Single-channel basis for conductance increase induced by isoflurane in Shaker H4 IR K+ channels

Jichang Li and Ana M. Correa

Department of Anesthesiology, School of Medicine, University of California, Los Angeles, California 90095-7115


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Volatile anesthetics modulate the function of various K+ channels. We previously reported that isoflurane induces an increase in macroscopic currents and a slowing down of current deactivation of Shaker H4 IR K+ channels. To understand the single-channel basis of these effects, we performed nonstationary noise analysis of macroscopic currents and analysis of single channels in patches from Xenopus oocytes expressing Shaker H4 IR. Isoflurane (1.2% and 2.5%) induced concentration-dependent, partially reversible increases in macroscopic currents and in the time course of tail currents. Noise analysis of currents (70 mV) revealed an increase in unitary current (~17%) and maximum open probability (~20%). Single-channel conductance was larger (~20%), and opening events were more stable, in isoflurane. Tail-current slow time constants increased by 41% and 136% in 1.2% and 2.5% isoflurane, respectively. Our results show that, in a manner consistent with stabilization of the open state, isoflurane increased the macroscopic conductance of Shaker H4 IR K+ channels by increasing the single-channel conductance and the open probability.

general anesthetics; volatile anesthetics; voltage-gated channels; mechanisms of action; single channels


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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GENERAL ANESTHETICS act on the central nervous system (CNS) to produce a reversible state of unconsciousness, amnesia, and analgesia. The molecular mechanism(s) of action are a matter of ongoing research, and, because of the multiplicity of systems and molecules targeted by anesthetic agents, the mechanisms are bound to be varied and complex. In terms of ion channels, it is well established that the CNS depressive action of volatile anesthetics entails modulation of neuronal activity involving GABAA-, nicotinic ACh (nACh)-, and glycine-mediated synaptic responses (1, 6, 9). Voltage-gated ion channels, on the other hand, are generally not considered to be highly sensitive sites for the action of volatile general anesthetics (10); nonetheless, it is recognized that these agents, at clinically relevant concentrations, alter signal transmission dependent on functional voltage-gated channels. Small perturbations in the function of voltage-gated channels affect patterns of neuronal firing and excitability; consequently, effects of general anesthetics on specific ion channels may, in fact, be critical to the establishment of general anesthesia.

Of the voltage-gated channels, K+ channels play an important role in neuronal excitability as they set the resting potential, keep fast action potentials short, terminate periods of intense activity, slow the rate of repetitive firing, and generally lower the excitability of the cell when open. Accumulating evidence indicates that some CNS anesthetic actions can be related to effects on K+ channels and that various anesthetic agents can affect different kinds of native and recombinant K+ channels (3, 4, 25, 28). The modulation of K+ channels by volatile anesthetics is diverse in nature, suggesting different molecular mechanisms of action. Whereas some K+ channels are unaffected by certain volatile anesthetics or alcohols (5, 20), in others some relatively specific effects have been described. Volatile anesthetics have been found to activate baseline K+ channels in rat cerebellar granule neurons (29) and rat motor neurons (25), cloned mammalian two-pore-domain K+ channels (21), and isoflurane in rat hippocampal and human neocortical neurons (2) or to block dShaw2 voltage-gated K+ channels (15). Differential effects of halothane, isoflurane, chloroform, and enflurane on voltage-gated and resting K+ channels have also been described (3, 21). In addition, cloning and mutational analysis of K+ channels have helped identify specific sites or regions (15, 21) in the channel proteins that are part of the effector sites of the anesthetic(s) or that at least participate in developing their effects, lending support to specific anesthetic-protein interactions.

Here we have used the H4 IR NH2-terminal mutant of the Shaker B K+ channel as a model to investigate the effect of the volatile anesthetic isoflurane on a specific class of K+ channels. In a previous study, Correa (3) showed that H4 IR Shaker K+ channels exhibit a differential response to three general anesthetics: halothane, chloroform, and isoflurane. In contrast to the depression of the macroscopic conductance (GK) produced by halothane and chloroform, isoflurane induced an increase in GK and a slowing down of current deactivation. It was concluded that isoflurane alters the macroscopic properties of the H4 IR channel by stabilization of the open state of the channel. To further investigate the molecular basis of the action of isoflurane, we have used the patch-clamp technique (14) to study the effect of this anesthetic agent on the single-channel properties of the Shaker H4 IR K+ channels expressed in Xenopus oocytes. We report here the results obtained under identical experimental conditions by using mean-variance analysis of a population of channels and performing single-channel studies. We have found that the increase in macroscopic current induced by isoflurane results from an increase in single-channel conductance and in the open probability, both of which are consistent with an overall higher GK and in line with the proposed isoflurane-induced stabilization of the open state of the channel (3).


    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
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Materials. Frogs (Xenopus laevis) were purchased from either Xenopus I or Xenopus Express. Isoflurane (Forane) was purchased from Ohmeda Carbide. All other chemicals were from Sigma.

K+ channel clones and cRNA transcription. The cDNA for the noninactivating mutant of the Drosophila Shaker B K+ channel (Delta N6-46) (17) used in this study was modified for maximal expression: the ZH4 IR pBSTA (26) was a generous gift of Drs. D. Starace and F. Bezanilla. The cDNA was transcribed with the commercial T7 mMessage mMachine kit (Ambion). The integrity of the transcribed RNA was assessed in agarose gels, and the concentrations were measured spectrophotometrically.

Oocytes. X. laevis oocytes were isolated from frog ovaries by following conventional protocols (13). Oocytes were defolliculated by collagenase treatment (collagenase type AI; GIBCO BRL) in Ca2+-free Ringer solution, followed by a progressive change to the normal frog Ringer solution containing 1.8 mM Ca2+. Defolliculated oocytes were kept in Ringer solution (standard oocyte solution) consisting of 100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM HEPES-Na, and 50 µg/ml gentamicin, pH 7.2. RNA samples (2-40 ng/µl) were injected into the oocytes within 24 h of isolation. The injected oocytes were used for study from 1-5 days after injection. Animal procedures were approved by the Animal Research Council at the University of California, Los Angeles (UCLA).

Recording solutions. Macroscopic and single-channel currents were recorded with 5 KMS [5 mM potassium methane sulfonate (KMS), 115 mM sodium methane sulfonate (NaMS), 1.8 mM CaCl2, and 10 mM HEPES-Na, pH 7.2] in the pipette and with 100 KMS (100 mM KMS, 10 mM HEPES-K, and 1 mM EGTA-K, pH 7.2) in the bath. The pipette solution in tail current experiments was 20 KMS (20 mM KMS, 60 mM NaMS, 40 mM N-methyl-D-glucamine methane sulfonate, 10 mM HEPES, and 1.8 mM CaCl2, pH 7.2). Anesthetic solutions were prepared by equilibrating the bath solutions for 30 min with isoflurane-containing air (Ohio clinical vaporizer) in an Equilibrator (R. S. Weber and Associates, Westlake Village, CA) to final concentrations of 1.2 vol% (0.4 ± 0.1 mM) and 2.5 vol% (0.8 ± 0.3 mM), approximately equivalent to 1 and 2 MAC (minimum alveolar concentration), respectively. Equilibrated solutions were collected into gas-tight syringes and delivered through Teflon tubing by a gravity-fed perfusion system. Perfusion with the bath solution alone had no effect on channel currents. Anesthetic concentrations were measured by head space sampling with a gas chromatograph (Hewlett-Packard HP 6890 with head space sampler HP 7694) from an aliquot of the anesthetic-containing solution withdrawn directly from the equilibration chamber.

Electrophysiology. Ionic currents were recorded with the patch-clamp technique using an Axopatch 200A amplifier (Axon Instruments) controlled by a custom-built acquisition system. Data were acquired by sampling at 100-200 kHz. Macroscopic and single-channel currents were recorded in cell-attached and inside-out patches. Patch electrodes were pulled (model P-97 micropipette puller, Sutter Instruments) from borosilicate capillaries (Warner Instrument). The shanks were coated with Sylgard (Dow Corning), and the tips were heat-polished to tip resistances of 1.5-4.0 MOmega for macroscopic current recording and 15-60 MOmega for single-channel current recording.

For nonstationary noise analysis, macroscopic currents were acquired filtered at 30 kHz from patches with 0.6-3.4 nA of current measured at 70 mV. The mean and variance of the mean were calculated using custom-made software; four hundred records were used to construct the mean under each experimental condition. Leak was subtracted off line, with subtraction pulses collected throughout the run. The variance of the records with respect to the mean current was computed by pairs to compensate for time-dependent shifts in the mean (23). The relationship between mean and variance is described by the equation: sigma 2 = i · I - I2/N, where for any given potential, sigma 2 is the variance, i is the single-channel current, I is the macroscopic mean current, and N is the number of channels. Values for N and i are then estimated from the fit of the variance vs. mean data to the equation above. Because at any given time t, I(t) = N · i · Po(t), where Po is the probability of opening, the apex of the parabola that relates the variance to the mean is at the point at which the probability of opening is 0.5; at either end, Po = 0 or Po = 1, the variance is zero. There are two important issues that need to be mentioned. First, to get the most accurate estimates of N and i, the Po should be >0.5, which is the reason we chose 70 mV to run the mean-variance analysis. Second, although not a quantitative measure, an increase in Po can be visualized directly from the plot of the variance vs. the mean. The limiting value of Po (see Tables 1 and 3) was calculated from I = N · i · Po, where, in this case, I is the value of the mean current at the end of the pulse, and the values of N and i are obtained from the fit of the variance-mean plot as described above.

                              
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Table 1.   Single-channel parameters from nonstationary noise analysis of patch macroscopic currents

Single-channel current recordings were filtered at 10 kHz and refiltered at 2.5 kHz for data analysis. Analysis was performed on the digitally stored data with custom-written software. Subtraction of capacitive transients and leak was done off line by using blank traces to construct the subtraction trace. In some single-channel experiments (see Table 4), the holding potential was set to 0 mV to reduce the activity during the pulse and allow better resolution to measure single-channel event size.

Fits were performed with NFIT and Sigma Plot 5.0. All experiments were performed at room temperature (~20-22°C). Data are presented as means ± SE. Statistical significance to P < 0.05 was assessed by paired t-test analysis.

Action potential simulations. Simulations were performed with the use of the program Nerve (http://pb010.anes.ucla.edu), written by Dr. Francisco Bezanilla (UCLA), used in the membrane action potential and propagated action potential modes. The program solves sets of differential equations developed by Hodgkin and Huxley from data obtained experimentally from the squid giant axon. The equations include the voltage dependence of nerve Na+ (fast inactivating) and K+ (delayed rectifier) conductances as described by Hodgkin and Huxley (16). Individual action potentials were elicited by brief stimuli: 0.25-ms, 10-µA current pulses. When paired stimuli were applied, channels were allowed to recover before the second stimulus of equal length and amplitude was applied. Trains of impulses were elicited by a sustained depolarizing current, large enough to initiate continuous spontaneous activity: 2 µA, with default GNa, or 5 µA, with reduced GNa. Changes in GK and GNa were introduced as percent changes in the conductances relative to the default values: 1%, 2%, 3%, 5%, 10%, 15%, 20%, and 30% increases in GK and 10% and 20% decrease in GNa. Except for the temperature, which was either the default value (6.3°C) or higher (10 and 15°C), all other variables and parameters were maintained at default values.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Macroscopic currents. Isoflurane, at 1.2% and 2.5%, induced an increase in macroscopic K+ currents measured in patches from oocytes injected with the cRNA for the Shaker H4 IR K+ channel. The increase was specific to the expressed channels because it was not observed in uninjected oocytes or in silent patches of injected oocytes, and it was specific to isoflurane treatment because simple exchange of the bath solution did not produce the effect. Figures 1 and 2 illustrate the effect of isoflurane on currents elicited by depolarizing pulses from a holding potential of -90 mV. In external 5 KMS (Fig. 1), the noninactivating K+ currents are outward throughout the voltage range tested (from -80 to 80 mV). The macroscopic current-voltage (I-V) plot in Fig. 1 is the average of six experiments performed before, during, and after treatment with 2.5% isoflurane. Treatment with isoflurane induced an increase in current amplitude for all test potentials positive to -40 mV; the effects, however, were greater and significant only at 45 mV and above, a range at which the control currents show evidence of reduced conductance. Negative to 45 mV, the average change (13 ± 0.3%, n = 6) was constant between -5 and 45 mV. The current magnitudes measured at the end of 6-ms pulses to 70 mV varied between 0.6 and 1.3 nA (n = 6) in the controls. Isoflurane (1.2% and 2.5%) significantly increased macroscopic current amplitudes, averaging 12% (n = 5, not shown) and 30% (n = 6, Table 1), respectively. The effect on the currents is shown throughout the pulse, and the increase was partially reversed by wash (Figs. 1 and 2).


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Fig. 1.   In patches, isoflurane induced an increase in macroscopic K+ currents (average increase: 30.4 ± 6.8%, n = 6 patches). The effect was partially reversible upon wash. Macroscopic current-voltage (I-V) plots show maximum current magnitude (mean ± SE, n = 6) at the end of the test depolarization vs. test pulse potential. Currents were recorded at voltages from -80 to 80 mV from a holding potential of -90 mV and were normalized to the control value () at 80 mV. Isoflurane (2.5%; ) caused an increase in current amplitude from potentials positive to -40 mV. The effect, however, is significant from 45 mV and higher. The apparent rectification of control K+ currents at very positive potentials was less evident with isoflurane and after wash. The effect of isoflurane was partially recovered with wash (triangle ). Inset: sample macroscopic currents recorded during pulses to 70 mV from a holding potential of -90 mV. Pipette solution, 5 potassium methane sulfonate (KMS); bath solution, 100 KMS (see text for detailed contents of KMS solutions). Hp, holding potential.



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Fig. 2.   Families of macroscopic currents: control (A), 2.5% isoflurane (B), and after wash (C). Isoflurane increased pulse and tail-current amplitudes; after wash, both pulse and tail currents partially recovered. D: normalized mean conductance (mean ± SE, n = 5) vs. voltage relationship obtained from the peak amplitude of the tail currents upon return to -90 mV after depolarizing test pulses to the indicated potentials. Data were normalized to the control values from +100 mV back to -90 mV. A substantial, partially reversible increase in conductance was induced by isoflurane at all potentials. Pipette solution, 20 KMS; bath solution, 100 KMS.

The consistent, reversible isoflurane-induced increase in the ionic currents seen during test depolarizations was also prominent in tail-current experiments like that illustrated in Fig. 2. Both 1.2% and 2.5% isoflurane had marked effects on tail-current amplitude and duration. As shown with the families of curves in Fig. 2, A-C, 2.5% isoflurane dramatically increased the tail currents and slowed down the current return to baseline; the effect was partly reversible upon wash (Fig. 2D, n = 5). The average increase in tail-current amplitude was 27 ± 0.2% (n = 5) and was constant from 30 to 100 mV. Likewise, the average increase in the macroscopic currents during the preceding test depolarizations was 31 ± 0.2% (n = 5) and was constant for the same voltage range. The time constants of the fast (tau f) and slow components of tail-current decay (tau s) increased in isoflurane in a concentration-dependent manner (Table 2); the values of tau s were 41% and 136% larger in 1.2% and 2.5% isoflurane, respectively.

                              
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Table 2.   Time constants of tail-current decay

The data in Figs. 1 and 2 (see also Tables 1 and 3) are consistent with our previous findings determined with the use of the cut-open oocyte voltage-clamp technique, validating the use of patches to investigate the biophysical, single-channel basis of the isoflurane-induced changes.

                              
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Table 3.   Single-channel parameters from noise analysis in tail-current experiments

Nonstationary noise analysis of macroscopic currents. Noise analysis is a powerful tool for use in investigating single-channel properties. It yields reliable quantitative estimates of single-channel parameters (24). For a homogeneous population of statistically independent channels, the mean current, I(t), and the current variance, sigma I(t)2, are given by the following equations: I(t) = N · I · Po(t) and sigma I(t)2 = N · i2 · Po(t)[1- Po(t)]. Provided that I and sigma 2 are estimated from various Po, i and N can be determined by fitting mean-variance data according to sigma 2 = i · I - I2/N. Figure 3 provides an example of the nonstationary macroscopic current mean-variance analysis performed to investigate the influence of isoflurane on single-channel properties. In this experiment, 2.5% isoflurane significantly increased the single-channel current from 1.09 pA to 1.35 pA. The limiting maximum open probabilities, obtained from the equation I = N · i · Po, were also increased by 23% (0.69 vs. 0.85, patch 6). The results from six such experiments are given in Table 1.


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Fig. 3.   Nonstationary noise analysis. A: averaged currents recorded from macropatches in response to a depolarization to 70 mV. Traces are averages of 400 recordings before (control) and after perfusion of 2.5% isoflurane to the bath, which resulted in an approximate increase in current of 30% measured at the end of the pulse. B: averaged ensemble variance of the experiment shown in A. Variance was computed by pairs (23). C: variance as a function of mean current. Continuous curves represent data fits to the equation sigma 2 = i · I - I2/N, where sigma 2 is the variance, i is the single-channel current, I is the mean macroscopic current; and N is the total number of channels. The fits yielded the indicated values for i and N. Data were filtered at 30 kHz. Pipette solution, 5 KMS; bath solution, 100 KMS.

On the average, the estimated single-channel current, i, and the calculated values of Po were significantly larger in isoflurane (17.4% and 20.7%, respectively; P > 0.05), while changes in N were not significant (Table 1) and were the least consistent. The effects were partially reversed with wash (Table 1). The effects were dependent on the concentration of isoflurane. In 1.2% isoflurane, the Po was 11% higher than control (0.72 ± 0.03 vs. 0.80 ± 0.02; n = 6, P < 0.05), while the increase in i of 5% (1.04 ± 0.05 vs. 1.09 ± 0.03 pA; n = 6, P > 0.05), although consistent, was not significant (data not shown).

Similar results were obtained in tail-current experiments. In 20 mM external K+, 2.5% isoflurane induced increases in I, i, and Po during the test pulse to 70 mV (Table 3) that were the same as those obtained with 5 mM K+ in the pipette. As shown in Fig. 2, the peak amplitude and time course of the currents upon repolarization to -90 mV were appreciably modified by anesthetic treatment. Mean-variance analysis of tail currents showed that the changes induced by 2.5% isoflurane (Table 3) are similar to those seen during the preceding pulse to 70 mV. The mean increase in i of 17.3%, although not significant (P > 0.05), compares well to the 17.5% increase observed during the test pulse to 70 mV (Table 3). The initial Po(t=0) (instantaneous Po immediately after the change to -90 mV) was 24.8% higher during perfusion with isoflurane relative to control (Table 3); this value is higher but in the same order of magnitude as the 21.5% increase during the pulse to 70 mV. Again, the change in N was not significant, and the effects were only partially reversed by wash.

Based on the estimated value of the single-channel-event amplitude obtained from the fit of the mean-variance data (Table 1), the calculated single-channel conductance, gamma o, was ~17% higher in isoflurane, proportional to the increase in i. To directly assess the change in gamma o, the amplitude of single-channel openings was determined from patches with few (1-3) channels.

Single-channel recording. The records shown in Fig. 4, top, are selected records that illustrate typical unitary Shaker H4 IR K+ currents in a patch with two channels during a test pulse to 70 mV. Visual inspection of the traces reveals that opening events in 2.5% isoflurane (Fig. 4, top right) were characterized by more stable openings, i.e., less flicker, than in control conditions (top left). At this potential, the amplitudes of control unitary currents averaged 1.4 pA (n = 3 patches, average of 100-150 events each) with 5 mM K+ in the pipette (Table 4). The unitary event sizes of these three patches averaged 1.7 pA after perfusion with 2.5% isoflurane (23% higher than control; Table 4). A similar result was obtained in patches exposed to 20 mM K+ (Table 4); the average single-channel current increased from 1.2 pA to 1.4 pA (n = 4 patches; 100-150 events per patch), a 20% increase.


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Fig. 4.   Top: selected single-channel traces recorded before (left) and after (right) perfusion of isoflurane (0.8 mM). Traces were recorded at 70 mV from a holding potential of -90 mV. Channel openings are represented by upward deflections. In isoflurane, event amplitudes increased and opening events exhibited less flickering. Data were filtered at 2.5 kHz. Bottom: single-channel I-V plots of unitary currents (mean ± SE) filtered at 2.5 kHz vs. test potential. Mean unitary currents were obtained from the average of ~150 single-channel events to the indicated test potentials. Isoflurane () increased by 19.4% the single-channel conductance (gamma o) calculated from the slope of the I-V plot. Lines are linear regressions to the data. Pipette solution, 5 KMS; bath solution, 100 KMS.


                              
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Table 4.   Single-channel parameters from single-channel recordings

The current-voltage plot in Fig. 4, bottom, was obtained from data recorded over a range of potentials from 10 to 70 mV. The plot illustrates the increase in unitary conductance observed when the patch was exposed to isoflurane 2.5%, in this example, an increase of ~18% (Fig. 4, bottom). Table 4 contains the values for gamma o obtained from the linear regression of single-channel I-V plots like that shown in Fig. 4 for seven patches under different conditions. On average, the single-channel conductance was a few percentages higher (2-4%) in external 20 KMS (patches 4-7) than in 5 KMS (patches 1-3). Most importantly, isoflurane induced a consistent increase in gamma o in all seven patches, averaging 22% in 5 KMS and 20% in 20 KMS.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have used the noninactivating mutant of the Shaker B K+ channel, the H4 IR, as a model to study anesthetic-induced changes in single-channel properties of a voltage-gated K+ channel. In a previous study, Correa (3) reported that isoflurane induced a significant increase in GK and a slowing down of current deactivation and proposed that the effect could be attributed to stabilization of the open state. To provide the single-channel basis for this phenomenon, we have used the patch-clamp technique to study macroscopic and single-channel currents under identical conditions.

The macroscopic currents recorded from patches containing several hundred to a few thousand channels had the same characteristics and the same response to isoflurane as the currents recorded with the cut-open oocyte voltage-clamp technique from a significantly larger area of the oocyte surface (3) and under different experimental conditions. In isoflurane, macroscopic currents were larger at all potentials at which channels open. In addition, the magnitude and the time course of the currents upon repolarization were enhanced. The effects were only partially reversible upon wash. Lack of full reversibility may result from partitioning of the anesthetic into the yolk of the oocyte, thus contributing a constant supply even after prolonged wash; this would be more critical when recording in the cell-attached mode. Limited reversibility may also arise from lack of direct access to the membrane patch and/or the pipette solution; anesthetic would thus remain in small amounts in the vicinity of the patch even after intensive exchange of bath solution.

We found that the difference in maximum current amplitude during test depolarizations after isoflurane treatment was only significant at potentials positive to 45 mV in external 5 KMS. At these potentials, control macroscopic currents show signs of rectification, which has been attributed to entry into a nonconducting state accessed from the open state (17, 22). The fact that the effect of isoflurane is more prominent at very positive potentials suggests that one action of the anesthetic could be the relief of such rectification by reducing the rate at which channels enter this nonconducting state. This effect would stabilize the open state of the channel by eliminating or making less frequent that exit route. An immediate prediction from this hypothesis is that the mean open time of the channel would be longer in the presence of the anesthetic. The single-channel records shown in Fig. 4 hinted at an increased dwell time in the open state at 70 mV in isoflurane. Preliminary analysis of single-channel records indicates that, in fact, the mean open time at 70 mV is ~30% longer in 2.5% isoflurane (1.17 ± 0.02 ms, 4,809 events) relative to the control (0.87 ± 0.01 ms, 5,580 events). Also, the mean close time is close to 9% shorter in isoflurane (0.34 ± 0.01 ms, 5,684 events in the control vs. 0.31 ± 0.01 ms, 5,051 events after isoflurane).

The effect of isoflurane appeared more prominent during repolarization, associated with channel closing, than during the depolarizing test pulses. Tail-current kinetics were slower in isoflurane, producing appreciable currents upon return to holding. The average effect in terms of the amplitude of the tail currents, however, was the same as that during the test pulse (27% vs. 31%, 30-100 mV), indicating that the change was proportional to the voltage and involved an increase in the macroscopic conductance. Because slower deactivation contributes significant current density during repolarization, this may be the most relevant physiological effect of the anesthetic, which could contribute to changes in firing patterns (see below).

An increase in macroscopic conductance could result from an increase in any of the following single-channel parameters: the single-channel conductance, gamma o; the number of channels in a patch, N; or the probability of opening, Po. The results presented in this paper, which were obtained with two independent techniques, support the idea that isoflurane induces an increase in the Po and gamma o of the channels already present in the membrane. Mean-variance analysis indicated that isoflurane induces an increase in the single-channel current amplitude. The value of i was consistently higher in isoflurane compared with control. Because the mean-variance analysis was done at 70 mV, a potential at which, as mentioned above, the increase in K+ currents could involve a reduction of the number of channels in the nonconducting state, the changes in K+ conductance would not necessarily be observed at more negative potentials. Evidence that the effect occurs at other potentials as well was provided by the observed increase in single currents of open-channel events at various potentials. Single-channel I-V plots demonstrated that i was larger for all potentials tested and that gamma o was increased by the anesthetic. If, in fact, isoflurane increases the conductance of the single channel, this finding would be rather unique for a voltage-gated channel. It is likely, however, that the increase in gamma o is apparent, a consequence of better resolved events, in view of the fact that channels flickered less in the presence of isoflurane (Fig. 4) relative to control conditions.

The second line of evidence that the effect of isoflurane takes place throughout the voltage range tested is provided by the tail-current experiments. The curve shown in Fig. 2D gives evidence that the conductance of the channels was affected for all potentials at which channels open. Moreover, maximum currents during the test pulses and peak tail currents upon repolarization were modified by the anesthetic to the same extent (~30% increase) in a voltage-independent manner for potentials more positive than 30 mV. Conversely, in external 5 KMS, the increase in macroscopic currents was smaller (13%) and was voltage independent only between -5 and 45 mV. It is tempting to hypothesize that the external [K+] can influence anesthetic interaction with the channel. K+-dependent K+ current inhibition by the intravenous general anesthetics has been reported in a study done on sympathetic nervous system-derived human neuroblastoma cells (SH-SY5Y) (11). Increases in the extracellular [K+] promoted delayed rectifier K+ current block by etomidate and propofol (but not ketamine), suggesting an active role of K+ on the extent of anesthetic effect. The results of mean-variance analysis reported here in Tables 1, 3, and 4, indicate that this is unlikely, because the change in i and Po induced by isoflurane was the same irrespective of the [K+]. Also, although gamma o was slightly larger in higher external K+, the anesthetic-induced increase in gamma o was independent of [K+]. Further experimentation is required to resolve this apparent discrepancy.

The value of i obtained through noise analysis (Tables 1 and 3) was smaller than the unitary amplitude at 70 mV measured from single-channel events (Table 4) even in the controls. Although not significant, this difference could result from the estimation of i, which is influenced by the open channel noise, normally giving an underestimate of the actual single-channel current (8). Because the size of an opening can be better resolved from the individual events, the magnitude of single-channel currents and the gamma o are more reliably determined from analysis of single channels. The values of gamma o reported here are consistent with those previously reported for this mutant (17).

The time course of tail currents was dramatically slower in isoflurane than in control conditions irrespective of the potential visited during the pulse. From the single-channel perspective, this means that isoflurane-treated channels take longer to go from the Po attained during the depolarizing pulse to that at the holding potential. Subsequent analysis of single-channel kinetics (in progress) should provide further insight to the steps, states, and rates that are modulated by the anesthetic.

Although the Shaker B H4 IR K+ channel is a model channel used to explore molecular mechanisms of action of anesthetics, the findings reported in this paper have implications for anesthesia because of the prominent role K+ channels play in setting the resting membrane potential and the rate of repolarization as well as determining, along with other voltage-gated channels, the overall excitability of neuronal tissue. Small changes in the activity of K+ channels can induce significant changes in the behavior of excitable cells. The concentrations of anesthetics used in this study, 1.2 and 2.5 vol%, are close to 1 and 2 MAC for isoflurane. In both concentrations we found an increase in open channel probability and a retardation of current decay back to baseline upon repolarization. Although clinically relevant concentrations of isoflurane are normally close to 1 MAC, the effects described here at concentrations >= 1 MAC are likely to occur during general anesthesia.

Volatile general anesthetics inhibit spontaneous and evoked neuronal activity in various parts of the CNS. It is well established that the molecular mechanisms of the depressive action of volatile anesthetics comprise the modulation of neuronal activity involving GABAA-, nACh-, and glycine-mediated synaptic responses and the modulation of several kinds of voltage-gated ionic channels (10). A number of publications have already indicated that volatile anesthetics modulate different kinds of native or recombinant potassium channels (12, 18, 19, 21, 27, 28, 30, 31). Our results indicate that isoflurane affects certain K+ channels during depolarization and repolarization. To evaluate the possible contribution of such changes at the cellular level, we performed simple action potential simulations with the program Nerve, which is based on the Hodgkin-Huxley model for action potential generation and propagation derived from the activity of squid nerve fast-inactivating Na+ channels and delayed rectifier K+ channels. Because the Shaker IR K+ channel has characteristics of a delayed rectifier K+ channel, we used the simulations to explore the influence of increases in GK, like those obtained in the present work, on the generation and propagation of action potentials. Increases in GK (from 1% to 30%) produced delays and amplitude reduction in successive action potentials triggered by paired stimuli or by maintained depolarizing currents. In fact, early termination of a train of impulses occurs with GK increases of 5% under sustained depolarization, producing effective filtering of action potential trains. Furthermore, the influence of increases in GK was more pronounced when a reduction of GNa, similar to that reported for isoflurane-treated mammalian Na+ channels (7), was included in the simulation. One could hypothesize that, combined, these effects could produce a significant alteration of nerve conduction and firing frequency. However interesting, this hypothesis remains to be tested in vitro.

Identification of general anesthetic binding sites on K+ channels is a matter of active research. Experiments involving mutational analysis on anesthetic-sensitive and -insensitive K+ channels have shed light on segments of the channel proteins that are required for anesthetic-channel interactions. Recent work on molecular determinants of 1-alkanol action on the dShaw2 (15) determined that the structural integrity of a 13-amino acid region in the cytoplasmic loop between membrane segments S4 and S5 is necessary for 1-alkanol action. Likewise, activation by volatile anesthetics of the human TREK-1 channel (21) depends on the COOH-terminal region of the channel. In view of the multiplicity of action of general anesthetics on various ion channels and the differential sensitivity to general anesthetics even within classes of channels, the molecular mechanisms of action are bound to be complex. Despite this complexity, the evidence clearly points toward direct anesthetic-protein interactions at specific sites, and although bilayer-mediated steps and modulation via other membrane-bound proteins may still play a crucial role, a solely bilayer-based anesthetic action is unlikely.

In conclusion, our results show that the volatile anesthetic isoflurane induces a reproducible increase of the macroscopic conductance of Shaker H4 IR K+ channels. Isoflurane modulates these K+ channels by effects on the single-channel properties. The increase in macroscopic conductance induced by isoflurane is associated with an increase in the single-channel conductance and the opening probability, which supports stabilization of the open state of the channel. The relevance of our findings at the cellular level needs yet to be addressed, but together with other data on volatile anesthetic action on voltage-gated channels, these results suggest that volatile general anesthetics may alter neuronal excitability during anesthesia by mechanisms that involve the fine modulation of the activity of voltage-gated ion channels.


    ACKNOWLEDGEMENTS

We thank Drs. D. Starace and F. Bezanilla for the cDNA of the ZH4 IR mutant of the Shaker B K+ channel, E. Grigorova for oocyte isolation, N. Shafaee for technical assistance, and Drs. D. Sigg and F. Bezanilla for guidance and helpful discussion.


    FOOTNOTES

This project was supported by National Institute of General Medical Sciences Grant GM-53781 (to A. M. Correa) and the Department of Anesthesiology, University of California, Los Angeles (UCLA).

Preliminary reports of these data have been published in abstract form (19) and presented in poster form at the Susumu Hagiwara Memorial Symposium, UCLA, 1999.

Address for reprint requests and other correspondence: A. M. Correa, Dept. of Anesthesiology, UCLA, BH-509A CHS, PO Box 951775, Los Angeles, CA 90095-7115 (E-mail: nani{at}ucla.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 19 April 2000; accepted in final form 22 November 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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