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The Role of Electrostatic Interactions in Governing Anesthetic Action on the Torpedo Nicotinic Acetylcholine Receptor

Raines, Douglas E., MD*†,; Claycomb, Robert J., BS

doi: 10.1213/00000539-200208000-00021
ANESTHETIC PHARMACOLOGY: Research Report
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Isoflurane and normal alkanols reduce the apparent agonist dissociation constant (Kd) of the nicotinic acetylcholine receptor (nAChR) at clinically relevant concentrations, whereas cyclopropane and butane do not. This suggests that electrostatic (hydrogen bonding and/or dipolar) interactions modulate anesthetic potency in this model receptor system. To further define the nature of these interactions, we quantified the potencies with which a heterologous group of general anesthetics reduces the nAChR’s apparent Kd for acetylcholine. We assessed the importance that an anesthetic’s molecular volume, ability to donate a hydrogen bond (hydrogen bond acidity), ability to accept a hydrogen bond (hydrogen bond basicity), and dipole moment play in determining aqueous potency. We found that aqueous anesthetic potency increases with molecular volume and decreases with hydrogen bond basicity but is unaffected by dipole moment and hydrogen bond acidity. These results suggest that anesthetics reduce the apparent agonist Kd of the nAChR by binding to a site that has a dipolarity and ability to accept hydrogen bonds that are similar to those of water, but a hydrogen bond-donating capacity that is less.

*Department of Anesthesia, Harvard Medical School; and †Massachusetts General Hospital, Boston, Massachusetts

Supported by the National Institutes of General Medical Sciences (Grant RO1-GM61927).

April 9, 2002.

Address correspondence and reprint requests to Dr. D. E. Raines, Department of Anesthesia, Massachusetts General Hospital, 32 Fruit St., Boston, MA 02114. Address e-mail to DRaines@partners.org.

The nicotinic acetylcholine receptor (nAChR) is the prototypical member of a structurally and functionally homologous, anesthetic-sensitive superfamily of ligand-gated ion channels that also includes the γ-aminobutyric acid-A receptor, the glycine receptor, and the 5-hydroxytryptamine-3 receptor (1,2). Because the structure and function of the nAChR have been better defined than those of any other ligand-gated ion channel, it has become a useful model for studying how anesthetics act at the molecular and receptor level (3–5). We have previously reported that isoflurane and normal alcohols reduce the nAChR’s apparent dissociation constant (Kd) for acetylcholine at physiologically relevant concentrations (5,6). Conversely, cyclopropane and butane do not reduce the nAChR’s apparent Kd for acetylcholine at concentrations that are sufficient to induce anesthesia (7). Because cyclopropane and butane have no hydrogen-bonding capacity or dipole moment, we suggested that these properties might modulate anesthetic action on the nAChR. In this study, we quantified the potencies with which a heterologous group of general anesthetics reduces the nAChR’s apparent agonist Kd in the hope of further defining the roles that these interactions play in modulating the agonist-enhancing effect of anesthetics on the nAChR.

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Methods

Torpedo nobiliana was obtained from Biofish Associates (Georgetown, MA). Diisopropylfluorophosphate and acetylcholine were purchased from Sigma Chemical Co. (St. Louis, MO). Isoflurane was purchased from Baxter Healthcare Corp. (Deerfield, IL). Xenon was obtained from BOC Gases (Murray Hill, NJ). All other anesthetics were obtained from Aldrich (Milwaukee, WI). The fluorescent agonist, [1-(5-dimethylaminonaph- thalene)sulfonamido] n-hexanoic acid β-(N-trimethyl- ammonium bromide) ethyl ester (Dns-C6-Cho), was synthesized according to the procedure of Waksman et al. (8).

Receptor membranes were obtained from freshly dissected T. nobiliana electric organs and prepared by using sucrose density gradient centrifugation as described by Braswell et al. (9). Acetylcholinesterase activity was inhibited by exposing membranes to 1 mM diisopropylfluorophosphate for 60 min. 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. The number of agonist binding sites was determined as previously described (10).

The nAChR’s apparent Kd for acetylcholine was determined by measuring the acetylcholine concentration dependence of the apparent rate of acetylcholine-induced desensitization. To avoid complications arising from anesthetic-induced channel blockade of ion flux, we used a double-agonist pulse fluorescence assay to follow the time course of desensitization by using a previously described protocol (5). In brief, receptor-rich membranes were first preincubated with acetylcholine for periods ranging from 15 ms to several minutes. The number of activatable (nondesensitized) receptors 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 a large, channel-activating concentration of acetylcholine (5 mM) and the fluorescent partial agonist Dns-C6-Cho. The apparent rate of acetylcholine-induced desensitization was then quantified by fitting a plot of the fluorescence amplitude versus preincubation time to an exponential equation. Where appropriate, each solution also contained anesthetic at the desired concentration. Solutions remained within a closed system made of Teflon and glass to minimize evaporative and absorptive loss. An excitation wavelength of 290 nm was provided by a 150-W xenon arc lamp, and the monochromator bandpass was 5 nm. Fluorescence emission of >500 nm was measured through a high-pass filter. Fluorescence intensity was recorded for 200–500 ms after the second mixing step. All experiments were performed at 20°C ± 0.3°C.

The nAChR’s apparent Kd for acetylcholine was derived from the acetylcholine concentration dependence of the apparent rate of desensitization by fitting plots of the apparent desensitization rate versus acetylcholine concentration to a Hill equation in the following form (5):

MATH where kapp is the experimentally determined apparent rate of desensitization at each acetylcholine concentration, kmax is the maximum apparent rate of desensitization induced by large acetylcholine concentrations, Kdapp is the nAChR’s apparent Kd for acetylcholine, and n is the Hill coefficient. An anesthetic’s SC50 is defined as the aqueous concentration that reduces the nAChR’s apparent Kd for acetylcholine in half and was quantified by fitting a plot of the log (apparent Kd for acetylcholine) versus anesthetic concentration, as previously reported (6).

Abraham et al. (11,12) have developed a scale of solute hydrogen bond acidity (α2H) and basicity (β2H) that allows one to objectively assess the abilities of anesthetic compounds to donate and accept, respectively, a hydrogen bond. Larger values of α2H and β2H indicate a greater hydrogen bond acidity and basicity, respectively. Table 1 lists the α2H and β2H values of all anesthetics used in this study. Inspection of this table reveals that ethane, propane, cyclopropane, butane, ethylene, and xenon are unable to participate in hydrogen bonding interactions as either donors or acceptors. Chloroform can act as a hydrogen bond donor because it has a hydrogen that is made acidic by the electron-withdrawing effect of neighboring chlorine atoms. However, chloroform cannot act as a hydrogen bond acceptor. Conversely, diethyl ether can act as a hydrogen bond acceptor but not a donor. Alkanols can act as both hydrogen bond donors and acceptors. Alkanethiols are structural analogs of alkanols; alkanethiols have a sulfur atom in place of an oxygen atom. This difference substantially reduces the abilities of alkanethiols to participate in hydrogen bonding interactions (13).

Table 1

Table 1

Although general anesthetics carry no net charge, many contain electronegative atoms that can induce an asymmetric electron distribution along covalent bonds. Such anesthetics possess a dipole moment and are frequently referred to as being dipolar. With the exceptions of isoflurane and cyclopropanemethanol, the dipole moments of all anesthetics used in this study are experimental values obtained from the literature (14). The dipole moments of isoflurane and cyclopropanemethanol and the molecular volumes of all compounds were estimated by using MacSpartan Pro (Wavefunction, Irvine, CA) on an Apple (Cupertino, CA) Macintosh G4 computer with ab initio molecular orbital calculations (3–21G basis set).

Minimum alveolar anesthetic concentration (MAC) values in a single species for all anesthetics were not available. However, species-dependent differences are expected to remain in a twofold range (15). MAC values for alkanols, alkanethiols, ethane, propane, cyclopropane, butane, cyclopentane, xenon, and diethyl ether are for rat, and those for chloroform and ethylene are for mouse (13,16–20). Each anesthetic’s MAC was converted to an aqueous 50% effective concentration (EC50) by using its aqueous/gas partition coefficient (13,16,17,20,21). The anesthetic EC50 for cyclopropanemethanol is the loss of righting reflex for tadpoles (22).

The significance that α2H, β2H, molecular volume (V), and dipole moment (Φ) play in determining anesthetic SC50 was assessed with the multiple linear regression approach described by Abraham et al. (23) and Abraham and Rafols (24) by using Equation 2: where a, b, c, and d are weighting coefficients that can be defined by multiple regression for the set of anesthetic compounds, and α2H, β2H, V, and Φ are given in Table 1.

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Results

Figure 1 plots the logarithm of the normalized apparent Kd for acetylcholine versus anesthetic concentration. It demonstrates that seven anesthetics (chloroform, diethyl ether, cyclopentane, propanethiol, butanethiol, pentanethiol, and cyclopropanemethanol) reduced the nAChR’s apparent Kd for acetylcholine in a concentration-dependent manner, without obvious signs of saturation over the concentration ranges studied and at pharmacologically relevant concentrations. For these drugs, the aqueous anesthetic concentration needed to reduce the apparent Kd in half (SC50) approximates the aqueous EC50 for in vivo anesthesia (SC50/EC50 = 1.0 ± 0.3;Table 2). An eighth anesthetic, butane, also reduced the nAChR’s apparent Kd for acetylcholine, but only at concentrations that exceed those required to induce anesthesia. Its SC50 was estimated by extrapolation and represented 120% of its aqueous saturated solubility. Five anesthetics did not reduce the nAChR’s apparent Kd for acetylcholine at saturated aqueous concentrations (xenon, ethane, ethylene, and propane) or at a concentration that was more than four times its EC50 for anesthesia (cyclopropane). For these anesthetics, SC50 values could not be reliably determined from these data.

Figure 1

Figure 1

Table 2

Table 2

Figure 2 demonstrates that although there was a linear relationship between the anesthetic potencies (1/SC50) and their molecular volumes (r2 = 0.64;n = 15 anesthetics), the aqueous potencies of relatively polar anesthetics (e.g., alcohols) were typically an order of magnitude lower than were those of nonpolar ones (e.g., alkanes and alkanethiols). The nature of this interaction was more specifically defined by assessing the contributions that anesthetic hydrogen bond acidity, hydrogen bond basicity, and dipole moment make in determining aqueous anesthetic potency, by using multiple linear regression analysis. We solved Equation 2 using the values of α2H, β2H, V, and Φ from Table 1 and the experimentally determined values for SC50 given in Table 2. This regression analysis indicated that neither anesthetic hydrogen bond acidity nor dipole moment contributed significantly to the aqueous potencies of these anesthetics, because the weighting coefficients a and d in Equation 2 were not significantly different from 0 (P = 0.3723 and P = 0.3251, respectively). However, anesthetic molecular volume and hydrogen bond basicity contributed very significantly (P < 0.0001 and P = 0.0012, respectively), producing the following equation:

Figure 2

Figure 2

The correlation between the observed potencies of polar and nonpolar anesthetics and those predicted by Equation 3 is plotted in Figure 3.

Figure 3

Figure 3

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Discussion

In this study, we have applied the multiple linear regression approach developed by Raines et al. (22) and Abraham et al. (23) to assess the contributions that hydrogen bond acidity, hydrogen bond basicity, molecular volume, and dipole moment make in determining aqueous anesthetic potency in a well characterized model receptor system, the nAChR. In this approach, a set of anesthetics is chosen that possesses a range of values for hydrogen bond acidity, hydrogen bond basicity, molecular volume, and dipole moment. Provided that these anesthetic chemical properties are not significantly cross-correlated, then each weighting coefficient in the regression equation provides a measure of the significance of each chemical property in defining anesthetic potency. A chemical property possessing a weighting coefficient that does not differ significantly from 0 does not influence anesthetic potency, whereas one that has a nonzero weighting coefficient does modulate potency. It is important to note that the anesthetic end point is expressed as an aqueous concentration. Therefore, if the putative anesthetic binding site and water, for example, accept hydrogen bonds equally well, then the weighting coefficient for anesthetic hydrogen bond acidity would be 0. In this case, an anesthetic’s ability to donate a hydrogen bond (i.e., its acidity) will not favor its partitioning into either the binding site or the water phase because both the binding site and water are equally capable of accepting a hydrogen bond. Consequently, this property will have no effect on the observed SC50. Conversely, if the binding site and water possess differential abilities to accept hydrogen bonds, then the weighting coefficient for α2H will be nonzero, and its sign will define whether aqueous anesthetic hydrogen bond acidity increases or decreases anesthetic potency.

Our results indicate that an anesthetic’s SC50 is independent of its hydrogen bond acidity and dipole moment but is strongly influenced by its molecular volume and hydrogen bond basicity. Specifically, SC50 decreases (potency increases) with increasing anesthetic molecular volume and decreasing basicity. Thus, if a single anesthetic binding site accounts for anesthetic action on the nAChR’s apparent Kd for acetylcholine, then our results suggest that it has a dipolarity and ability to accept a hydrogen bond that are similar to those of water but a hydrogen bond-donating capacity that is significantly less.

Five anesthetics in our study (xenon, ethane, ethylene, propane, and cyclopropane) failed to reduce the nAChR’s apparent Kd for acetylcholine either at a saturated aqueous concentration or at a concentration that was well in excess of its EC50 for anesthesia. It is noteworthy that Equation 3 predicts that xenon, ethane, ethylene, and propane will have SC50 values that are much greater than their anesthetic EC50 values and aqueous saturated solubilities (Table 3). This equation also predicts that cyclopropane’s SC50 will be nearly five times its anesthetic EC50 and will equal 65% of its saturated solubility. In this study, we did not assess the effect of a cyclopropane concentration this large, but we have previously observed that a near-saturating aqueous concentration of cyclopropane (88% saturation; 9.3 mM) induced a small (33%) reduction in the nAChR’s apparent Kd for acetylcholine (7).

Table 3

Table 3

In their previous study, Abraham et al. (23) used the multiple linear regression analysis approach to assess the importance of anesthetic chemical features in defining anesthetic potencies for acting on another model protein, the firefly luciferase enzyme. They found that the inhibitory potencies of anesthetics correlated positively with anesthetic molecular volume and negatively with hydrogen bond basicity, whereas neither hydrogen bond acidity nor dipolarity/polarizability influenced anesthetic aqueous potency at all. This is the same pattern that we observed for anesthetic-induced reductions in the apparent Kd of the nAChR, suggesting that the chemical characteristics of the molecular sites responsible for inhibiting firefly luciferase and reducing the apparent agonist Kd of the nAChR are similar.

In summary, we have quantified the aqueous potencies with which a heterologous group of general anesthetics reduces the apparent agonist Kd of the nAChR, and we analyzed the data with multiple linear regression analysis. We observed that aqueous potency increases with anesthetic molecular size, decreases with hydrogen bond basicity, and is unaffected by either hydrogen bond acidity or dipole moment. This result suggests that a single site responsible for this action would possess a hydrogen bond-accepting capacity and dipolarity that are similar to those of water but a hydrogen bond donating capacity that is less.

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