Background

Isoflurane increases the apparent agonist affinity of ligand-gated ion channels. This action reflects a reduction in the receptor's agonist dissociation constant and/or the preopen/open channel state equilibrium. To evaluate the effect of isoflurane on each of these kinetic constants in the nicotinic acetylcholine receptor, the authors analyzed isoflurane's actions on (1) the binding of the fluorescent agonist Dns-C6-Cho to the nicotinic acetylcholine receptor's agonist self-inhibition site and (2) the desensitization kinetics induced by the binding of the weak partial agonist suberyldicholine.

Methods

The dissociation constant for Dns-C6-Cho binding to the self-inhibitory site was determined using stopped-flow fluorescence spectroscopy. The values of the kinetic constants for agonist binding, channel gating, and desensitization were determined by modeling the suberyldicholine concentration-dependence of the apparent rate of desensitization.

Results

Isoflurane did not significantly alter the dissociation constant for Dns-C6-Cho binding to the self-inhibitory site even at a concentration as high as 1.5 mM, the highest concentration studied. At this concentration, isoflurane substantially reduced the dissociation constant for suberyldicholine binding to its channel opening site by 97% from 17 +/- 5 microM to 0.5 +/- 0.2 microM, whereas the preopen/open channel state equilibrium was reduced only from 19.1 to 5 +/- 1.

Conclusions

Isoflurane increases the apparent agonist affinity of the nicotinic acetylcholine receptor primarily by reducing the agonist dissociation constant of the site responsible for channel opening rather than altering channel gating kinetics.

Topics:
agonists, desensitization, isoflurane, receptor desensitization, dissociation constant, affinity

THE nicotinic acetylcholine, γ-aminobutyric acidA(GABAA), glycine, and 5-hydroxytryptamine3receptors form a structurally and functionally related superfamily of ligand-gated ion channels. 1–3Upon agonist binding to a closed resting state, all members of this superfamily undergo a rapid conformational transition to an open channel state(s) that permits ion flux, followed by a slower conformational transition to a closed desensitized state(s). 4–10The processes of agonist binding and unbinding, channel gating, and desensitization may be most simply represented by the following generic kinetic scheme:SCHEME 1 

where A is the agonist, R is the resting state, AR is the preopen state, AO is the open channel state, AD is the desensitized state, K  dis the agonist dissociation constant for the resting state, K  ois the equilibrium constant between the preopen and open channel states ([AR]/[AO]), and k  desis the rate constant for desensitization. This simple scheme accounts for the observation that both ion flux and apparent desensitization rates increase before plateauing with agonist concentration and for the existence of partial agonists. Partial agonists are defined as agonists that open few channels even at receptor saturating concentrations (K  o> 1), whereas efficacious agonists open most channels at high concentrations (K  o< 1).

Volatile general anesthetics increase ion flux and/or apparent desensitization rates induced by low concentrations of agonist, shifting agonist concentration–response curves to the left. 11–16Within the context of scheme 1, this increase in apparent agonist affinity may reflect a decrease in either K  dor K  o. Unfortunately, direct measurements of K  dand K  ousing rapid agonist application techniques are difficult because the rate constants that define these equilibria are typically fast. 7,17,18 

The nicotinic acetylcholine receptor (nAcChoR) from Torpedo  is the only member of this superfamily that can be obtained in the multimilligram quantities required for detailed biophysical and biochemical analysis. Such studies have led to the development and testing of more precise kinetic schemes that provide a powerful framework for defining the effects of general anesthetics on a ligand-gated ion channel 19:SCHEME 2 

Scheme 2for the nAcChoR differs from generic scheme 1in several important ways. First, two agonist molecules must bind to the nAcChoR to open its channel. Second, an agonist molecule may also bind to the nAcChoR’s open channel state at a site that is distinct from those that induce channel opening, leading to inhibition of ion flux (A2BA). This is referred to as agonist self-inhibition and typically (but not always) occurs at high agonist concentrations. 20Finally, nAcChoRs may desensitize from all states that have agonist molecules bound to the two sites that cause channel opening (A2R, A2O, and A2BA). Ion flux studies suggest that the rate constants for Torpedo  nAcChoR desensitization in scheme 2, k  des1, k  des2, and k  des3, are similar and in the range of 2–7 s−1. 21–23The rates of desensitization from unliganded and singly liganded states and the rates of resensitization are much slower and not considered in this scheme. 24,25 

Provided that k  des1and k  des2are similar, scheme 2dictates that at noninhibiting concentrations, partial and efficacious agonists induce nAcChoR desensitization via  different pathways; efficacious agonists induce desensitization primarily through the open state (A2O → D), whereas partial agonists do so primarily through the preopen state (A2R → D). Consequently, efficacious agonists desensitize nAcChoRs with an apparent affinity that is strongly dependent on the value of K  o, whereas partial agonists do not. We reasoned that this critical difference in desensitization kinetics could be exploited to determine whether isoflurane increases the nAcChoR’s apparent agonist affinity primarily by reducing the nAcChoR’s agonist dissociation constant or, alternatively, by reducing the equilibrium constant between the preopen and open channel states.

In this study, we first validated scheme 2for describing the kinetics of nAcChoR desensitization induced by the very efficacious agonist acetylcholine (K  o= 0.039) and the weak partial agonist suberyldicholine (K  o= 19.1) over a range of agonist concentrations that spans five orders of magnitude. 19,26We confirmed that k  des1and k  des2are similar but found that k  des3is approximately twofold to fourfold faster. We then analyzed the effects of isoflurane on the kinetics of acetylcholine- and suberyldicholine-induced desensitization within the context of this scheme. We determined that over a wide range of agonist concentrations, a reduction in the value of K  d2, the microscopic dissociation constant for agonist binding to the lower affinity resting state site, primarily accounts for the effect of isoflurane on the apparent rates of nAcChoR desensitization.

Materials

Torpedo nobiliana  was obtained from Biofish Associates (Georgetown, MA), and diisopropylfluorophosphate, acetylcholine, and suberyldicholine were obtained from Sigma Chemical Co. (St. Louis, MO). The fluorescent 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.  27Isoflurane was purchased from Anaquest (Murray Hill, NJ).

Methods

Preparation, Characterization, and Isoflurane Exposure of nAcChoR-rich Membranes.

Receptor membranes were obtained from Torpedo nobiliana  electric organs and purified by sucrose density gradient centrifugation essentially as described by Braswell et al.  28Membranes were stored 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) at −80°C and thawed on the day of use. Acetylcholinesterase activity was inhibited by exposing membranes to 3.0 mM diisopropylfluorophosphate for 30 min before dilution with Torpedo  physiologic solution to obtain the desired receptor concentration. The number of agonist binding sites was determined from Dns-C6-Cho titrations as previously described. 29Solutions containing isoflurane were prepared from saturated Torpedo  physiologic solutions at room temperature assuming a saturated solubility of 15 mM. 30All kinetic experiments were performed at 20 ± 0.3°C.

Determination of the Dissociation Constant Defining Dns-C6-Cho Binding to the nAcChoR’s Agonist Self-inhibitory Site.

Receptor-rich membranes were loaded into one of the stopped-flow spectrofluorometer syringes (Applied Photophysics, Leatherhead, United Kingdom), and 10 mM acetylcholine along with Dns-C6-Cho (2–120 μM) were loaded into the other. Where appropriate, all solutions also contained isoflurane at the desired concentration. The two syringes were rapidly mixed (1 ms mixing time; 1:1 vol:vol) and the fluorescence intensity was recorded for 1 s. The excitation wavelength was 290 nm, and the monochromator bandpass was 5 nm. Fluorescence emission > 500 nm was measured through a high pass filter. In a typical experiment, three individual runs were signal averaged to reduce noise. In our experiments, the rapid addition of 5 mM acetylcholine (final concentration) saturates the two agonist binding sites on resting state nAcChoRs, leading to channel opening. Dns-C6-Cho, a fluorescent agonist that has a relatively high affinity for the agonist self-inhibitory site, then binds to the self-inhibitory site on the open channel state and induces a receptor conformational transition. 25,27This conformational transition produces an approximately exponential fluorescence enhancement whose rate was determined by fitting the recorded fluorescence trace to an exponential equation along with a linear component. 12The linear component of the traces represented < 10% of the total amplitude and was not analyzed in detail. The observed rate of this fluorescence enhancement, k  obs, varies with Dns-C6-Cho concentration according to the following equation 25:

where K  Bis the Dns-C6-Cho dissociation constant for the agonist self-inhibitory site, and k  fand k  bare the forward and backward rate constants, respectively, describing the conformational transition that is induced by Dns-C6-Cho binding to this site.

Determination of the Apparent Rate of Agonist-induced Desensitization.

The apparent rate of agonist-induced desensitization was determined with a double agonist pulse assay using the stopped-flow spectrofluorometer in the sequential mixing configuration as previously described. 12This assay permits the measurement of desensitization rates even in the presence of isoflurane, an anesthetic whose predominant effect is to block ion flux. Briefly, receptor membranes were loaded into one of the spectrofluorometer’s premix syringes, and agonist was loaded into the other premix syringe. The solutions were rapidly mixed (1:1 vol:vol) and allowed to preincubate for the desired time. The number of nondesensitized receptors that remained after preincubation with agonist was quantitated from the amplitude of the fluorescence enhancement observed when the nAcChoR/agonist solution is mixed with 10 μM Dns-C6-Cho and sufficient acetylcholine to open all remaining resting state nAcChoRs (5 mM). The apparent rate of desensitization was determined from an exponential fit of a plot of the fluorescence amplitude versus  preincubation time. At least 10 preincubation time points were used for the determination of each apparent rate. Where appropriate, all solutions also contained isoflurane at the desired concentration. The excitation wavelength was 290 nm, and the monochromator bandpass was 5 nm. Fluorescence emission > 500 nm was measured through a high pass filter. Fluorescence intensity was recorded for 500 ms after the second mixing step. In a typical experiment, three to five individual runs were signal averaged to reduce noise.

Statistical Analysis and Curve Fitting.

Data points on all figures represent the average of at least three determinations, and the error bars indicate the SD. Where the data points are larger than the errors, the error bars have been omitted. Data were fit with the analysis program Igor 3.01 (Wavemetrics, Lake Oswego, OR). The reported errors for the fitted parameters are the SDs derived from iterative curve fits.

Effect of Isoflurane on the Binding of the Fluorescent Agonist Dns-C6-Cho to the Agonist Self-inhibitory Site

Figure 1Ashows representative fluorescence traces recorded when receptor-rich membranes were rapidly mixed with 5 mM acetylcholine and either 1 μM, 5 μM, or 40 μM Dns-C6-Cho (final concentrations). For clarity, the traces have been normalized to the same maximal fluorescence amplitude. In this experiment, the rates of the fluorescence enhancement in the presence of 1 μM, 5 μM, and 40 μM Dns-C6-Cho were 9.4 ± 0.3 s−1, 14.1 ± 0.2 s−1, and 46.9 ± 0.5 s−1, respectively. Figure 1Bplots the rate of the fluorescence enhancement versus  Dns-C6-Cho concentration in the absence of isoflurane and in the presence of 0.5 mM, 1 mM, or 1.5 mM isoflurane. At all Dns-C6-Cho concentrations, the rate of the fluorescence enhancement in the presence of isoflurane was within 29% of the control rate obtained in the absence of isoflurane. The dissociation constant defining Dns-C6-Cho binding to the agonist self-inhibition site, K  B, was determined to be 26 ± 5 μM in the absence of isoflurane from a fit of the control data in figure 1Bto . The value of K  Bdid not vary systematically with isoflurane concentration and was 44 ± 5 μM, 26 ± 2 μM, and 36 ± 5 μM in the presence of 0.5 mM, 1.0 mM, and 1.5 mM isoflurane, respectively (fig. 1B, inset;table 1).

Agonist Concentration–Dependence of the Apparent Rate of Desensitization

Figure 2Aplots the apparent rate of nAcChoR desensitization as a function of acetylcholine concentration. Over the concentration range shown (1 μM to 1 mM), acetylcholine opens channels but does not inhibit ion flux. 20The apparent rate of desensitization increased with acetylcholine concentration before reaching a plateau of 3 s−1by 100 μM. At much higher, self-inhibiting concentrations of acetylcholine (> 30 mM), the apparent rate of desensitization increased further, reaching 7.1 ± 0.5 s−1by 300 mM acetylcholine (fig. 2B). Figures 3A and 3Bplot the apparent rate of nAcChoR desensitization as a function of suberyldicholine concentration using the same receptor preparation that was used in the acetylcholine experiments shown in figures 2A and 2B. The same membrane preparation was used in figures 2 and 3to compare more easily acetylcholine- and suberyldicholine-induced desensitization kinetics without the potentially confounding effects of variability between membrane preparations. Figure 3Aplots the apparent rate of desensitization as a function of suberyldicholine concentration over a concentration range that opens channels but does not inhibit them. 20The apparent rate of desensitization increased with suberyldicholine concentration before reaching a plateau of 3–4 s−1by 100 μM. At the high, self-inhibiting suberyldicholine concentrations emphasized in figure 3B(> 1 mM), the apparent rate of desensitization increased further to 11 ± 0.6 s−1by 100 mM.

Analysis of the Agonist Concentration–Dependence of the Apparent Rates of Desensitization within the Context of

Scheme 2 

Scheme 2predicts that the apparent rate of desensitization will vary with agonist concentration as follows (see appendix):

Agonist binding and electrophysiologic studies have shown that the value of K  d1is 100–1,000-fold lower than that of K  d2in Torpedo  nAcChoRs. 25,31,32Therefore, although the apparent rate of desensitization is highly dependent on K  d2, it is essentially independent of K  d1; for all agonist concentrations that are sufficiently high to produce appreciable binding to the lower affinity site, the higher affinity sites on resting state nAcChoRs will be nearly saturated with agonist. This explains our previous observation that the Hill coefficients for acetylcholine and carbamylcholine concentration–response curves for desensitization are near 1 rather than 2. 12In addition, because the maximal (plateau) apparent rates of desensitization induced by suberyldicholine and acetylcholine at noninhibiting concentrations are similar (3–4 s−1) in the absence of isoflurane and well as in its presence (see next section), the rate constants for desensitization via  the preopen and open states (k  des1and k  des2, respectively) are assumed to be similar in the absence of isoflurane and well as in its presence. Therefore, for our desensitization experiments, may be closely approximated as:

where the rate constants for desensitization from the preopen and open states are assumed to be equivalent and equal to k  des12.

Previous ion flux studies using Torpedo  nAcChoRs in native membranes have determined that in the absence of anesthetic, K  ovalues for acetylcholine and suberyldicholine are 0.039 and 19.1, respectively. 19,26The values of the remaining four kinetic constants were determined by iterative curve fitting of the biphasic data in figures 2 and 3to while keeping K  oconstant and equal to 0.039 (for acetylcholine) or 19.1 (for suberyldicholine). The values of the kinetic constants derived from these fits are given in table 2. For comparison, table 2also gives their values as previously determined using ion flux techniques assuming a similar kinetic model. The nearly complete overlap of the experimental data points by the curve fits to in figures 2 and 3and the close agreement between the binding constants determined using the analysis of desensitization kinetics and those previously determined using ion flux techniques confirms the validity of scheme 2and for describing the kinetics of agonist binding, channel opening, and desensitization.

Analysis of the Effect of Isoflurane on the Apparent Rates of Desensitization within the Context of

Scheme 2 

In a previous study, we analyzed the effect of isoflurane on acetylcholine-induced desensitization kinetics and determined that isoflurane increases the nAcChoR’s apparent affinity for acetylcholine. 12This is reflected in figure 4Aas an isoflurane-induced leftward shift in the concentration–response curve at noninhibiting acetylcholine concentrations. At a concentration of 1 mM, isoflurane reduced the nAcChoR’s apparent dissociation constant for acetylcholine (defined as the acetylcholine concentration that induces a half-maximal apparent desensitization rate) by 12-fold from 44 ± 4 μM to 3.6 ± 0.6 μM. By fitting the control data in figure 4Ato with K  oheld constant at 0.039, we determined the equilibrium and rate constants for this receptor preparation in the absence of anesthetic and report the results in the figure legend (dotted curve). Because we did not use acetylcholine at concentrations that are sufficiently high to inhibit channels in that study, we have taken the values for K  Band k  des3from table 2, which were obtained using another preparation. The solid and dashed curves in figure 4demonstrate the effect of reducing either K  d2and K  oby 12-fold. The complete overlap of the solid and dashed curves demonstrates that a 12-fold reduction in either K  d2or K  ocan equally well account for the 12-fold increase in the nAcChoR’s apparent affinity for acetylcholine. Smaller reductions in both K  d2and K  ocould similarly account for the increase in apparent affinity, indicating that there is no unique solution to when the very efficacious agonist acetylcholine is used as the desensitizing agonist (curves not shown). Conversely, predicts that equivalent reductions in K  d2and K  owill have dramatically different effects on the agonist concentration–dependence of the apparent rate of desensitization when using the weak partial agonist suberyldicholine (fig. 4B). Specifically, predicts that at channel-opening suberyldicholine concentrations, an isoflurane-induced reduction in K  d2will cause a relatively larger increase in the apparent desensitization rate than an identical reduction in K  o, whereas at channel-inhibiting suberyldicholine concentrations, the reverse is true. The significance of this observation is that it implies that the effects of isoflurane on K  d2and K  omay be distinguished by analyzing the kinetics of nAcChoR desensitization induced by suberyldicholine over a wide concentration range.

Figure 5plots the apparent rate of desensitization as a function of suberyldicholine concentration in the presence of 1.0 mM isoflurane along with the control rates obtained in the same preparation in the absence of anesthetic. Isoflurane substantially increased the apparent rates of desensitization induced by low concentrations of suberyldicholine but had a relatively small effect on that induced by high concentrations. The values of the rate constants in the presence of 1.0 mM isoflurane were determined by iterative curve fitting of the data in figure 5to . Because isoflurane concentrations even as high as 1.5 mM did not significantly affect the binding of the fluorescent agonist Dns-C6-Cho to the agonist self-inhibitory site on the nAcChoR (fig. 1B), we concluded that isoflurane does not significantly change K  Bfrom its control value determined in the absence of anesthetic. Therefore, K  Bwas held constant and equal to 0.5 mM for this membrane preparation, and the values of the remaining four kinetic constants were permitted to vary. The results of this fit are plotted in figure 5A, and the kinetic constants are given in table 3(preparation 2). This analysis indicated that the kinetic constant most affected by isoflurane was K  d2, which was reduced by more than an order of magnitude from 16 ± 7 μM to 0.9 ± 0.3 μM, whereas K  owas reduced only in half from 19.1 to 10 ± 1. The rate constants for desensitization, k  des12and k  des3, remained essentially unchanged.

Using two other receptor preparations, we also examined the effects of 0.5 and 1.5 mM isoflurane on the kinetics of suberyldicholine-induced desensitization (fig. 6and table 3). Using the approach previously detailed, we determined that 0.5 mM isoflurane reduced K  d2from 18 ± 9 μM to 9 ± 2 μM, whereas K  owas reduced from 19.1 to 13 ± 1. At a concentration of 1.5 mM, isoflurane reduced K  d2by 34-fold from 17 ± 5 μM to 0.5 ± 0.1 μM but reduced K  oonly from 19.1 to 5 ± 1. The rate constants for desensitization were little affected by isoflurane in either of these receptor preparations.

Isoflurane Minimally Affects the Binding of a Fluorescent Agonist to the Agonist Self-inhibitory Site

In the presence of channel-opening concentrations of acetylcholine (5 mM), the fluorescent agonist Dns-C6-Cho binds rapidly and with relatively high affinity to the nAcChoR’s agonist self-inhibitory site. By measuring the Dns-C6-Cho concentration–dependence of the observed rate of the resulting fluorescence enhancement, we determined that the dissociation constant of Dns-C6-Cho for this site (K  B) is 26 ± 5 μM in the absence of isoflurane. As predicted by scheme 2, this value equals the concentration of Dns-C6-Cho that is required to reduce by half the ion flux induced by maximally activating concentrations of acetylcholine (24 μM). 33In the presence of isoflurane, the observed rates of the fluorescence enhancement, K  B, k  f, or k  bdid not change systematically with isoflurane concentration. Consequently, we conclude that isoflurane has no significant effect on the kinetics of Dns-C6-Cho binding to the agonist self-inhibitory site. Furthermore, because we used isoflurane at concentrations that also inhibit ion flux in this study, the lack of any significant effect by isoflurane on the binding of Dns-C6-Cho to the agonist self-inhibition site strongly suggests that isoflurane and agonists (at self-inhibiting concentrations) inhibit ion flux by binding to distinct sites. 34 

Agonist Concentration–Dependence of the Apparent Rates of Desensitization and the Effect of Isoflurane

In our desensitization studies, we elected to use two agonists whose efficacies are markedly different; 96% of all receptors that bind two acetylcholine molecules open, whereas only 5% of receptors that bind two suberyldicholine molecules open. In addition, and importantly, self-inhibition is induced by both of these agonists only at concentrations that far exceed those required to open channels (in the absence of a transmembrane potential). 20These features allow us to readily interpret our desensitization data in terms of distinct kinetic pathways in scheme 2:

SCHEME 3 

At the noninhibiting concentrations shown in figures 2A and 3A, an agonist may induce desensitization via  pathways 1 and/or 2. For a given agonist, the principal kinetic pathway leading to the desensitized state is dependent on K  oand the relative values of k  des1and k  des2. In Torpedo  nAcChoRs, ion flux studies suggest that these two rate constants are not significantly different. Conversely, in mouse muscle nAcChoRs, k  des1<<k  des2as almost all desensitization proceeds through the open state. 35Our observation that the apparent rates of desensitization induced by noninhibiting concentrations of acetylcholine and suberyldicholine increase and then plateau at similar values of 3–4 s−1supports the conclusion that k  des1and k  des2are equal in nAcChoRs derived from Torpedo.  Thus, our kinetic model dictates that suberyldicholine will desensitize 95% of all receptors from the preopen state (pathway 1), whereas acetylcholine will desensitize 96% from the open state (pathway 2).

At the higher, self-inhibiting concentrations emphasized in figures 2B and 3B, both suberyldicholine and acetylcholine desensitize receptors from the self-inhibited state (pathway 3). 20Ion flux studies indicate that the apparent affinity of acetylcholine and suberyldicholine for the self-inhibitory site is on the order of 100 mM and 10 mM, respectively, in the absence of a transmembrane potential. 20At these agonist concentrations, we consistently observed that the apparent rate of desensitization induced by either suberyldicholine or acetylcholine increased beyond the plateau rate observed at noninhibiting agonist concentrations, indicating that in scheme 2, k  des3is greater than either k  des1or k  des2.

Having established that scheme 2can account for both ion flux and desensitization kinetics induced by a weak partial agonist and a very efficacious agonist over a very wide range of concentrations in the absence of anesthetic, we examined the effect of isoflurane on the agonist concentration–dependence of the apparent rate of desensitization within the context of that scheme. We demonstrated that our previously reported isoflurane-induced reduction in the nAcChoR’s apparent acetylcholine dissociation constant may be modeled as a quantitatively identical reduction in either K  d2or K  oor smaller reductions in the values of both. 12This occurs because for very efficacious agonists, the apparent agonist dissociation constant for desensitization (via  the open state) approximates the product of K  d2and K  o. Conversely, because suberyldicholine desensitizes nearly all nAcChoRs without ever opening them, the apparent agonist dissociation constant for desensitization (via  the preopen state) is relatively insensitive to changes in K  oand approximates K  d2. A fit of our 1 mM isoflurane data in figure 5to revealed that K  d2is reduced by 18-fold, whereas K  ois reduced only in half. Thus, within the context of scheme 2, the increase in the nAcChoR’s apparent affinity for suberyldicholine is explained almost entirely as an isoflurane-induced reduction in the agonist dissociation constant. By studying a range of isoflurane concentrations, it was evident that K  d2decreases with isoflurane concentration, consistent with our previous observation that isoflurane increases the apparent agonist affinity in a concentration-dependent manner. 12 

Dilger et al.  have used electrophysiologic techniques to define the effects of isoflurane on acetylcholine-activated ion flux through nAcChoRs expressed in BC3H-1 cells. 36They also observed that isoflurane increases the nAcChoR’s apparent agonist affinity. 36From measurements of macroscopic currents induced by the very rapid application of agonist and the analysis of single channel data, they tentatively concluded that this increase in apparent agonist affinity reflected a reduction in the agonist dissociation constant rather than a change in channel gating kinetics. 36,37Using a completely different approach, we have confirmed their tentative conclusion that isoflurane increases the apparent agonist affinity of the Torpedo  nAcChoR primarily by reducing the agonist dissociation constant. Although the specific kinetic step altered by isoflurane has not yet been elucidated in studies of the GABAAreceptor, a preliminary study by Li et al.  suggested that halothane reduces the rate at which GABA dissociates from it receptor binding site. 38Thus, volatile anesthetics may act similarly on both the nAcChoR and the GABAAreceptor by altering agonist binding kinetics.

1.
Stroud RM, McCarthy MP, Shuster M: Nicotinic acetylcholine receptor superfamily of ligand-gated ion channels. Biochemistry 1990; 29:11009–23
2.
Changeux JP: The TiPS lecture. The nicotinic acetylcholine receptor: An allosteric protein prototype of ligand-gated ion channels. Trends Pharmacol Sci 1990; 11:485–92
3.
Devillers TA, Galzi JL, Eisele JL, Bertrand S, Bertrand D, Changeux JP: Functional architecture of the nicotinic acetylcholine receptor: A prototype of ligand-gated ion channels. J Membr Biol 1993; 136:97–112
4.
Rang HP, Ritter JM: On the mechanism of desensitization at cholinergic receptors. Mol Pharmacol 1970; 6:357–82
5.
Cash DJ, Subbarao K: gamma-Aminobutyric acid (GABA) mediated transmembrane chloride flux with membrane vesicles from rat brain measured by quench flow technique: Kinetic homogeneity of ion flux and receptor desensitization. Life Sci 1987; 41:437–45
6.
Oh DJ, Dichter MA: Desensitization of GABA-induced currents in cultured rat hippocampal neurons. Neuroscience 1992; 49:571–6
7.
Jones MV, Westbrook GL: Desensitized states prolong GABAA channel responses to brief agonist pulses. Neuron 1995; 15:181–91
8.
Harty TP, Manis PB: Kinetic analysis of glycine receptor currents in ventral cochlear nucleus. J Neurophysiol 1998; 79:1891–901
9.
van Hooft JA, Vijverberg HP: Selection of distinct conformational states of the 5-HT3 receptor by full and partial agonists. Br J Pharmacol 1996; 117:839–46
10.
Yakel JL, Shao XM, Jackson MB: Activation and desensitization of the 5-HT3 receptor in a rat glioma x mouse neuroblastoma hybrid cell. J Physiol (Lond) 1991; 436:293–308
11.
Raines DE, Rankin SE, Miller KW: General anesthetics modify the kinetics of nicotinic acetylcholine receptor desensitization at clinically relevant concentrations. A NESTHESIOLOGY 1995; 82:276–87
12.
Raines DE, Zachariah VT: Isoflurane increases the apparent agonist affinity of the nicotinic acetylcholine receptor. A NESTHESIOLOGY 1999; 90:135–46
13.
Longoni B, Demontis GC, Olsen RW: Enhancement of gamma-aminobutyric acidA receptor function and binding by the volatile anesthetic halothane. J Pharmacol Exp Ther 1993; 266:153–9
14.
Mihic SJ, McQuilkin SJ, Eger EI II, Ionescu P, Harris RA: Potentiation of gamma-aminobutyric acid type A receptor-mediated chloride currents by novel halogenated compounds correlates with their abilities to induce general anesthesia. Mol Pharmacol 1994; 46:851–7
15.
Downie DL, Hall AC, Lieb WR, Franks NP: Effects of inhalational general anaesthetics on native glycine receptors in rat medullary neurones and recombinant glycine receptors in Xenopus oocytes. Br J Pharmacol 1996; 118:493–502
16.
Jenkins A, Franks NP, Lieb WR: Actions of general anesthetics on 5-HT3receptors in N1E-115 neuroblastoma cells. Br J Pharmacol 1996; 117:1507–15
17.
Liu Y, Dilger JP: Opening rate of acetylcholine receptor channels. Biophys J 1991; 60:424–32
18.
Matsubara N, Billington AP, Hess GP: How fast does an acetylcholine receptor channel open? Laser-pulse photolysis of an inactive precursor of carbamoylcholine in the microsecond time region with BC3H1 cells. Biochemistry 1992; 31:5507–14
19.
Wu G, Miller KW: Ethanol enhances agonist-induced fast desensitization in nicotinic acetylcholine receptors. Biochemistry 1994; 33:9085–91
20.
Forman SA, Firestone LL, Miller KW: Is agonist self-inhibition at the nicotinic acetylcholine receptor a nonspecific action? Biochemistry 1987; 26:2807–14
21.
Forman SA, Miller KW: High acetylcholine concentrations cause rapid inactivation before fast desensitization in nicotinic acetylcholine receptors from Torpedo. Biophys J 1988; 54:149–58
22.
Takeyasu K, Shiono S, Udgaonkar JB, Fujita N, Hess GP: Acetylcholine receptor: Characterization of the voltage-dependent regulatory (inhibitory) site for acetylcholine in membrane vesicles from Torpedo californica electroplax. Biochemistry 1986; 25:1770–6
23.
Walker JW, Takeyasu K, McNamee MG: Activation and inactivation kinetics of Torpedo californica acetylcholine receptor. in reconstituted membranes Biochemistry 1982; 21:5384–9
24.
Boyd ND, Cohen JB: Kinetics of binding of [3H]acetylcholine and [3H]carbamoylcholine to Torpedo postsynaptic membranes: Slow conformational transitions of the cholinergic receptor. Biochemistry 1980; 19:5344–53
25.
Raines DE, Krishnan NS: Transient low-affinity agonist binding to Torpedo postsynaptic membranes resolved by using sequential mixing stopped-flow fluorecence spectroscopy. Biochemistry 1998; 37:956–64
26.
Wu G, Tonner PH, Miller KW: Ethanol stabilizes the open channel state of the Torpedo nicotinic acetylcholine receptor. Mol Pharmacol 1994; 45:102–8
27.
Waksman G, Fournie ZMC, Roques B: Synthesis of fluorescent acyl-cholines with agonist properties: Pharmacological activity on Electrophorus  electroplaque and interaction in-vitro  with Torpedo  receptor-rich membrane fragments. Febs Lett 1976; 67:335–42
28.
Braswell LM, Miller KW, Sauter JF: Pressure reversal of the action of octanol on postsynaptic membranes from Torpedo. Br J Pharmacol 1984; 83:305–11
29.
Heidmann T, Changeux J-P: Fast kinetic studies on the interaction of a fluorescent agonist with the membrane-bound acetylcholine receptor from Torpedo marmorata . Eur J Biochem 1979; 94:255–79
30.
Firestone LL, Miller JC, Miller KW: Table of physical and pharmacological properties of anesthetics, Molecular and Cellular Mechanisms of Anesthetics. Edited by Roth SH, Miller KW. New York, Plenum Press, 1986, pp 455–70
31.
Sine SM, Claudio T, Sigworth FJ: Activation of Torpedo acetylcholine receptors expressed in mouse fibroblasts: Single channel current kinetics reveal distinct agonist binding affinities. J Gen Physiol 1990; 96:395–437
32.
Prinz H, Maelicke A: Ligand binding to the membrane-bound acetylcholine receptor from Torpedo marmorata : A complete mathematical analysis. Biochemistry 1992; 31:6728–38
33.
Rankin SE: Lipid-protein interactions and nicotinic acetylcholine receptor function, Biological Sciences (thesis). Oxford University, 1995, p 67–8
34.
Dilger JP, Vidal AM, Mody HI, Liu Y: Evidence for direct actions of general anesthetics on an ion channel protein: A new look at a unified mechanism of action. A NESTHESIOLOGY 1994; 81:431–42
35.
Auerbach A, Akk G: Desensitization of mouse nicotinic acetylcholine receptor channels: A two-gate mechanism. J Gen Physiol 1998; 112:181–97
36.
Dilger JP, Brett RS, Mody HI: The effects of isoflurane on acetylcholine receptor channels: 2. Currents elicited by rapid perfusion of acetylcholine. Mol Pharmacol 1993; 44:1056–63
37.
Dilger JP, Brett RS, Lesko LA: Effects of isoflurane on acetylcholine receptor channels: 1. Single-channel currents. Mol Pharmacol 1992; 41:127–33
38.
Li X, Pearce RA: Alteration of GABAA receptor kinetics by halothane is consistent with slowing the agonist microscopic unbinding rate (abstract). A NESTHESIOLOGY 1998; 89:A730

Appendix

The general approach for deriving the agonist concentration–dependence of the apparent desensitization rate has been previously reported. 19According to scheme 2, the rate of formation of desensitized receptors is equal to the sum of the rates of desensitization from the A2R, A2O, and A2DA states:

The equilibrium constants in scheme 2are defined as:

Substituting the equilibrium constants into A1 yields:

The total receptor concentration equals the sum of the concentrations of all of the individual receptor conformational states

This may be rewritten as

By rearrangement:

Substituting equation A3into A2yields:

Under conditions of excess agonist over receptor sites, receptor desensitization is a pseudo–first-order process characterized by an apparent rate constant, appk  des:

Rearrangement yields:

Substituting equation A4into equation A5yields equation 2in the Results section.