There is increasing evidence that direct interactions between volatile anesthetics and channel proteins may result in general anesthesia. Using voltage-clamp techniques, the authors examined the effect of two general anesthetics (ketamine and halothane) on a rat brain potassium channel of known amino acid sequence, and further assessed whether the inhibition of the channel is altered by a partial deletion of the C-terminal sequence of this channel.
Xenopus laevis oocytes were microinjected with either Kv2.1 or delta C318 (a mutated channel in which the last 318 amino acids of the C-terminus have been deleted) cRNA, and channel function in translated channels was observed before, during, and after exposure to graded concentrations of ketamine (25, 50, and 75 micrometers) and halothane (1%, 2%, and 4%).
Ketamine and halothane reduced Kv2.1 and delta C318 peak current amplitude in a dose-dependent and reversible fashion. The inhibition of current was voltage dependent for halothane but not for ketamine. Halothane accelerated the time constant of current inactivation, whereas ketamine affected this parameter minimally in both channel types. Use dependence of ketamine and halothane action was observed in both Kv2.1 and the mutant channel, attributable to augmentation of C-type inactivation.
Although both ketamine and halothane inhibit potassium currents through the Kv2.1 channel, their mechanisms of action at this potential target may be different. Deletion of the C-terminal sequence resulted in decreased sensitivity to both anesthetics. Although it is not clear whether anesthetics interact directly with the C-terminus, which is thought to reside intracellularly, this portion of the channel protein clearly influences the actions of both anesthetics.
Key words: Anesthesic action: potassium channels. Anesthetics, intravenous: ketamine. Anesthetics, volatile: halothane. Animals, Xenopus laevis: oocytes. Techniques: voltage clamp.
DESPITE their widespread clinical use, the mechanisms of action of general anesthetics remain unresolved. There is an extensive body of discordant literature about whether general anesthetics act on membrane proteins directly, or secondarily, after primary action on membrane lipids. Recent evidence is in favor of direct interactions between volatile anesthetics and channel proteins as the underlying mechanism of general anesthesia. Both voltage-gated and ligand-gated membrane channels are suggested targets for general anesthetic action. [1-7]The effects of general anesthetics on potassium channels are well reported. [1,3,5-7,8]Recently, Tinklenberg et al. studied the effects of isoflurane on drosophila carrying Shaker mutations, which affect IA(transient current) potassium channels. The rank order of behavioral insensitivity to isoflurane was correlated with the degree to which potassium conductance through the IAchannel was impaired, suggesting an involvement of IAin the anesthetic action of isoflurane. Inhibition of potassium channels, especially in central neurons, has been suggested to underlie some of the excitatory effects and emergence phenomena observed with dissociative anesthetics such as phencyclidine and ketamine. [9-11]Voltage-clamp studies have identified a diverse group of potassium channels with distinct characteristics in nerve cells. The cDNA encoding many of these potassium channels have been sequenced. [12-14]The subfamilies of voltage-gated potassium channels originally discovered in drosophila are conserved in mammals. The currents carried through these channels influence a number of functions in the central nervous system, including modulation and shaping of action potentials, temporal firing patterns in neurons, and cellular mechanisms affecting memory and learning. In this article, we examine the actions of two general anesthetics of widely differing structure, ketamine and halothane, on Kv2.1, a rat brain potassium channel, which produces a very slowly inactivating current on membrane depolarization. The proposed membrane arrangement of a typical voltage-gated potassium channel is shown in Figure 1. We extend our studies to examine a mutant of Kv2.1, delta C318, in which the last 318 amino acids of the C-terminus have been deleted (Figure 1). This mutant channel has previously been shown to be more sensitive to ethanol inhibition than the parent channel. The use of mutagenized channels allows elucidation of the relationship between protein structure and anesthetic action. A striking aspect of the most recent work on voltage-gated potassium channels is the modularity of channel protein construction, whereby particular stretches of the amino acid sequence appear to underlie specific physiologic functions such as voltage dependency of gating and the kinetics of inactivation. [12,18]This finding provides the possibility that modulation of channel activity by different anesthetics may be understood in terms of interactions of the drug with particular subdomains of the protein, with predictable physiologic consequences. While activation and inactivation of Kv2.1 still can occur after mutation of the C-terminal, the terminal has considerable effects on these kinetic parameters.
Materials and Methods
This study was approved by University of Massachusetts Medical Center's Animal Care and Use Committee. Ribonucleotide triphosphates, m sup 7 G(5')ppp(5')G and DNAse (RNAse free) were purchased from Pharmacia LKB (Piscataway, NJ). RNAsin was obtained from Promega (Madison, WI). Restriction enzymes were purchased from New England Biolabs (Beverly, MA). T3 or T7 RNA polymerase were purchased from either New England Biolabs or Bethesda Research Laboratories (Gaithersburg, MD). N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid], pyruvic acid (sodium salt), collagenase (Type IA) penicillin/streptomycin solution, and theophylline were obtained from Sigma Chemical (St. Louis, MO).
In Vitro Transcription
Runoff transcriptions were carried out in a standard transcription buffer (40 mM Tris HCl, pH 7.5; 6 mM MgCl2, 2 mM spermidine HCl, 5 mM NaCl) containing 5 micro gram linearized DNA template, 500 micro Meter rNTPS, 500 micro Meter m7G(5')ppp(5')G, 64 units RNasin, 2 mM DTT, and 20 units RNA polymerase in a 50-micro liter volume. After 1 h at 37 degrees C, the templates were digested with DNase I for 30 min at 37 degrees C. The cRNAs were extracted in phenol-chloroform and precipitated with ethanol, dried, and then taken up in diethyl pyrocarbonate-treated water for injection into Xenopus laevis oocytes. The cRNAs were checked for concentration and size on formaldehyde gels prepared with 1% agarose.
Adult female Xenopus laevis frogs were maintained in artificial pond water at room temperature on a 12 h/12 h light/dark cycle. Dissected stage V and VI oocytes were defolliculated in Calcium2+ -free culture medium (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM N- [2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] [pH 7.5] containing 2 mg/ml collagenase). Oocytes were maintained in this medium, with continuous shaking at room temperature, for 2-4 h. After defolliculation, the culture medium was replaced with normal Calcium2+ -containing recording medium (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM N- [2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]; pH 7.5) with 2.5 mM Na pyruvate, 0.5 mM theophylline, 100 units/ml penicillin and 100 micro gram/ml streptomycin.
Oocytes were injected with Kv2.1 and delta C318 cRNAs using a 10-micro liter Drummond micropipette modified for microinjection (Drummond Scientific, Broomall, PA). The micropipette was backfilled with mineral oil and loaded with 2 micro liter cRNA in diethyl pyrocarbonate-treated distilled water. The cRNA concentrations injected varied between 10 and 50 ng/50 nl. After injection of approximately 50 nl of cRNA, the oocytes were placed in culture medium in 24-well plates and incubated at 18 degrees Celsius for at least 2 days, before recording. Media was changed every 1 or 2 days. Oocytes remained viable for up to 2 weeks after injection. The same batch of oocytes were injected with the cRNA for both Kv2.1 and the deletion mutant when making comparisons, whenever possible.
Individual oocytes were impaled with two microelectrodes for conventional two-electrode voltage clamping using a Dagan model 8500 voltage-clamp amplifier (Dagan, Minneapolis, MN) to provide voltage compliances sufficient to clamp the oocyte potential. The microelectrodes were filled with 3M KCl and had resistances of 1 or 2 M omega. The resting membrane potential of the oocyte was measured differentially with respect to another electrode in the extracellular bath fluid independent of the bath ground. Oocytes with resting potentials more depolarized than -50 mV were discarded. The recording chamber had a volume of 300 micro liter, and the oocytes were superfused with at least 15-18 ml of the recording medium in the presence or absence of ketamine or halothane at a rate of 2-2.5 ml/min, before channel characteristics were examined. Voltage-clamp records were collected and analyzed using PClamp 5.51 software (Axon Instruments, Foster City, CA). Capacitative and leak currents were subtracted off-line using a P/2 protocol. The time constant of current inactivation (taudecay) was computed by least-squares linear regression.
Ketamine hydrochloride powder supplied by Parke Davis (Morris Plains, NJ) and halothane supplied by Halocarbon Laboratories (River Edge, NJ) were used in this study.
Halothane Preparation and Delivery
All halothane solutions were prepared in a measured volume glass bottle, and placed on a rotating mixer (Blood mixer, Drummond Scientific) for equilibration overnight at room temperature. The method described by Renzi and Waud was used to determine the aqueous gas partition coefficient for halothane at 22 degrees C, the temperature at which experiments were conducted. The partition coefficient determined for halothane was 1.49+/-0.06 (n = 19). Equilibrated solutions of 0.675, 1.35, and 2.7 mM halothane corresponded to 1%, 2%, and 4% gaseous halothane, respectively. Gas chromatography was used to confirm the bath concentrations of halothane.
Halothane solution was delivered via tygon tubing, fittings, valves (Omnifit, UK) and metal extensions resistant to absorption of halothane. The concentrations of halothane administered to oocytes were confirmed by periodic collection and comparison of superfusate with freshly prepared halothane standards, using gas chromatography.
After subtraction of linear leakage and capacitative currents, amplitude and taudecaydata were analyzed by paired and unpaired Student's t test. Linear regression analysis by analysis of variance was used for data obtained during repetitive stimulation protocols. All data are presented as mean+/-standard error of the mean (SEM). A probability of < 0.05 was considered to be significant.
Oocytes microinjected with mRNAs encoding Kv2.1 and delta C318 channels exhibited depolarization-evoked potassium current. The size of the potassium current recorded after a 250 ms pulse from a holding potential of -50 or -60 mV was 22.90 micro A+/-1.74 (n = 48) for Kv2.1, and 32.97 micro A+/-1.84 (n = 53) for delta C318 (P < 0.01). Control measurements made on uninjected oocytes revealed endogenous currents that were 103-fold smaller than those of cRNA injected oocytes. The following parameters of potassium current were examined for their sensitivity to ketamine and halothane: (1) peak current amplitude, (2) taudecay, (3) voltage dependence of channel activation, (4) response to repetitive depolarizations, and (5) voltage dependence of anesthetic-induced channel inhibition. The terms, deletion mutant for Delta C318, and parent channel for Kv2.1, are used interchangeably throughout.
Effect on Kv2.1 Current Amplitude
Peak current carried through both Kv2.1 and Delta C318 channels was reduced in a concentration-dependent manner by both ketamine and halothane (Figure 2). Peak currents at all concentrations of ketamine and halothane tested differ significantly from control currents recorded in the absence of anesthetics (P < 0.001). Current traces recorded during a step to +50 mV are shown in Figure 2. Half-maximal reduction of peak current occurred within 15-30 s of the introduction of halothane, and maximal inhibition was reached after 4-5 min of drug exposure. The onset of ketamine inhibition of peak current was apparent after 1-2 min and peak inhibition was evident after 6-7 min of drug exposure. The effects of both ketamine and halothane were almost completely reversible in the majority of oocytes examined (Figure 2and Figure 3). Reversal of drug effects was complete within 5-6 min and 7-9 min after the start of washout periods of halothane and ketamine, respectively.
C-Terminal Deletion Decreases Anesthetic Effect on Peak Current Amplitude
The reduction in Kv2.1 peak current was 14.1+/-2.0%, 20.8 +/-2.6% and 27.9+/-2.9% in the presence of 25, 50, and 75 micro Meter ketamine, respectively (P < 0.001 vs. control for each concentration), whereas current through Delta C318 was inhibited by only 4.0+/-2.1%, 9.7+/-2.0%, and 14.5+/-3.1% at the same concentrations (P < 0.05 vs. control for 75 micro Meter). The deletion mutant was significantly less sensitive to ketamine than the parent channel at all three concentrations tested (P < 0.001). Delta C318 also was significantly less sensitive to halothane than was the parent channel at 1.35 and 2.7 mM concentrations (P < 0.001). Kv2.1 peak current was reduced by 19.2+/-2.0% in 0.675 mM, 29.5+/-1.6% in 1.35 mM, and 50.7+/-2.62% in 2.7 mM halothane (P < 0.001 vs. control for all 3 concentrations), whereas Delta C318 was inhibited by 13.7+/-1.9%, 20.7+/-1.7%, and 36.9+/-2.2% at similar halothane concentrations (P < 0.001 vs. control; Figure 3). The differences in current inhibition by halothane or ketamine on these two channels were not due the difference in the control current size, because Delta C318 currents equal to or smaller than Kv2.1 currents were still less sensitive to either drug.
Voltage Dependence of Channel Inhibition
The two drugs were strikingly different with respect to the effect of membrane potential on drug action. Conductance-voltage plots are shown in Figure 4for both channels, in the absence and presence of ketamine or halothane. The data have been fitted to the Boltzmann equation Equation 2where Vmis the membrane potential, V1/2 is the voltage at which conductance becomes half-maximal, and k is a slope factor. The steady-state activation curve for Kv2.1 achieved half-maximal conductance at 8.50+/-2.62 mV with a slope parameter of 13.83+/- 1.69. The corresponding curve for Delta C318 yielded a V1/2 of 5.07 +/-2.62 mV, slightly more negative than that of Kv2.1, with a similar k of 13.64+/-2.39. As a result, the threshold for activation of Delta C318 was shifted to a more hyperpolarized potential, consistent with previous findings. .
For both channels, ketamine inhibited conductance without altering either V1/2 or k significantly. In the presence of halothane, however, inhibition of both channels was accompanied by a concentration-dependent depolarizing shift in V1/2, while k was unchanged at all concentrations. In 2.70 mM halothane, the half-maximal conductance of Kv2.1 was shifted to 24.88+/-4.91 mV, while the V sub 1/2 for Delta C318 was shifted to 15.34+/-2.55 mV.
The voltage dependency of block is further illustrated by expressing the data as the fraction of channels blocked by each drug, according to the relationship: In [(gctrl/gdrug) - 1], as a function of the voltage step (Figure 5). Ketamine decreased the conductance of both the Kv2.1 and Delta C318 channels to the same degree at all potentials examined. In contrast, inhibition of peak conductance by halothane was significantly greater at less depolarizing potentials. The voltage dependency of halothane's effects on peak current amplitude did not result from a voltage dependence of halothane-induced acceleration of inactivation (see later), because the effects of halothane on the rate of inactivation were voltage-independent in all cases examined (data not shown).
Effect on Decay Kinetics
The effect of the two anesthetics on taudecayduring a 10-s depolarizing pulse differed greatly (Figure 6). Halothane had a potent effect on this channel parameter, accelerating inactivation for both clones tested. The taudecayfor the Kv2.1 current at 0.675, 1.35, and 2.7 mM halothane was significantly reduced by 56.3+/-2.0%, 62.7 +/-3.0%, and 79.4+/-2.0%, respectively (P < 0.001 vs. control) while the same concentrations of halothane reduced taudecayin Delta C318 by 44.5+/-5.5%, 52.1+/-3.5%, and 70.5 +/-1.5% (P < 0.001 vs. control). In contrast, ketamine had minimal effects on this parameter in both Kv2.1 and Delta C318. As with halothane, ketamine's effects on taudecaywere slightly greater in Kv2.1 than in Delta C318 (Figure 7). Effects on decay kinetics were fully reversible by washout of the anesthetic.
Effect of Ketamine and Halothane using a Repetitive Stimulation Protocol
Repetitive stimulation of potassium channels with a series of short depolarizing voltage pulses results in a cumulative inactivation of potassium current, which is dependent on the frequency of the voltage steps (Figure 8(A)). Cumulative inactivation occurs even under conditions where no significant inactivation is evident within each short pulse. This cumulative inactivation, which has been termed C-type inactivation, is a state-dependent and voltage-independent process that can occur from a closed state, before the channel opens. [20-22]It is less well defined structurally and functionally. C-type inactivation is thought to be influenced by the presence of the cytoplasmic C-terminal domain of the channel (Figure 8(A)), as well as by the ionic composition of the external milieu and amino acid sequence, especially at amino acid position 449 in the channel pore region. Both 50 micro Meter ketamine and 0.675 mM halothane enhanced the development of cumulative inactivation in each of the clones (Figure 8(B)). The slopes generated during repetitive pulse protocols in Kv2.1 and Delta C318 in the presence of halothane and ketamine were significantly different from control slopes measured before introduction of the anesthetics (linear regression by analysis of variance, P less or equal to 0.0001). Moreover, the enhancement of cumulative inactivation by halothane and ketamine was greater in Kv2.1 than in Delta C318 (Figure 8(B)).
The two potassium channels studied behaved similarly for both anesthetics in a number of respects. In particular, the deletion of 318 amino acids from the C-terminus of the channel, thought to reside on the cytoplasmic side of the membrane protein, resulted in a lesser inhibition of peak potassium current by either ketamine or halothane. Another similarity in the actions of both anesthetics was the augmentation of the cumulative inactivation that is apparent during repetitive stimulation. While the structural correlate of this phenomenon may not be a single discrete site, the C-terminus of the channel protein has been implicated in its generation. [21,22]In support of this, removal of the C-terminus resulted in a decrease in cumulative inactivation.
Some aspects of the actions of ketamine and halothane on these potassium channels were different. One of the major findings of the current study is that the kinetics of Kv2.1 current decay were significantly accelerated by halothane, whereas they were much less affected by ketamine. In each case, the acceleration of decay was less pronounced in the deletion mutant than in the parent channel.
Surprisingly, the two anesthetics also differed regarding the effects of transmembrane voltage on anesthetic action. Both Kv2.1 and Delta C318 currents were inhibited in a voltage-dependent fashion by halothane, whereas the reduction of current by ketamine was independent of membrane potential. Ketamine reduced the maximal conductance of both channels in a voltage-independent manner without affecting their steady-state activation kinetics. This suggests that ketamine is not acting as an open channel blocker. Rather, ketamine's binding site(s) may be located outside of the membrane electric field. Alternatively, because ketamine is only 50% protonated at the pH of our experiments (pKa7.5), the population of ketamine molecules expected to be in the uncharged state may represent the active drug component. It has been proposed that the high-affinity phencyclidine receptor in the brain is a voltage-gated potassium channel. Phencyclidine action on these channels appears to involve different mechanisms for different channel types, occurring via hydrophobic routes not requiring open channels for some delayed rectifier potassium channel currents, while requiring open channels to block IA, a transient potassium current. Unlike ketamine, halothane, which is uncharged, did show a voltage-dependent inhibition of potassium conductance, shifting both channels' V1/2 in the depolarizing direction. The most economical explanation for this finding is that voltage-induced changes in the conformation of the channel affect either the access or efficacy of halothane action. The state dependence of anesthetic action is well described for local anesthetic effects on sodium channels. This difference in the action of ketamine and halothane suggests different sites of action for the two drugs. Neither drug altered the slope of the steady-state activation curves for either channel, suggesting that drug-channel interactions did not affect the movement of gating charges within the membrane potential field.
The inhibition of potassium currents noted in the current study would presumably lead to increased neuronal excitability. If this effect is indeed relevant to the generation of general anesthesia, the most likely explanation would be by activation of inhibitory pathways within the brain. In addition, increased neuronal excitability attributable to inhibition of potassium conductances may contribute to the excitatory phase of general anesthesia induction.
It is difficult to extrapolate data from model systems such as frog oocytes used here to the complex intact animal and determine whether the concentrations of anesthetic employed are relevant for the mammalian anesthetic state. Effects of temperature on anesthetic requirements are particularly difficult to incorporate into such extrapolations. In humans, approximate plasma ketamine concentrations achieved during induction and maintenance of anesthesia are 100 micro Meter and 10 micro Meter, respectively. In rats, plasma concentrations greater than 50 micro Meter are reported to produce general anesthesia. [27,28]Thus, the potassium channel effects observed at the concentrations used in this study may be relevant during induction of anesthesia in both humans and rats (the species from which the channels were cloned). The concentration of unbound ketamine in plasma will be slightly lower than the applied concentrations in our studies, because approximately 12% will be bound by plasma proteins. The threshold concentrations of ketamine for actions on potassium channels are higher than the concentrations necessary for action at the NMDA receptor complex, [24,28]which will presumably play the principal role in the generation of anesthesia.
At 37 degrees C, halothane EC50or minimum alveolar concentration in the rat is 0.29 mM or 1.03%, respectively. We observed significant inhibition of taudecayand peak current amplitude at 0.675 mM halothane, which is 2.3 times higher than the EC50for the rat at 37 degrees C. However, because the EC50for volatile anesthetics decreases with decreasing temperature, the threshold values for potassium channel inhibition, which we observed at room temperature, are higher than 2.3 times the EC50. Importantly, the EC50concentrations for general anesthesia are much less affected by temperature when expressed as aqueous concentrations than when expressed as partial pressures. Previously, we have published results that show that Kv2.1 current amplitude is reduced at lower concentrations than used in this study (by approximately 14% in the presence of 0.5% halothane). .
In humans, higher inspired volatile anesthetic concentrations often are required during induction, compared to maintenance, of general anesthesia, suggesting possible clinical relevancy for the range of halothane concentrations employed in this study. In addition, small changes in potassium currents can lead to relatively large differences in physiologic processes, such as transmitter release and signal transduction. [31,32]It also is suggested that cumulative inactivation may play a prominent role in a number of functions, including frequency modulation of transmitter release. [20,33].
In conclusion, while both ketamine and halothane inhibit currents through the Kv2.1 channel, their mechanisms of action at this potential target for the production of anesthesia appear to be different. Most strikingly, halothane significantly accelerated the decay of potassium current through Kv2.1 and the deletion mutant, whereas this parameter was less affected by ketamine. Moreover, the voltage dependency of anesthetic action was significantly different for the two drugs. Deletion of the C-terminal sequence resulted in qualitatively similar effects on the actions of both anesthetics. While it is not possible to state that the anesthetics interact directly with the C-terminus, thought to reside intracellularly, this portion of the channel protein clearly influences the actions of each of these anesthetics. Interestingly, the same C-terminus deletion increases sensitivity to ethanol. .
It should be noted that our current level of understanding of sequence-function relationships in Kv2.1 is not adequate to draw definitive conclusions regarding the sites of action for the anesthetics studied. For example, while deletions in either the N- or C-termini may disrupt both activation and inactivation of these potassium channels, more extensive deletions of both termini can restore channel gating. .
The authors thank Dr. Rolf H. Joho and Dr. Arthur Brown, for the gifts of Kv2.1 and Delta C318 cDNAs, respectively. They also acknowledge Dr. S. Heard, for statistical analysis; S. St. Martin, in preparation of the manuscript; Andrew Wilson and Michael Perkins, for expert technical assistance; and Dr. B. Waud, in establishing the protocol to determine volatile anesthetic concentrations. Dr. Kulkarni and Dr. Zorn contributed equally to the manuscript.