Anesth Analg 2004;99:409-415
© 2004 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000131969.46439.71
ANESTHETIC PHARMACOLOGY
Halothane and Propofol Modulation of 
-Aminobutyric AcidA Receptor Single-Channel Currents
Akira Kitamura, MD PhD*,
,
Ryoichi Sato, MD PhD*,
William Marszalec, PhD*,
Jay Z. Yeh, PhD*,
Ryo Ogawa, MD PhD, and
Toshio Narahashi, DVM PhD*
*Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois; and
Department of Anesthesiology, Nippon Medical School, Tokyo, Japan
Address correspondence and reprint requests to Toshio Narahashi, DVM, PhD, Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611. Address e-mail to narahashi{at}northwestern.edu
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Abstract
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Halothane and propofol enhance the activity of the 
-aminobutyric acid (GABA) system, which is one of the most important systems in the mechanism of anesthesia. To determine whether halothane and propofol enhance GABAergic responses by the same mechanism, we performed single-channel patch-clamp experiments with rat cortical neurons in primary culture. Each of the open-time and closed-time distributions of GABAA receptor single channels was expressed by a sum of fast and slow time constants. Neither halothane nor propofol changed the single-channel conductance. Halothane increased the probability of the channel being open via a prolongation of the slow phase of open time, whereas propofol increased the channel open probability via a shortening of the slow phase of closed time. Thus, although both halothane and propofol augmented the channel open probability, thereby causing an increase in charge transfer during inhibitory transmitter action, they acted by different mechanisms.
IMPLICATIONS: Although both halothane and propofol increase -aminobutyric acid (GABA)-mediated synaptic inhibition, thereby causing anesthesia, the underlying mechanisms at the single GABA receptor channels have been shown to be different by using the patch-clamp technique.
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Introduction
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Many receptors and ion channels are altered by volatile anesthetics; the -aminobutyric acid (GABA) and glutamate systems play important roles in producing anesthesia (1). The GABAA receptor activity is enhanced and the glutamate receptor activity is inhibited by volatile anesthetics. Also, neuronal nicotinic acetylcholine receptors (nAChRs) are inhibited by anesthetics (2–4). The nAChRs, located in presynaptic/preterminal areas of interneurons in the brain, modulate the release of various neurotransmitters, including glutamate, GABA, acetylcholine, dopamine, and norepinephrine (5–7). Thus, anesthetic inhibition of nAChRs is expected to modulate the activity of the GABAergic and glutamatergic systems.
Many general anesthetics exert three possible actions on GABAA receptors: potentiation, inhibition, and direct activation (8–10). With the exception of xenon and nitrous oxide (11,12), volatile anesthetics enhance GABA-activated currents, whereas inactive compounds do not affect GABA-induced responses (13). Physiologically, this enhancement seems to manifest itself as a prolongation of the synaptic current. A positive correlation was found between the mean effective concentration values for anesthetic action and those for prolongation of GABA-induced currents (14). Thus, enhancement of GABAA-mediated inhibitory responses is most likely related to clinical anesthesia. In the presence of halothane, enflurane, isoflurane, or sevoflurane, evoked and miniature inhibi- tory postsynaptic currents (mIPSCs) were slightly suppressed, yet the decay time constant was greatly increased, resulting in an increase in total charge transfer (8,12,15). Thus, halothane slows inhibitory postsynaptic current decay by slowing dissociation of the agonist from the receptor (16).
We recently compared the effects of halothane and propofol on spontaneous mIPSCs and miniature excitatory postsynaptic currents (mEPSCs) in rat cortical neurons in primary culture (17). The frequencies of mIPSCs and mEPSCs were slightly suppressed by halothane but not by propofol. The only changes common to both of these anesthetics were a prolongation of the decay phase of mIPSCs and an increase in charge transfer during inhibitory transmitter action. Thus, this effect on the inhibitory systems appears to be important in causing anesthesia, yet the question remains whether halothane and propofol increase the mIPSC charge transfer by the same mechanism. This study addressed this issue by using single-channel current-recording techniques.
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Methods
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Cortical neurons were cultured by a method previously used for hippocampal cells (18). In brief, 17-day fetuses were removed from a pregnant Sprague-Dawley rat under methoxyflurane anesthesia. This procedure was approved by the Animal Care and Use Committee of Northwestern University and followed their guidelines. Small wedges of frontal cortex were excised and treated for 25 min at 37°C in a phosphate-buffered saline solution containing 0.25% trypsin (wt/vol) (Sigma-Aldrich, St. Louis, MO). After mechanical trituration by repeated passages through a Pasteur pipette, the dissociated cells were suspended in Dulbecco’s modified Eagle’s medium with the addition of 10% (vol/vol) Ham’s F-12 supplement, 2 mM glutamine, and 20 U of penicillin/20 ng of streptomycin per milliliter. The cells were placed into 35-mm culture wells at a concentration of 200,000/3 mL. Each well contained 5 12-mm glass coverslips with a confluent layer of glia that had been plated 2 to 4 wk previously. This cortical/glial coculture was maintained in a humidified atmosphere of 93% air and 7% CO2. Cells used in these experiments were cultured for 14 to 28 days. All current recordings were made from pyramidal-shaped neurons 30–50 µm in diameter.
Cell-attached patches were used for recording single-channel currents. GABA at a concentration of 10 µM was added to the pipette solution to activate the GABAA receptor. Six cells were used for each type of single-channel experiment. Conductance measurements on cell-attached patches have shortcomings involving determination of the resting membrane potential and interpretation of current/voltage relationships due to unequal intracellular and extracellular concentrations of Cl– ions. Nonetheless, we used cell-attached patches because this configuration eliminates problems associated with patch excision, such as rundown of channel acti- vity, alterations in receptor-channel kinetics, and activation of previously inactive Cl– channels (19,20). For single-channel recordings from cell-attached patches, pipette tips were coated with silicone elastomer to reduce capacitance and to enhance the signal-to-noise ratio. The tip resistance was 8 to 10 M
in extracellular medium. The lag time between the control recording of channel activity and the response to each drug application was 5 to 10 min. The extracellular pipette solution for cell-attached single-channel current recording contained (mM): CsCl 140, CaCl2 1, and HEPES 5. The pH was titrated to 7.3 with NaOH.
The currents were recorded with a 2-kHz low-pass Bessel filter and stored on tape. For analysis, recordings were replayed, low-pass-filtered at 1 kHz through an eight-pole Bessel filter, and digitized at a sampling rate of 200 µs per point. Data were analyzed by using the routines available in pClamp 6 software (FETCHAN and pSTAT; Axon Instruments, Union City, CA). Each opening was examined directly; in case of drift or artifacts, the baseline level and the current amplitude were corrected before the data were stored. Openings and closings were discerned by using a 50% threshold-crossing algorithm for event detection. Measurements derived from the channel transitions were collected into histograms to allow analysis of single-channel kinetics. Mean dwell times were determined from the sum of exponential fits to the distributions of open and closed times. When a sufficiently long continuous recording was achieved (
10 min), the number of active channels (N) in the patch could be predicted by using this equation (21):
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where Pobs is the probability of observed opening, T is the length of the observed record, mo is the mean open life time, and ms is the mean closed time. When multiple channel activities were observed in a patch (more than two channels open simultaneously), an averaging technique was used to obtain the channel open probability according to this equation:
where I is the average total channel current amplitude, N is the number of channels, Po is the probability of individual channel opening, and i is the unitary amplitude of the single-channel current; i was obtained by directly measuring the individual channel current magnitude. I was obtained by calculating the average level of channel open events. When a sufficiently long recording was achieved, Po could be obtained from Equation 2.
The open and closed time distributions were calculated by Equation 3:
where A1 and A2 are the proportions of fast and slow components, respectively; t is the time; and 
1 and 2 are the fast and slow time constants, respectively.
The glass coverslips that contained neurons were placed into a microscope-mounted recording chamber (0.5-mL volume) to which control and drug-containing solutions were perfused at 1–2 mL/min. Saturated solutions of halothane (Ayerst Laboratories, New York, NY) were made by stirring halothane in the external solution for 8 h in a sealed glass container with minimal air space. Halothane test solutions were prepared by diluting the saturated halothane solution with external solution immediately before the experiment by using sealed glass containers and glass pipettes. By using F-19 nuclear magnetic resonance spectroscopy (GE-NMR Instruments, Premont, CA), the saturated solution was found to contain 18.0 mM halothane, a value identical to that determined previously (22). The solution diluted 80 times from the saturated solution was found to contain 0.23 mM halothane. Propofol (2,6-diisopropylphenol) was dissolved in dimethyl sulfoxide at a concentration of 50 mM and was diluted to 3 µM in the external solution. The dimethyl sulfoxide concentration (0.006%, vol/vol) contained in the test solution had no effect on the GABA-induced current.
All analyses, including curve-fitting, were performed with pClamp software. Unless otherwise stated, data are presented as mean ± SD. Differences between sample means were determined by using Student’s t-test or one-way analysis of variance. A P value <0.05 was considered statistically significant.
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Results
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Figure 1, A and C, illustrates examples of single-channel currents recorded at a holding potential of –70 mV in the presence of 10 µM GABA in the pipette solution. Downward deflections indicate inward single-channel currents. The currents occurred with brief single-channel openings interrupted by brief closures or with more complex events consisting of long bursts of single-channel currents. When the current amplitude was plotted as a function of patch potential, a single-channel conductance of 25.8 ± 2.5 pS (n = 5) was obtained.
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Figure 1. Single-channel currents of a cortical neuron activated by 10 µM -aminobutyric acid (GABA) before (A; control) and during (B) application of 0.6 mM halothane. Single-channel currents recorded from another neuron: control (C) and during application of 3 µM propofol (D). Currents were recorded from cell-attached patches at a holding potential of –70 mV.
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With coapplication of 0.6 mM halothane and 10 µM GABA, the channel activity increased, sometimes resulting in simultaneous openings of several channels (Fig. 1, A and B). However, the single-channel current amplitude remained unchanged, and no significant change was observed in single-channel conductance on application of halothane (data not shown).
The mechanism responsible for the increase in channel activity was investigated by analyzing the events in two ways: by calculating the probability of a channel being open and by analyzing the distribution of mean open and closed times of the channel. The mean open time increased from the control level of 4.21 ± 2.28 ms to 8.97 ± 3.98 ms after application of 0.6 mM halothane. Figure 2 shows the distribution of open and closed times of the channel at –70 mV. Open time distribution was best described by a 2-exponential function; time constants in the control were 1.6 ms for the fast component (of) and 53.0 ms for the slow component (os) (Fig. 2A). These time constants changed to 4.7 ms (of) and 220.5 ms (os) on application of 0.6 mM halothane (Fig. 2B). On average, halothane significantly prolonged only the slow open time constant, without changing the proportion (Table 1). Similarly, at least two exponential terms were required to fit closed time distributions in the control, with a time constant for the fast component (cf) of 3.3 ms and that for the slow component (cs) of 107.5 ms (Fig. 2C). These time constants changed to 4.5 ms (cf) and 86.5 ms (cs) after exposure to 0.6 mM halothane (Fig. 2D). However, these changes in both time constants were not significant (Table 1).
The probability of channel opening (Po) was significantly increased from 0.08 to 0.41 by application of halothane, primarily because of an increase in the slow open time (Table 1). All of the effects of 0.6 mM halothane on single channels were reversible on washing with a halothane-free solution.
Currents in the control and after coapplication of 3 µM propofol are shown in Figure 1, C and D, respectively. Propofol also augmented the activity of GABA-induced single-channel currents, but it caused more burst activity than halothane. Similar to halothane, however, no effect of propofol on single-channel current amplitude or conductance was observed (data not shown). The distributions of open and closed times of the channel at –70 mV were best described by a 2-exponential function (Fig. 3). Open time constants in the control were 3.0 ms for of and 68.6 ms for os (Fig. 3A). These time constants were changed on application of 3 µM propofol to 1.4 ms (of) and 58.8 ms (os) (Fig. 3B). However, on average, neither the fast nor the slow time constant changed significantly by application of propofol (Table 1). Closed time distributions in the control had time constants of 2.1 ms for cf and 112.9 ms for cs (Fig. 3C). After exposure to 3 µM propofol, time constants of 2.1 ms (cf) and 12.5 ms (cs) were obtained (Fig. 3D). On average, only the slow closed time constant was significantly shortened by propofol, without changing the proportion (Table 1). Po was significantly increased from 0.08 to 0.33 by application of propofol, primarily because of a decrease in cs (Table 1). All effects of 3 µM propofol on single-channel variables were reversed by washing with propofol-free solution.
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Discussion
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Whereas both halothane and propofol prolong the decay phase of mIPSCs and inhibitory postsynaptic currents and augment the charge transfer during the inhibitory transmitter action, these single-channel patch-clamp experiments clearly showed differences between halothane and propofol in their mechanism of action on the GABAA receptor. Halothane at 0.6 mM (2.6 minimum alveolar anesthetic concentration [MAC]) (23) increased the open time and the probability of channels being open. However, propofol at 3 µM decreased the slow component of the closed time and increased the probability of channel openings. Therefore, although both halothane and propofol prolong the inhibitory postsynaptic current decay phase, the underlying mechanism is different. However, neither of them affected single-channel conductance.
Our previous study also showed an increase in the mean open time of GABAA receptor channels of rat dorsal root ganglion neurons in the presence of 4 MAC halothane (24). The present study with rat cortical neurons confirmed and extended the previous data to a more clinically relevant concentration (2.6 MAC) and to analysis of 2 components of open time. Drastic prolongation of the slow open time by halothane was reflected in the prolongation of the falling phase of mIPSCs (17). Propofol at 1.7–2 MAC was reported to increase the channel open probability in mouse hippocampal neurons (25) and bovine chromaffin cells (26). Our results also showed an increase in the probability of channel openings and a decrease in slow closed time. These effects appeared in the propofol-induced prolongation of the decay phase of mIPSCs (17,25).
Although volatile anesthetics including halothane, isoflurane, enflurane, and sevoflurane modulate other ligand-gated channels, such as glutamate receptors and nAChRs, it is becoming abundantly clear that the GABAA receptor channel is one of the most important sites of action of anesthetics (1). In contrast, the IV anesthetics propofol and etomidate augment the activity of GABAA receptors almost exclusively, without effects on glutamate receptors and nAChRs (1).
In our previous study (17), halothane at 0.3, 0.6, and 1.2 mM (1–4 MAC) and propofol at 1, 3, and 10 µM increased the duration and charge transfer of mIPSCs. Neither drug, however, affected the duration of mEPSCs. Although this single-channel study was limited to 0.6 mM halothane and 3 µM propofol, it is reasonable to assume that these anesthetics at clinically relevant concentrations modulate the single-channel activity of GABAA receptors, thereby augmenting the charge transfer during inhibitory synaptic transmission. In a previous study with human embryonic kidney cells transfected to express the 
1ß22s GABA receptor subunit combination, halothane was also found to increase the duration of GABA currents by reducing the rate constant for agonist-receptor unbinding (16).
In this study, the histograms of channel open and closed times could be fitted to a sum of two exponential functions, suggesting that the channel could be in at least two open and closed states. This property has been described in GABAA receptors (27–30). A wide variability in the open time of GABA-activated Cl– channels may occur, because the power spectra of GABA-induced fluctuations are often dominated by lower-frequency, long-duration events that approximate burst length duration rather than reflecting the open-time distribution (29,31). Moreover, in single-channel recordings, many patches might contain multiple channels, resulting in the recording of multiple superimposed events that complicate kinetic analysis.
Our findings regarding open and closed times suggest that GABA-activated Cl– channels in rat cortical neurons have complex channel kinetics with at least two open and closed states. However, we do not have sufficient data to exclude alternative hypotheses that GABA may open a single class of Cl– channels when the receptor has bound either one or two GABA molecules or that GABA may activate a nonhomogenous population of two-state (open/closed) Cl– channels with different open and closed times in the presence of halothane and propofol. This study shows that halothane and propofol exert the same effects on GABA-induced single-channel currents in that both increase the probability of channels being open. However, the mechanism that underlies the increase in Po is not the same (i.e., halothane via an increase in the open time and propofol via a decrease in the closed time). Thus, although both halothane and propofol augmented the channel open probability, thereby causing an increase in charge transfer during inhibitory transmitter action, they acted by different mechanisms.
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Acknowledgments
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This work was supported by Grant AA07836 from the National Institutes of Health.
The authors thank Nayla Hasan for technical assistance and Julia Irizarry for secretarial assistance.
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Accepted for publication March 2, 2004.
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Abstract
Introduction
