized state(s). As required by this scheme and demonstrated by our data, Torpedo nAchR apparent desensitization rates and GABAAR currents increase with agonist concentration before reaching a plateau. An apparent agonist Kdmay then be defined from such agonist concentration–response curves as the agonist concentration required to induce a half-maximal response. In the current study we used two different methods for determining the apparent agonist Kd. For the Torpedo nAchR, we used a nonflux fluorescence assay that determines the apparent agonist Kdfrom the apparent rate of acetylcholine-induced desensitization measured over a wide range of acetylcholine concentrations. This technique (rather than ion flux techniques) was chosen to measure the apparent agonist Kdbecause many anesthetics not only increase the Torpedo nAchR’s apparent agonist affinity, but also inhibit ion flux through it. 8,31,32Such inhibition can mask anesthetic-induced agonist potentiation when electrophysiologic or radiotracer flux techniques are used. 9For the GABAAR (which is not as potently blocked by anesthetics), we used a standard electrophysiologic approach and calculated the apparent agonist Kdfrom the GABA concentration dependence of the measured peak current. We chose cyclopropane and butane as representative nonhalogenated alkane anesthetics because human potency data are available for both of these anesthetics and because concentrations in excess of those required to induce anesthesia may be readily achieved.
We have previously demonstrated that in the Torpedo nAchR, isoflurane and normal alcohols increase the apparent rate of desensitization induced by low concentrations of agonist. 33Subsequent studies using a wide range of agonist concentrations demonstrated that these anesthetics induce a leftward shift in acetylcholine–response curves for desensitization. 8,9This is indicative of a reduction in the Torpedo nAchR’s apparent agonist Kd. In these studies, we found that anesthetics reduced the apparent agonist Kdby approximately half at clinical anesthetic concentrations. Furthermore, we observed that the apparent agonist Kddecreased logarithmically with anesthetic concentration without clear evidence of saturation even at relatively high concentrations. Consequently, high concentrations of isoflurane and normal alcohols can reduce the Torpedo nAchR’s apparent agonist Kdby 10–100-fold. One of the principle results of this study is that even at high concentrations that are more than sufficient to induce anesthesia, cyclopropane and butane fail to potentiate the effects of low agonist concentrations or to reduce the apparent agonist Kdof the Torpedo nAchR.
At clinically relevant concentrations, a wide range of general anesthetics also potentiate the actions of low GABA concentrations on GABAARs. 11In common with the Torpedo nAchR, studies using a range of agonist concentrations indicate that this potentiation reflects a reduction in the GABAAR’s apparent agonist Kd. 3,34Because GABA is a major inhibitory neurotransmitter in the central nervous system, it is thought that the central nervous system–depressant effects of general anesthetics may result from their abilities to potentiate GABA’s actions on GABAARs. In light of our data demonstrating that Torpedo nAchRs are unaffected by clinically relevant concentrations of cyclopropane and butane, and given the similarity in structure and function between the nAchR and GABAAR, we hypothesized that GABAARs might also be insensitive to clinically relevant concentrations of these cyclopropane and butane. To test this hypothesis, we studied the effect of 1.6 MAC concentrations of cyclopropane and butane on the apparent agonist affinity of the GABAAR. Figures 4A and Bdemonstrate that at these concentrations, both cyclopropane and butane fail to reduce the GABAAR’s apparent agonist Kd. Thus, not only are Torpedo nAchRs and GABAARs both sensitive to agonist potentiation by isoflurane and normal alcohols, they are both relatively insensitive to agonist potentiation by cyclopropane and butane. This correlation between anesthetic action on the Torpedo nAchR and GABAAR suggests that the underlying molecular mechanism(s) responsible for agonist potentiation in both superfamily members is the same. Furthermore, the inability of cyclopropane and butane to reduce the apparent agonist Kdof the GABAAR at a concentration that induces anesthesia strongly suggests that other molecular mechanisms likely account for their in vivo anesthetic activities. Although we have not yet studied the activities of nonhalogenated anesthetics on other superfamily members (5-hydroxytryptamine3or glycine receptors), the observation that they do not potentiate agonist actions on either the nAchR or the GABAAR suggests that they also fail to potentiate these LGICs. As these anesthetics potently inhibit neuronal nAchRs and NMDA receptors, these ion channels must be considered to be potentially relevant targets of nonhalogenated alkane anesthetics.
A recent study by de Sousa et al. 35identified xenon as another anesthetic that does not potentiate the GABAAR at a clinically relevant concentration (1.1 MAC). In an accompanying editorial, Harrison 36suggested that xenon may fail to potentiate agonist action because it is unable to participate in hydrogen-bonding interactions with the receptor. Such interactions, which are possible for ether, alcohol, and nearly all halogenated alkane anesthetics, have been suggested to augment binding affinity to a model protein by an order of magnitude. 37Our data demonstrating that at 1.6 MAC, cyclopropane and butane have no effect on the GABAAR’s apparent Kdis generally consistent with Harrison’s suggestion as these two anesthetics are also incapable of forming hydrogen bonds. Alternatively, Cafiso et al. and Pohorille et al. have suggested that an anesthetic’s dipole moment mediates its interactions with membrane targets. 38–41Because nonhalogenated alkanes (and xenon) are among the very few anesthetics that do not possess a permanent dipole moment, we cannot rule out a critical role for the dipole moment in modulating anesthetic action on the Torpedo nAchR and GABAAR.
In summary, our studies show that, at concentrations that are sufficient to induce anesthesia, neither cyclopropane nor butane significantly increases the apparent agonist affinities of two representative members of the superfamily of LGICs, the Torpedo nAchR and the GABAAR. Such findings strongly suggest that agonist potentiation of LGICs does not account for the anesthetic activities of nonhalogenated alkane anesthetics.
Dns-C6-Cho was synthesized by S. Shaukat Husain, D.Phil. (Research Associate in Anesthesia, Massachusetts General Hospital, Boston, Massachusetts); GABAAreceptor cDNAs were supplied by Paul J. Whiting, Ph.D. (Research Scientist, Merck Sharp & Dohme Research Labs, Essex, United Kingdom); neuronal nicotinic receptor subunit cDNAs were gifts from James Boulter, Ph.D. (Associate Professor of Psychiatry and Biobehavioral Sciences, Salk Institute, San Diego, California); NMDA receptor subunit cDNAs were gifts from Steven Triestman, Ph.D. (Professor of Pharmacology, University of Massachusetts Medical Center, Worcester, Massachusetts).