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Anesth Analg 2008; 106:846-857
© 2008 International Anesthesia Research Society
doi: 10.1213/ane.0b013e318162ca7c
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ANESTHETIC PHARMACOLOGY

Molecular Actions of Propofol on Human 5-HT3A Receptors: Enhancement as Well as Inhibition by Closely Related Phenol Derivatives

Martin Barann, PhD, Isabelle Linden, PhD, Stefan Witten, PhD, and Bernd W. Urban, PhD

From the Department of Anesthesiology and Intensive Care Medicine, University of Bonn, Bonn, Germany.

Address correspondence and reprint requests to Dr. Martin Barann, Department of Anesthesiology and Intensive Care Medicine, University of Bonn, Sigmund-Freud Str. 25, 53127 Bonn, Germany. Address e-mail to martin.barann{at}ukb.uni-bonn.de.


   Abstract
Top
 Abstract
Introduction
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
BACKGROUND: 5-Hydroxytryptamine type 3 (5-HT3) receptors are excitatory ligand-gated ion channels which are involved in postoperative nausea and vomiting. They are depressed by the anesthetic propofol, which, in contrast, enhances the activity of inhibitory ligand-gated ion channels such as {gamma}-aminobutyric acid type A receptors and glycine receptors. To investigate the molecular mechanisms responsible for these contrasting actions, we examined the kinetics of the action of propofol and its lesser hydrophobic derivatives 2-isopropylphenol and phenol on human 5-HT3A receptors.

METHODS: Human embryonic kidney 293 cells containing stably transfected cDNA of the human 5-HT3A receptor subunit were patch clamped (excised outside-out patches). Drugs were applied with a fast solution exchange system (within 2 ms) and their concentrations were determined by high performance liquid chromatography.

RESULTS: When applied in equilibrium (60 s before and during the 5-HT pulse), propofol inhibited human 5-HT3A receptors (IC50 = 18 ± 1.0 µM). In equilibrium, the less hydrophobic 2-isopropylphenol was surprisingly a similarly potent inhibitor of human 5-HT3A receptors (IC50 = 17 ± 3.2 µM), whereas phenol was considerably less potent (IC50 = 1.6 ± 0.2 mM). Varying the duration of drug application before currents were elicited, and then applying 5-HT still in the presence of the drug revealed that fast and slow processes contributed to the (equilibrium) effects of propofol ({tau}IN-1 = 35 ms and {tau}IN-2 = 4.8 s), 2-isopropylphenol ({tau}IN-1 = 64 ms and {tau}IN-2 = 6.6 s), and phenol ({tau}IN-1 < 10 ms, {tau}IN-2 = 20.4 s). When applied transiently together with 5-HT (open channel application), propofol depressed currents and accelerated the 5-HT-induced desensitization significantly, whereas, in contrast, 2-isopropylphenol and phenol increased currents and slowed desensitization. Slowed desensitization was also observed for 5-hydroxyindole (1 mM), a 5-HT derivative, but not for benzene. The fast effects of phenol, 2-isopropylphenol, and propofol were more pronounced when the 5-HT concentration was decreased from 30 to 3 µM, whereas the slow effects were not sensitive to 5-HT.

CONCLUSIONS: At least two separate inhibitory actions on 5-HT3A receptors could be identified for propofol, whereas the enhancing action seen for the two related smaller phenol derivatives could no longer be detected. 5-HT-dependent and 5-HT-independent interactions could be distinguished for all three drugs. Propofol was less potent than expected from its hydrophobic properties. Underlying mechanisms appear to involve the phenolic hydroxyl group, hydrophobic interactions, and steric restrictions.


   Introduction
Top
 Abstract
 Introduction
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There is good evidence that several, weak interactions with proteins are characteristic of anesthetic action, involving specific polar interactions and nonspecific hydrophobic interactions.1 Although not differing much in their hydrophobicity, intravenous anesthetics appear to be generally more potent than inhaled anesthetics, suggesting that additional polar interactions are responsible for the greater anesthetic potency.1 As a necessary step towards rational anesthetic drug design, functional groups that might increase anesthetic potency should be identified and distinguished from those portions of the molecule that have a potential of causing undesirable clinical side effects, such as emesis. Before this can be done, all chemical groups of an anesthetic molecule have to be characterized in terms of their ability to interact with functional proteins.

Propofol is clinically the most extensively used IV anesthetic. Compared with other IV anesthetics, such as barbiturates or etomidate, the molecular structure of propofol is simple and symmetric (Fig. 1), consisting of a plain benzene ring and a phenolic hydroxygroup located between two identical alkyl residues. Thus, any systematic study of IV anesthetic interactions may start with this clinically important drug. For reasons not yet understood, propofol, on the molecular level, suppresses ion flux through excitatory ion channels but it enhances ion flow through inhibitory ion channels1–4 Propofol has the advantage over inhaled anesthetics of causing less emesis, if any at all.5


Figure 124
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  		Figure 1. (a) Structures of phenols and benzene with their respective experimental log P values, taken from the PhysProp Database (Syracuse Research Corporation; http://www.syrres.com/esc/physdemo.htm), see also Tetko et al.16 (b) Structure of 5-hydroxyindole (left) and 5-HT (right).

 

The 5-HT3 receptor is a ligand-gated excitatory ion channel6,7 which is located in the periphery and within the central nervous system. It plays a major role in the modulation of nausea and vomiting,8,9 consistent with the fact that 5-HT3 receptor antagonists are commonly used antiemetic drugs.10 The amino acid sequences of the pentameric 5-HT3 receptors show homologies with excitatory nicotinic acetylcholine receptor channels,11 and, to some extent, even with inhibitory {gamma}-aminobutyric acid type A (GABAA) receptors and glycine receptors. 5-HT3A receptors are the only functional homopentameric 5-HT3 receptors, and because of their well defined stoichiometry, they provide a useful model studying the molecular actions of drugs such as general anesthetics and cannabinoids.12,13

A previous study has shown that propofol suppresses 5-HT3 receptors (mouse) by more than one action, reducing the peak current amplitude and accelerating the desensitization process.14 In the present study, we used an experimental approach that allowed us to differentiate kinetically between different actions of propofol on human 5-HT3A receptors. Using the patch-clamp technique (excised outside-out patch mode) and a fast solution exchange system, we aimed (a) to test whether the human 5-HT3A receptor is also a sensitive target for propofol; (b) to identify separate components of action by, first, applying propofol to different conformations of the 5-HT3 receptor and, second, by comparing its actions with structurally related derivatives (Fig. 1); and, thus, (c) to shed light on the different responses to propofol by excitatory and inhibitory ligand-gated ion channels.


   METHODS
Top
 Abstract
 Introduction
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture
A human embryonic kidney (HEK)-293 cell line containing stably transfected human 5-HT3A receptors was used.12 HEK-293 cells were grown as monolayer on culture plates (Nunc) in DMEM Nutrient Mix F12 medium containing 10% heat inactivated fetal calf serum, penicillin (100 IU/mL), streptomycin (100 µg/mL), geneticin (0.75 mg/mL), and glutamine (292 µg/mL). Cells were cultured at 37°C in humidified atmosphere (5% CO2). For patch clamp experiments, cells were subcultured in monodishes (Nunc, 35 mm diameter) 7–11 days before an experiment.

Drugs and Solutions
5-HT (the creatinine sulfate salt) was obtained from Sigma (München, Germany). Propofol (free substance, without vehicle) was obtained from RBI (MA, USA), propofol derivatives, 5-hydroxyindole, and benzene from Sigma (München, Germany). 5-HT solutions were prepared daily from 25 mM aqueous stock solutions stored at –20°C. Propofol and 2-isopropylphenol were prepared daily from ethanol stock (1 M) by dilution (e.g., 1:10,000 resulting in 100 µM) in extracellular buffer and stirring for 3 h in glass bottles. The highest resulting ethanol concentrations at the highest drug concentrations were 2.2 mM (propofol) and 6.6 mM (2-isopropylphenol); they had no effect on 5-HT control currents. Ethanol concentrations were proportionally lower at lower drug concentrations. Benzene, phenol, and 5-hydroxyindole were soluble in the extracellular buffer without any further vehicle. All concentrations of the lipophilic drugs propofol and 2-isopropylphenol (log P = 3.79 and 2.88, respectively) were measured by high performance liquid chromatography [HPLC,15]. For this purpose, samples of drug solution were collected from the tubes directed to the patches of the application system. It was found, for example, that near the IC50 value an expected concentration of 25 µM propofol resulted in (mean ± standard deviations) 14 ± 2.5 µM (n = 40) propofol and 30 µM 2-isopropylphenol resulted in 21 ± 4.7 µM (n = 11).

Electrophysiology
In previous studies, we described human 5-HT3A receptors as activated by 5-HT in a concentration-dependent manner (EC50 values, 5–9 µM) and that the resulting currents were sensitive to the 5-HT3 receptor antagonist, ondansetron. For the experiments in this study, we used identical conditions, including electrophysiological methods, solution application systems,12,13 and a concentration of 30 µM 5-HT (unless specified otherwise) because it elicits a reproducible, nearly maximal (85%) signal.

Before starting patch-clamp recordings, the culture medium was replaced by "extracellular" solution of the following composition: NaCl 150 mM; KCl 5.6 mM; CaCl2 1.8 mM; MgCl2 1 mM; HEPES 10 mM; d-Glucose 20 mM; pH 7.4. d-Glucose was omitted in the extracellular solution used for superfusion of the excised patches. Patch pipettes were filled with intracellular solution containing: KCl 140 mM; EGTA 10 mM; MgCl2 5 mM; HEPES 10 mM; pH 7.4 and had resistances of 2–4 M{Omega}. Experiments were performed at room temperature (20°C–26°C).

Patch pipettes were manufactured from Borosilicate glass capillaries (Kwik-FilTM, World Precision Instruments, USA) using a pipette puller (List L/M-3P-A, List Electronic, Darmstadt, Germany). The seal resistances (excised outside-out patches) were 1–6 G{Omega}. For current measurements, we used a patch-clamp amplifier (EPC-7, List) in combination with an external low pass filter set at 1 kHz (Frequency Devices, MA). Data were digitally recorded at a sampling rate of 2 kHz with a Digidata 1200 (Axon Instruments, Foster City, CA) interface. Clampex-6 software (Axon) was used for the recording protocols. Five-hundred milliseconds before 5-HT exposure, the membrane potential was stepped from a holding value of 0 mV to –100 mV. These conditions were chosen to optimize the stability of the excised patches and the reproducibility of results.

Fast Solution Exchange System
A multitube perfusion system with an exchange rate (solution exchange) below 2 ms (RSC 200, Biologic, France) was used.12 The drug application systems were equipped with five separate tubes and inert materials, such as Teflon tubing and glass, to avoid loss of hydrophobic drugs.15

Drug Application Modes
Three different protocols of drug application were used12:

  1. Equilibrium application: Continuous exposure to the drug 60 s before and during the application of 5-HT;
  2. Open channel application: No drug application prior to the 5-HT pulse, only simultaneous application of drug and 5-HT;
  3. Closed channel application: Pre-exposure to the drug 60 s before but not during the 5-HT application.

Limitations of Data Aquisition
Although more than one measurement could be performed on a typical patch, the interval between measurements had to be at least 1 min because of the slow kinetics of recovery from desensitization. To correct for small rundown of currents when present and to discard inconsistent data, the measurement of each data point involved a three-fold repetition of the sequence: control, drug test, recovery (=control for next drug test), lasting a minimum of six min. The limited but varying life times of such patches (normally ranging from 5 to 25 min, in a few instances 1 h) resulted in inhomogeneous data sets with regard to the number of different data points measured on each patch. However, each data point was measured on at least three different patches. As the recording of entire concentration–response curves on single patches was not possible, mean IC50 values and their standard deviations could not be calculated. Instead, IC50 values and estimates of their standard errors were calculated from fitting a single concentration–response curve to the entire data set (Graph Pad Prism 3.02).

Highly hydrophobic substances have a tendency to absorb in the tubing,15 and, therefore, drug concentrations could not be randomized. Most experiments were started with the lowest hydrophobic drug concentrations and continued with increasingly larger concentrations. Otherwise, the superfusion system would have had to be extensively rinsed and rechecked for remaining drug after each experiment, requiring unrealistic experimental time for each experiment. Instead, a second, very different superfusion system and set-up was used to check whether there was any indication that the sequence (history) in which experiments were performed had any impact on the outcome. Whenever evidence for dependence on "history" was found, the underlying reasons were investigated. Only in artifactual situations was a dependence on history found, otherwise there was no evidence or suggestion that the sequence in which experiments were performed matters.

As this was an exploratory study, the number of experiments needed to reach statistical significance could not be estimated in advance with a power analysis.

Partition Coefficients and Predictions of Potency from Meyer-Overton Correlations
The following Meyer-Overton correlations1 have been used to estimate anesthetic potencies arising only from nonspecific, hydrophobic actions. For ligand-gated ion channels pooled for a wide range of anesthetics (Fig. 12b in Ref. 1), the parameters were: log (IC50) = –1.568 to 0.9898 * log (Poctanol/water); for ligand-gated ion channels pooled for inhaled anesthetics only (Fig. 12d in Ref. 1): log (IC50) = –1.178 to 0.8975 * log (Poctanol/water); for voltage-gated ion channels pooled for IV anesthetics only (Fig. 12c, published in Ref. 1): log (IC50) = –0.8064 to 0.9306 * log (Poctanol/water). Experimental octanol/water partition coefficients, Poctanol/water, are taken from the PhysProp Database (Syracuse Research Corporation; http://www.syrres.com/esc/physdemo.htm), see also Tetko et al.16

Data Analysis and Statistics
Analysis of the original current traces (baseline adjustment, determination of peak currents and current kinetics) was performed with Clampfit 8 software (Axon Instruments, Foster City, CA). Graph Pad Prism 3.03 software (Graph Pad, CA) was used to create graphics. The concentration–response curves were fitted by the Hill equation, i = [ICn50/(cn + ICn50)], i is the remaining peak current as fraction of the maximal (control) current, c is the drug concentration, n is the Hill coefficient, and IC50 is the drug concentration causing half-maximal effect. Potencies are expressed as IC50 values plus standard error as calculated by Graph Pad Prism 3.03. Differences between single data points were tested for significance with either paired or unpaired t-tests (Excel and Prism 3.03). Differences were considered significant when P values for the respective test were <0.05. Values are reported as means ± standard deviations (sd), unless stated otherwise.


   RESULTS
Top
 Abstract
 Introduction
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Propofol (0.5–160 µM) did not cause an activation of human 5-HT3A receptors in the absence of 5-HT when it was applied to patches which before had shown a pronounced response to 5-HT. Propofol inhibited 5-HT-induced currents reversibly with an IC50 = 18 ± 1 µM (Figs. 2a and 3) when applied under equilibrium condition (see Methods). Next, we examined the kinetics of washing in the propofol effect. Pre-exposing the patch to propofol for various durations (32 ms to 60 s) before applying 5-HT in the presence of propofol resulted in a wash-in time course of propofol that was characterized by a fast ({tau}IN-1 = 35 ms) and a slow ({tau}IN-2 = 4.8 s) process (Fig. 2b).


Figure 224
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  		Figure 2. (a, c, e; left panels) Effects of propofol, 2-isopropylphenol, and phenol (equilibrium application) on 5-HT (30 µM)-induced currents (original traces from three patches). Note that all three drugs caused suppression of the peak current; however, propofol speeded current decay, whereas 2-isopropylphenol and phenol slowed current decay (statistical analysis in Fig. 5). (b, d, f; right panels) Time courses of wash-in of drugs. Currents induced by 5-HT (30 µM) in the presence of drug are plotted against the time duration for which the 5-HT3A receptors were pre-exposed to the respective drug. Data points (circles) represent percent of the control currents after 60 s wash with propofol-free buffer (means ± sd, n = 4–10 different experiments). A fast and slow inhibitory process was observed for propofol, and a fast potentiating and a slow inhibitory effect was detected for 2-isopropylphenol. Phenol also showed a slow inhibitory effect, whereas potentiation was immediate. Wash-in curves for propofol and 2-isopropylphenol were fit biexponentially: i = imax1 * exp(–t/{tau}IN-1) + (100-imax1 – Plateau) * exp(–t/{tau}IN-2) + Plateau. The wash-in curve for phenol was fit monoexponentially: i = imax * exp(–t/{tau}IN) + Plateau.

 


Figure 324
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  		Figure 3. Concentration–response curves for three phenols on 5-HT (30 µM)-induced peak-currents. The bottoms and tops of the fits were set constant at 0% and 100%, respectively. Drugs were applied in equilibrium (means ± sd, n = 4–10 different patches).

 
In an attempt to further characterize the slow and the fast wash-in component of the propofol effect, 2-isopropylphenol and phenol were examined. Both substances are straight derivatives of propofol, in which the balance of polar and hydrophobic properties is shifted towards lesser hydrophobicity. In the absence of 5-HT, neither 2-isopropylphenol (0.01–1 mM) nor phenol (0.1–3 mM) caused direct activation of 5-HT3A receptors in patches which in a preceding control had shown pronounced response to 5-HT. When applied continuously (equilibrium condition), both drugs inhibited 5-HT-induced currents as can be seen in the traces of Figure 2c and e. As a third similarity with propofol, wash-in experiments (drug application for varying durations before 5-HT and during 5-HT exposure) revealed that fast and slow processes (Fig. 2d and f) contribute to the effects of 2-isopropylphenol ({tau}IN-1 = 64 ms and {tau}IN-2 = 6.6 s) and phenol ({tau}IN-1 <10 ms, {tau}IN-2 = 20.4 s).

The concentration–response curves of all three drugs could be fitted to Hill-equations (Fig. 3 and Table 1). The IC50 values for propofol and 2-isopropylphenol were about the same (18 ± 1, 17 ± 3.2 µM, respectively), whereas the slope of the concentration–response curve for propofol (nHill = 2 ± 0.2) was about two times steeper than that of 2-isopropylphenol (nHill = 1 ± 0.2, Table 1). Phenol was considerably less potent (IC50 = 1.6 ± 0.2 mM).


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  		Table 1. Parameters of the Hill Equations Derived from the Concentration–Response Curves (Inhibition of Peak-Currents Induced by 30 µM 5-HT) of Phenols

 

However, propofol (≥6 µM) increased the speed of the current desensitization (e.g., by 51% at 18 µM) as did many other anesthetics before,12 whereas both 2-isopropylphenol (≥20 µM) and phenol (≥1 mM) caused an extensive slowing of current decay (e.g., by 214% at 21 µM 2-isopropylphenol and by 188% at 1 mM phenol). This can be seen in the traces of Figure 2 and it is substantiated by the subsequent statistical analysis (see below).

As these observations may suggest different processes, an attempt was made to separate these (Fig. 4). Fast actions were separated by coapplying propofol and the phenol derivatives simultaneously with 5-HT but not before (open channel application). In this manner, their effects on peak currents could be investigated within a time frame of approximately 20 ms, whereas their effects on current desensitization could be observed for a period of 50 ms to several 100 ms. Slow drug actions were separated by exposing drugs to the patches for 60 s exclusively before 5-HT was applied (closed channel application). The combined effects of open channel and closed channel application (fast and slow actions of propofol) were studied by applying drugs 60 s before and also during the activation of the 5-HT3A receptor by 5-HT (equilibrium application).


Figure 424
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  		Figure 4. Effects of three phenols on 5-HT (30 µM)-induced currents, using three different drug application modes (original current traces). Traces for propofol were obtained from two patches (separate patch for the closed channel application); those for 2-isopropylphenol and phenol from single patches, respectively.

 

The fast actions (open-channel) of 2-isopropylphenol and phenol (Fig. 4), in contrast to propofol (which caused significant peak amplitude reduction and acceleration of the desensitization at concentrations ≥6 µM, Fig. 5), consisted of an increase of the peak amplitude and a pronounced slowing of the desensitization process at drug concentrations ≥20 µM and 1 mM, respectively (Fig. 5). The slow action (closed-channel application) of 2-isopropylphenol and phenol left the time constants of desensitization unchanged (Fig. 5), whereas both substances suppressed the peak amplitudes in a concentration-dependent manner (IC50 values = 14 ± 1.1 µM and 1.04 ± 0.1 mM, respectively).


Figure 524
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  		Figure 5. Effects of phenols on the current decay-kinetics of 5-HT (30 µM)-induced currents using three different drug application modes (note the different scaling of the y-axes). Results are expressed as percentage of the time constants {tau}off of the control currents in the absence of drug (100%, indicated by dashed lines); the mean for all {tau}off values of the respective control currents was 117 ± 73 ms, n = 43, which is a typical value for human 5-HT3A receptors, see discussion. Data are shown as means ± sd, n = 3–10 different patches. *Significant difference from the control value in the absence of drug (P < 0.05, paired t-test).

 

However, whenever the fast process was visible (both in the open-channel and in the equilibrium application), the desensitization time constants changed with concentration in the same manner (Fig. 5): they accelerated with increasing propofol concentration (≥6 µM) and they were successively slowed by increasing concentrations of either 2-isopropylphenol (≥20 µM) or phenol (≥1 mM). During closed-channel application (slow action), desensitization time constants were not affected by any of the three drugs (Fig. 5). No significant effects of propofol, 2-isopropylphenol, and phenol on the current activation kinetics were observed in any application mode.

An additional series of experiments tested for each application mode separately whether the drug effects depended on the agonist (5-HT) concentration (Fig. 6). We compared the effects of propofol and its derivatives when currents were elicited either by a nearly saturating concentration of 5-HT (30 µM) or a concentration well below the EC50 for 5-HT (5–9 µM) but high enough (3 µM) to evoke currents amenable to reproducible curvefitting. The slow (closed channel application) inhibitions of current amplitudes by any of the three substances, propofol (14 µM), 2- isopropylphenol (21 µM), or phenol (1 mM), were not significantly different when the 5-HT concentration was changed (Fig. 6, bottom row), in contrast to the fast (open channel application) actions of the three drugs. Here, in the case of propofol, 3 µM 5-HT-evoked currents were much more potently suppressed (by 58%) than 30 µM 5-HT-evoked currents (by 14%), whereas 2-isopropylphenol or phenol produced much larger current increases at 3 µM 5-HT (by 206% and 197%, respectively) than at 30 µM 5-HT (by 11% and 13%, respectively (Fig. 6, middle row).


Figure 624
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  		Figure 6. Effects of three phenols on 3 µM and 30 µM 5-HT-induced currents, using three different drug application modes. Any of the three substances propofol, 2-isopropylphenol or phenol caused suppression of current in the closed channel application, which did not depend on the concentration of 5-HT. Note the different current scales in the open channel or in the equilibrium applications, since propofol caused inhibition while isopropylphenol or phenol produced current potentiation. Significance (P < 0.05, unpaired t-test) refers to the difference between relative drug effects on current amplitudes elicited by either 3 µM or 30 µM 5-HT (each reduction normalized to the respective 3 and 30 µM 5-HT control in the absence of drug).

 

In the equilibrium application, the inhibition by 14 µM propofol was larger than in either of the other application modes (open channel and closed channel), irrespective of the 5-HT concentration (Fig. 6, left panels). Although increases in current amplitudes by 2-isopropylphenol and phenol were still present for current peaks evoked with the lesser 5-HT concentration in the equilibrium application (Fig. 6, upper trace), they were reduced compared with the open channel application. However, at the higher 5-HT (30 µM) concentration in the equilibrium application, in the presence of either 2-isopropylphenol (21 µM) or phenol (1 mM), inhibition of current amplitudes (by 37% and 30%, respectively) was observed (Fig. 6, upper row), in contrast to the current increases in the open channel application.

When the slow and the fast effects on current amplitudes were multiplied for each drug, then the product obtained both for the lesser 3 µM 5-HT concentration (Fig. 6, upper row, dotted line on the left) and the higher 30 µM 5-HT concentration (Fig. 6, upper row, dotted line on the right) came very close to the effect measured in the equilibrium application (Fig. 6, upper row, respective bars through which dotted line runs).

A phenolic group is also part of the agonist of the 5-HT3 receptor. When ethylamine is removed from 5-HT, 5-hydroxindole is obtained, which no longer directly activates currents through 5-HT3A receptors (data not shown). When this aromatic alcohol with its phenolic OH group (1 mM) was applied to the 5-HT3 receptor in the open-channel mode, it also lead to a slowing of the current desensitization time constant (Fig. 7, left panel). On the other hand, benzene (3 mM) no longer showed a slowing of desensitization but rather an acceleration (Fig. 7, right panel).


Figure 724
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  		Figure 7. Effect of 5-hydroxyindole (1 mM) and benzene (3 mM) on 5-HT-induced currents in two outside-out patches. 5-Hydroxyindole or benzene was exclusively applied together with the 1.2 s 5-HT pulse. Note that the traces for control and wash superimpose. 5-Hydroxyindole decreased the current decay kinetics almost two-fold (i.e., slow down), which remained monoexponential (time constant {tau}off). In contrast, benzene accelerated the current decay. Typical experiments from four experiments (each) are shown.

 


   DISCUSSION
Top
 Abstract
 Introduction
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Comparison with Previous Study
The present study confirms that human 5-HT3A receptors are also inhibited by 30 µM 5-HT are also inhibited by propofol with a potency similar to that recorded for murine 5-HT3 receptors.14

The slope of the concentration–response curve for propofol (at 30 µM 5-HT) is steeper for the human receptor (Table 1). Other differences between human and murine receptors are the biphasic wash-in of the propofol effect reported here and the fact that the fast wash-in time constant (35 ms) is almost a factor of 3 faster than the current desensitization time constant, {tau}off, of the 5-HT3A receptor [this study: {tau}off = 117 ± 73 ms, n = 43 patches; entire data pool from our studies: {tau}off = 112 ± 100 ms, n = 1760 patches17], whereas the slow wash-in time constant is considerably slower than {tau}off. This latter observation goes against the hypothesis proposed in an earlier paper14 that propofol triggers an unmodified desensitization mechanism in the absence of 5-HT with the same kinetics as does 5-HT.

Slow Action
Propofol and its derivatives clearly interact with human 5-HT3A receptors, even when they are applied to the receptor exclusively in the absence of any 5-HT. Although not with the same, but with much slower, kinetics than for the unmodified desensitization process hypothesized above, the compounds investigated in this study may still shift activable 5-HT3A receptors into an anesthetic-related desensitized state. The fraction of channels inhibited by the slow action (closed channel application) may be in this state. The actions consist of a reduction in the peak current of the test pulse for all three substances (Fig. 4), without any effect on the current decay time constant (Fig. 5), even though the duration of superfusion by propofol or its derivatives had lasted long enough so that the reduction in peak current had reached a steady state.

The IC50 of phenol for slow action (closed channel application, Table 1) is very close to the one predicted by the Meyer-Overton relation for nonspecific inhibition by phenol (predicted, see Methods: 970 µM; measured: 1035 µM). For 2-isopropylphenol, the prediction under-estimates the actual potency by a factor of 3 (predicted: 38 µM; measured: 14 µM), whereas for propofol it over-estimates the potency by a factor of 4 (predicted: 5 µM; measured: 22 µM). This may be an indication that more than added hydrophobicity is involved in the actions of compounds made more hydrophobic by the addition of purely hydrophobic isopropyl-groups. By adding an isopropyl-group to phenol, the resulting 2-isopropylphenol becomes more potent than predicted by lipophilicity. In contrast, when the second isopropyl-group is added, turning 2-isopropylphenol into propofol, the IC50 remains unchanged, although hydrophobicity alone should have made propofol roughly nine times more potent. This observation, together with the differences in the slopes of the respective concentration–response curves, suggests additional interactions to be involved that are antagonistic to hydrophobic actions. However, as has been found before for many other anesthetics,1 on average the three substances follow the correlation with hydrophobicity very well, as propofol scatters by approximately the same factor to the right as 2-isopropylphenol scatters to the left.

Fast Action
Exclusive exposure of 5-HT3A receptors to propofol or its derivatives during the 5-HT pulses (open channel application) sufficed to produce effects on the amplitudes of the elicited currents as well as on their desensitization time constants (Figs. 4–6). Propofol suppressed the amplitudes of the currents and caused them to desensitize more rapidly, whereas phenol and 2-isopropylphenol, in contrast, increased their amplitudes and slowed desensitization in a concentration-dependent manner. Increases in peak amplitudes have been observed for volatile anesthetics18–21 and alcohols,22–24 whereas retardation of desensitization has been reported for morphine.25

The effects on the amplitudes were more clearly seen for lower 5-HT concentrations, whereas the effects on the time constants of desensitization were obvious at both 5-HT concentrations. These observations may, at least partly, be explained by the kinetics of wash-in (Fig. 2), as the fast action may not have fully developed by the time the current peak is reached (about 20 ms at 30 µM 5-HT), whereas desensitization can be observed for a duration of several 100 ms. Whether or not additional competition between these drugs and 5-HT is involved, could only be decided in an additional study, which would be difficult and extensive, as the 5-HT activation curve itself is bell-shaped,26 which is typical for ligand-gated ion channels.27,28

Additivity of Fast and Slow Actions
The effects of propofol and its derivatives on the desensitization time constants persisted as long as they were administered together with 5-HT (Figs. 4 and 5), whether or not they had been given before the 5-HT application as well (equilibrium or open channel application). Thus, the concentration-dependent acceleration (propofol) or retardation (propofol derivatives) of the desensitization time constant requires the coapplication with 5-HT, i.e., an open channel conformation during drug application.

There is additional evidence that fast and slow actions appear to be independent and additive: When the effects of the open-channel application and the closed-channel applications on current amplitudes are multiplied, the resulting product (see dotted lines in Fig. 6, upper panels) is close to the measured result under equilibrium condition (bars in the same figure). This observation supports the hypothesis of at least two separate and superimposing processes postulated in our previous paper.14

Comparison with Other Ligand-Gated Ion Channels
Comparing the concentrations at which propofol interacts with other ion channels, one finds them all to lie within a similar range, as observed in this study (IC50 = 18 µM; more potent at lower 5-HT concentrations, Fig. 6). IC50 values of 20 µM,29 between 4.6 and 23 µM,30 and between 6 and 25 µM31 have been reported for human brain, skeletal muscle, and rat brain of sodium channels, respectively. The voltage-dependent potassium channel from SH-SY5Y human blastoma cell lines was suppressed with an IC50 = 44 µM.32 For the neuronal type {alpha}4β2 nicotinic acetylcholine receptor channel, IC50 values of 19 µM33 or 4.5 µM34 have been found, whereas the muscle subtype {alpha}β{gamma}{Delta} was half-suppressed by 46 µM.34 Depending on subtype, GABAA receptors are enhanced by 2.6 µM ({alpha}1β3{gamma}235), 4.6 µM ({alpha}6β3{gamma}235), 8.7 µM ({alpha}2β136), or 39.7 µM ({alpha}1β2{gamma}2S37) propofol. Enhancement is also observed for the glycine receptor channel {alpha}1β with an EC50 = 12.5 µM.38 All these IC50 and EC50 values scatter around the value of 5 µM predicted by the Meyer-Overton correlation for nonspecific interactions of propofol (see Methods).

Active Functional Groups
Just as has been postulated for the interaction between serotonin and its receptor site,39 the interaction between propofol or its phenol derivatives and the 5-HT3 receptor may be a combination of hydrophobic and specific polar interactions. Evidence for hydrophobic contributions is the relatively close prediction of the IC50 values for closed channel inhibition from Meyer-Overton correlations (see Methods). Candidates for specific interactions are hydrogen bonds formed by the phenolic hydroxygroup, van der Waals interactions with the {pi}-electrons of the aromatic ring of the phenol and its derivatives, a lipophilic pocket of limited dimensions, or a combination of some or all of these possibilities.

The phenolic —OH group seems to be involved in the effect of increasing current amplitudes and slowing desensitization as suggested by the failure of benzene to cause either effect (Fig. 7). The only difference between phenol and benzene is the —OH group, which benzene is lacking. There is other circumstantial evidence that phenolic groups of a molecule may slow the desensitization of 5-HT3 receptors. Morphine, which also contains a phenolic —OH group, causes a pronounced slowing of 5-HT3A receptor current decay.25 5-Hydroxyindole, a 5-HT derivative lacking the ethylamine group, has lost the intrinsic activity of eliciting currents through 5-H3A receptor, but it does slow current decay of 5-HT3 receptors [Ref. 40 and Figure 7].

When first synthesizing and exploring phenol derivatives suitable for general anesthesia, James and Glen41 already noted not only the importance of their lipophilic character and H-bond donor/acceptor properties but they also realized that steric considerations mattered. They found that potency and kinetics appeared to be a function of both the lipophilic character and the degree of steric hindrance exerted by ortho substituents. The phenolic group, in conjunction with aliphatic substituents in the ortho position, has been implicated in the direct activation of chloride currents through GABAA receptors in the absence of GABA.42 Additional evidence that steric considerations are important comes from the observation by Krasowski et al.37 that 2,6-di-sec-butylphenol did enhance GABAA receptor, whereas 2,6-di-tert-butylphenol did not. In addition, hydrophobic groups of a drug may show specificity, for example, when they exceed the molecular volume available in a hydrophobic pocket. Thus, fewer hydrophobic sites may be available to propofol than to either 2-isopropylphenol or to phenol.

This may be why inhaled anesthetics with too large a molecular volume no longer cause net enhancement of 5-HT3 receptors.43 Thus, steric limitations compromising the simultaneous interaction of two or more functional groups and/or too large a hydrophobic volume may be why propofol, in this and other studies, does not enhance 5-HT3A receptors,22,44,45 while 2-isopropylphenol and phenol (this study) do.

Implications for Anesthetic Mechanisms
James and Glen41 finally settled for propofol to be tested in human trials, because of all the phenol derivatives they had tested, propofol had proved to be the most potent hypnotic. In our study, we find that propofol is about equally as potent as 2-isopropylphenol in suppressing 5-HT3A receptor currents in the equilibrium application, although propofol is much more hydrophobic. This implies that propofol in comparison with 2-isopropylphenol has lost some potential for interactions that cause current suppression, even though propofol already lacks the counteracting ability for enhancement, i.e., it causes neither increases in current amplitudes nor in the desensitization time constant.

There is other evidence for a lesser specificity of propofol compared with other IV drugs. The IC50 (18 µM) for propofol differs little from those listed in a previous section for voltage-gated ion channels. It differs by only a factor of two from that predicted by hydrophobicity for voltage-gated ion channels (see Methods), and not by about an order of magnitude as would be the case when average values for IV anesthetic action on ligand-gated and on voltage-gated ion channels are compared.1

In fact, in some sense, propofol is not unlike inhaled anesthetics, which are considered less specific in their molecular actions than IV anesthetics, because here we find propofol to be more potent than inhaled anesthetics by a factor of only 1.5 (see Methods), whereas on average, IV anesthetics are an order of magnitude more potent on ligand-gated ion channels than inhaled anesthetics.1 When the various ion channels were compared in a previous section, it was evident that they all respond to propofol within a factor of 10 in the low micromolar concentration range, rather than in the low nanomolar or picomolar range, as would be expected for a specific pharmacological drug, supporting the empirical hypothesis that the less specific drug may be the more useful general anesthetic. Thus, both diethylether and propofol lack specificity compared with other inhaled anesthetics and IV anesthetics, respectively, yet each has been clinically one of the most successful anesthetics in their respective class.

The fast time resolution resulting from the use of outside-out patches in conjunction with a fast solution exchange system made it possible to discover an enhancing component of anesthetic action in some phenol derivatives, a component normally masked by an overall suppression in the equilibrium condition. Thus, the different responses by excitatory and inhibitory ligand gated ion channels to propofol may simply reflect an altered balance of inhibiting and enhancing effects rather than a qualitative difference between these two channel types. Kinetic (as an alternative to mathematical) separation of anesthetic interactions, as demonstrated in this study, may prove to be a useful tool in identifying relevant functional groups and characterizing these interactions.

The fast time resolution has also shown that electrophysiological competition experiments are very difficult to interpret when drug action takes longer than the time required for the agonist to act. In our system, the peak of the current is reached typically after 20 ms, whereas the fast component of drug action described here (Fig. 2) may be considerably slower. When there is a part of the fast component that is present only in the open channel conformation, then neither the open channel nor the equilibrium application would allow steady-state measurements of the current peak. In this case, it would be extremely difficult to experimentally separate kinetic from competitive effects. On the other hand, the experiments involving drug action in the absence of the agonist clearly demonstrate that there is at least a large component of drug action that is not competitive with the agonist. In vivo it would not be possible to overcome this component by synapses that are flooded with transmitter agonists. As to the fast component, clearly, a synapse releasing a great deal of agonist would be suppressed by propofol less than a synapse that released less 5-HT.

In conclusion, at least two separate inhibitory actions on 5-HT3A receptors could be identified for propofol, whereas the enhancing action seen for the two related smaller phenol derivatives could no longer be detected. 5-HT-dependent and 5-HT-independent interactions could be distinguished for all three drugs. Propofol was less potent than expected from its hydrophobic properties. Underlying mechanisms appear to involve the phenolic hydroxyl group, hydrophobic interactions, and steric restrictions.


   ACKNOWLEDGMENTS
 
We thank Dr. J.P. Dilger for carefully reading this manuscript and discussions, Ms von dem Bussche for maintaining the cell cultures, and Ms M. Meiboom for performing experiments with 5-hydroxyindole.


   Footnotes
 
Accepted for publication November 13, 2007.

Supported by the DFG (BA 1454).

I.L. and S.W. contributed equally to this work.


   REFERENCES
Top
 Abstract
 Introduction
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Urban BW, Bleckwenn M, Barann M. Interactions of anesthetics with their targets: non-specific, specific or both?. Pharmacol Ther 2006;111:729–70[Web of Science][Medline]
  2. Belelli D, Callachan H, Hill-Venning C, Peters JA, Lambert JJ. Interaction of positive allosteric modulators with human and Drosophila recombinant GABA receptors expressed in Xenopus laevis oocytes. Br J Pharmacol 1996;118:563–76[Web of Science][Medline]
  3. Orser BA, McAdam LC, Roder S, MacDonald JF. General anaesthetics and their effects on GABA(A) receptor desensitization. Toxicol Lett 1998;100–101:217–24
  4. Pistis M, Belelli D, Peters JA, Lambert JJ. The interaction of general anaesthetics with recombinant GABAA and glycine receptors expressed in Xenopus laevis oocytes: a comparative study. Br J Pharmacol 1997;122:1707–19[Web of Science][Medline]
  5. Borgeat A, Stirnemann HR. [Antiemetic effect of propofol]. Anaesthesist 1998;47:918–24[Web of Science][Medline]
  6. Kilpatrick GJ, Jones BJ, Tyer MB. The identification and distribution of 5-HT3 receptors in rat brain using radioligand binding. Nature 1987;330:746–8[Medline]
  7. Costall B, Naylor RJ. 5-HT3 receptors. Curr Drug Targets CNS Neurol Disord 2004;3:27–37[Medline]
  8. Carpenter DO. Neural mechanisms of emesis. Can J Physiol Pharmacol 1990;68:230–6[Web of Science][Medline]
  9. Minami M, Endo T, Hirafuji M, Hamaue N, Liu Y, Hiroshige T, Nemoto M, Saito H, Yoshioka M. Pharmacological aspects of anticancer drug-induced emesis with emphasis on serotonin release and vagal nerve activity. Pharmacol Ther 2003;99: 149–65[Web of Science][Medline]
  10. Tramer MR, Reynolds DJ, Stoner NS, Moore RA, McQuay HJ. Efficacy of 5-HT3 receptor antagonists in radiotherapy-induced nausea and vomiting: a quantitative systematic review. Eur J Cancer 1998;34:1836–44[Web of Science][Medline]
  11. Hille B. Ligand-gated channels of fast chemical synapses. In: Hille B, ed. Ion channels of excitable membranes. Sunderland: Sinauer, 2001:169–99
  12. Barann M, Meder W, Dorner Z, Brüss M, Bönisch H, Göthert M, Urban BW. Recombinant human 5-HT3A receptors in outside out patches of HEK 293 cells; basic properities and barbiturate effects. Naunyn Schmiedebergs Arch Pharmacol 2000;362: 255–65[Web of Science][Medline]
  13. Barann M, Molderings G, Brüss M, Bönisch H, Urban BW, Göthert M. Direct inhibition by cannabinoids of human 5-HT3A receptors: probable involvement of an allosteric modulatory site. Br J Pharmacol 2002;137:589–96[Web of Science][Medline]
  14. Barann M, Dilger JP, Bönisch H, Göthert M, Dybek A, Urban BW. Inhibition of 5-HT3 receptors by propofol: equilibrium and kinetic measurements. Neuropharmacology 2000;39:1064–74[Web of Science][Medline]
  15. Barann M, Friederich P, Retzmann K, Dybek A, Urban BW. Loss of propofol during in vitro experiments. Anesthesiology 2000; 93:310–1[Web of Science][Medline]
  16. Tetko IV, Tanchuk VY, Kasheva TN, Villa AE. Internet software for the calculation of the lipophilicity and aqueous solubility of chemical compounds. J Chem Inf Comput Sci 2001;41:246–52[Web of Science][Medline]
  17. Linden I. Molecular effects of general anesthetics on human 5-HT3A receptors, their kinetics and relevance for postoperative emesis (German). PhD Thesis. Faculty for Science and Mathematics, University of Bonn, 2007;36
  18. Zhang L, Oz M, Stewart RR, Peoples RW, Weight FF. Volatile general anaesthetic actions on recombinant nACh alpha 7, 5-HT3 and chimeric nACh alpha 7-5-HT3 receptors expressed in Xenopus oocytes. Br J Pharmacol 1997;120:353–5[Web of Science][Medline]
  19. Suzuki T, Koyama H, Sugimoto M, Uchida I, Mashimo T. The diverse actions of volatile and gaseous anesthetics on human-cloned 5-hydroxytryptamine3 receptors expressed in Xenopus oocytes. Anesthesiology 2002;96:699–704[Web of Science][Medline]
  20. Stevens RJ, Rusch D, Davies PA, Raines DE. Molecular properties important for inhaled anesthetic action on human 5-HT3A receptors. Anesth Analg 2005;100:1696–703[Abstract/Free Full Text]
  21. Jenkins A, Franks NP, Lieb WR. Actions of general anaesthetics on 5-HT3 receptors in N1E-115 neuroblastoma cells. Br J Pharmacol 1996;117:1507–15[Web of Science][Medline]
  22. Machu TK, Harris RA. Alcohols and anesthetics enhance the function of 5-hydroxytryptamine3 receptors expressed in Xenopus laevis oocytes. J Pharmacol Exp Ther 1994;271:898–905[Abstract/Free Full Text]
  23. Lovinger DM, Sung KW, Zhou Q. Ethanol and trichloroethanol alter gating of 5-HT3 receptor-channels in NCB-20 neuroblastoma cells. Neuropharmacology 2000;39:561–70[Web of Science][Medline]
  24. Barann M, Ruppert K, Göthert M, Bönisch H. Increasing effect of ethanol on 5-HT3 receptor-mediated 14C-guanidinium influx in N1E-115 neuroblastoma cells. Naunyn Schmiedebergs Arch Pharmacol 1995;352:149–56[Web of Science][Medline]
  25. Wittmann M, Peters I, Schaaf T, Wartenberg HC, Wirz S, Nadstawek J, Urban BW, Barann M. Effects of morphine on human 5-HT3A receptors. Anesth Analg 2006;103:747–52[Abstract/Free Full Text]
  26. Barann M. Pharmacological studies on the cation channel of the 5-HT3 receptor and the fast, voltage-dependent sodium channel in cultured neuronal cells (German). PhD Thesis. Faculty for Science and Mathematics, University of Bonn, 1993;70
  27. Daly JW, Nishizawa Y, Edwards MW, Waters JA, Aronstam RS. Nicotinic receptor-elicited sodium flux in rat pheochromo-cytoma PC12 cells: effects of agonists, antagonists and non-competitive blockers. Neurochem Res 1991;16:489–500[Web of Science][Medline]
  28. Forman SA, Firestone LL, Miller KW. Is agonist self-inhibition at the nicotinic acetylcholine receptor a nonspecific action?. Biochemistry 1987;26:2807–14[Web of Science][Medline]
  29. Frenkel C, Urban BW. Human brain sodium channels as one of the molecular target sites for the new intravenous anaesthetic propofol (2,6-diisopropylphenol). Eur J Pharmacol 1991;208:75–9[Web of Science][Medline]
  30. Haeseler G, Stormer M, Bufler J, Dengler R, Hecker H, Piepenbrock S, Leuwer M. Propofol blocks human skeletal muscle sodium channels in a voltage-dependent manner. Anesth Analg 2001; 92:1192–8[Abstract/Free Full Text]
  31. Rehberg B, Duch DS. Suppression of central nervous system sodium channels by propofol. Anesthesiology 1999;91:512–20[Web of Science][Medline]
  32. Friederich P, Urban BW. Interaction of intravenous anesthetics with human neuronal potassium currents in relation to clinical concentrations. Anesthesiology 1999;91:1853–60[Web of Science][Medline]
  33. Flood P, Ramirez-Latorre J, Role L. Alpha 4 beta 2 neuronal nicotinic acetylcholine receptors in the central nervous system are inhibited by isoflurane and propofol, but alpha 7-type nicotinic acetylcholine receptors are unaffected [see comments]. Anesthesiology 1997;86:859–65[Web of Science][Medline]
  34. Violet JM, Downie DL, Nakisa RC, Lieb WR, Franks NP. Differential sensitivities of mammalian neuronal and muscle nicotinic acetylcholine receptors to general anesthetics [see comments]. Anesthesiology 1997;86:866–74[Web of Science][Medline]
  35. Krasowski MD, O'Shea SM, Rick CE, Whiting PJ, Hadingham KL, Czajkowski C, Harrison NL. Alpha subunit isoform influences GABA(A) receptor modulation by propofol. Neuropharmacology 1997;36:941–9[Web of Science][Medline]
  36. Krasowski MD, Koltchine VV, Rick CE, Ye Q, Finn SE, Harrison NL. Propofol and other intravenous anesthetics have sites of action on the gamma-aminobutyric acid type A receptor distinct from that for isoflurane. Mol Pharmacol 1998;53:530–8[Abstract/Free Full Text]
  37. Krasowski MD, Jenkins A, Flood P, Kung AY, Hopfinger AJ, Harrison NL. General Anesthetic Potencies of a Series of Propofol Analogs Correlate with Potency for Potentiation of gamma-Aminobutyric Acid (GABA) Current at the GABA(A) Receptor but Not with Lipid Solubility. J Pharmacol Exp Ther 2001; 297:338–51[Abstract/Free Full Text]
  38. Ahrens J, Haeseler G, Leuwer M, Mohammadi B, Krampfl K, Dengler R, Bufler J. 2,6 Di-tert-butylphenol, a nonanesthetic propofol analog, modulates alpha1beta glycine receptor function in a manner distinct from propofol. Anesth Analg 2004; 99:91–6[Abstract/Free Full Text]
  39. Beene DL, Price KL, Lester HA, Dougherty DA, Lummis SCR. Tyrosine residues that control binding and gating in the 5-hydroxytryptamine3 receptor revealed by unnatural amino acid mutagenesis. J Neurosci 2004;24:9097–105[Abstract/Free Full Text]
  40. Kooyman AR, van Hooft JA, Vijverberg HPM. 5-Hydroxyindole slows desensitization of the 5-HT3 receptor-mediated ion current in N1E-115 neuroblastoma cells. Br J Pharmacol 2006; 108:287–9
  41. James R, Glen JB. Synthesis, biological evaluation, and preliminary structure-activity considerations of a series of alkylphenols as intravenous anesthetic agents. J Med Chem 1980;23:1350–7[Web of Science][Medline]
  42. Mohammadi B, Haeseler G, Leuwer M, Dengler R, Krampfl K, Bufler J. Structural requirements of phenol derivatives for direct activation of chloride currents via GABA(A) receptors. Eur J Pharmacol 2001;421:85–91[Web of Science][Medline]
  43. Stevens RJ, Rusch D, Davies PA, Raines DE. Molecular properties important for inhaled anesthetic action on human 5-HT3A receptors. Anaesth Analg 2006;100:1696–703[Web of Science]
  44. Patten D, Foxon GR, Martin KF, Halliwell RF. An electrophysiological study of the effects of propofol on native neuronal ligand-gated ion channels. Clin Exp Pharmacol Physiol 2001; 28:451–8[Web of Science][Medline]
  45. Lopreato GF, Banerjee P, Mihic SJ. Amino acids in transmembrane domain two influence anesthetic enhancement of serotonin-3A receptor function. Brain Res Mol Brain Res 2003;118:45–51[Medline]




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