Department of Anesthesiology, School of Medicine, University of California, Los Angeles, California 90095-1778
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The ShakerB K+ channel was used as a model voltage-gated channel to probe the interaction of volatile general anesthetics with gating mechanisms. The effects of three anesthetics, chloroform (CHCl3), isoflurane, and halothane, were studied using recombinant native and mutant Shaker channels expressed in Xenopus oocytes. Gating currents and macroscopic ionic currents were recorded with the cut-open oocyte voltage-clamp technique. The effects of CHCl3 and isoflurane on gating kinetics of noninactivating mutants were opposite, whereas halothane had no effect. The effects on ionic currents were also agent dependent: CHCl3 and halothane produced a reduction of the macroscopic conductance, whereas isoflurane increased it. The results indicate that the gating machinery of the channel is mostly insensitive to the anesthetics during activation until near the open state. The effects on the conductance are mainly due to changes in the transitions in and out of the open state. The data give support to direct protein-anesthetic interactions. The magnitude and nature of the effects invite reconsideration of Shaker-like K+ channels as important sites of action of general anesthetics.
Xenopus oocytes; chloroform; halothane; isoflurane; volatile anesthetics
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE CLOSE CORRELATION FOUND between the partition
coefficient of certain hydrophobic compounds and their anesthetic
potency led to the hypothesis that the effectiveness of an anesthetic was directly associated with its solubility in the lipid bilayer. Under
this proposal, anesthetics would affect the function of membrane
proteins through the perturbation of the physical properties of the
bilayer (16). Today there is no doubt that some membrane proteins are
specific targets for general anesthetics (e.g., see Ref. 7), and there
is no question as to the relevance of anesthetic action on the function
of ligand-gated channels, particularly -aminobutyric acid-activated
channels (e.g., see Ref. 19). Still matters of debate are whether
voltage-gated channels are targets of anesthetics at clinically
relevant concentrations and to what extent they play significant roles
in the development of the anesthetized state (8, 11). There are
numerous reports on the effects of general anesthetics on the function
of voltage-gated Na+ (1, 10, 29),
Ca2+ (3, 13, 21, 26), and
K+ channels (17, 28, 29, 32), but
studies on the molecular mechanisms involved are few. This is in part
because there are numerous types of voltage-gated channels in excitable
cells, which complicates studies on specific channels. Most
voltage-gated ion channels are blocked by volatile anesthetics, and in
the case of Ca2+ channels this is
due to a reduction in the open probability without a change in the
single-channel conductance (e.g., see Ref. 21).
Changes in the gating properties of a channel can be examined further by analysis of gating currents. Reports of this nature are scarce. In 1982, Fernandez et al. (10) studied the effect of chloroform (CHCl3) on the gating currents of the Na+ channel of the squid giant axon. They found that CHCl3 (60 mM) abolished Na+ currents and blocked a significant fraction of the fast component of the gating charge with no change in kinetics. In contrast, the movement of the dipicrylamine, which distributes passively across the bilayer following the membrane potential, was accelerated by CHCl3. They concluded that, although the protein was sensing the presence of the anesthetic, the gating machinery of the channel was moving within the channel protein and not in direct contact with the bilayer, i.e., the effect of CHCl3 was not due to perturbations of the properties of the lipid matrix.
With the recent cloning of the genes that encode voltage-gated ionic channels and with the advent of mutations that are helpful in the study of ionic and gating currents, questions about the extent, nature, and relevance of the interaction of anesthetics with membrane proteins can be readdressed. In this paper, I report data obtained using the ShakerB K+ channel to investigate the effects of general anesthetics on the gating mechanisms of the channel in a system in which the channel can be studied in virtual isolation. The ShakerB K+ channel encodes an A-type K+ current implicated in spike generation and modulation of neuronal output in the central nervous system (5, 14). It is a good ion channel model for the purpose of this study because it is functionally relevant to general anesthesia, because it is amenable to electrophysiological studies and therefore has been well studied, and because it can be easily modified using molecular biological tools. In this study, the heterologous expression system of the Xenopus oocyte was used. Gating and ionic currents through inactivating and noninactivating Shaker K+ channels were studied by exposing the channels to three volatile anesthetics: CHCl3, halothane, and isoflurane. This study addressed whether general anesthetics interact with the gating machinery of the ShakerB K+ channel, whether there is anesthetic specificity in the effects, and which molecular mechanisms are involved. The results indicate that 1) the anesthetics can differentially modify gating current kinetics, 2) the effects seen at the level of gating currents are consistent with those observed in the macroscopic conductance, 3) the anesthetics exert their effects concomitantly with channel opening, and 4) the anesthetics affect channel function by altering the kinetics of transitions near the open state.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials
Frogs (Xenopus laevis) were purchased from either Xenopus I or Alcon. Halothane was from Halocarbon Laboratories, and isoflurane (Forane) was from Ohmeda Carbide. CHCl3 (chemical grade) and methane sulfonic acid (99%) were from Aldrich Chemical. All other chemicals were purchased from Sigma.K+ Channel Clones and cRNA Transcription
The cDNA for the Drosophila ShakerB wild-type K+ channel was a generous gift of Dr. D. Papazian. All other clones used in this study were generously provided by Dr. L. Toro. They were the noninactivating mutant of the ShakerB channel, H4 IR, and the nonconducting mutant of the ShakerB channel, W434F, either with intact inactivation (W434F) or with inactivation removed (W434F IR). cDNAs for the various clones were transcribed with the commercial T7 mMessage mMachine kit from Ambion. The integrity of the transcribed RNA was assessed in agarose gels, and concentrations were determined spectrophotometrically.Oocytes
Xenopus oocytes were isolated from frog ovaries, following conventional protocols described elsewhere (12). Oocytes were defolliculated by collagenase treatment (collagenase type AI, GIBCO BRL) in zero-Ca2+ Ringer, followed by a progressive change to the normal frog Ringer containing 1.8 mM Ca2+. Defolliculated oocytes were kept in Ringer solution (standard oocyte solution) that consisted of (in mM) 100 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, and 5 HEPES, pH 7.2. RNA samples (0.05-0.1 µg/µl) were injected into the oocytes within 1 day after isolation. The oocytes were tested for expression levels from 1 to 4 days after injection, depending on the K+ channel clone. All animal procedures were approved by the Animal Research Council at the University of California, Los Angeles.Recording Solutions
The external solution (in mM) was 120 potassium methane sulfonate, 1.8 CaCl2, and 10 HEPES (KMSext; pH 7.2), 120 sodium methane sulfonate, 1.8 CaCl2, and 10 HEPES (NaMSext; pH 7.2), or 60 sodium methane sulfonate, 60 potassium methane sulfonate, 1.8 CaCl2, and 10 HEPES (NaKMSext; pH 7.2). The internal solution (in mM) was either 120 potassium methane sulfonate, 1 EGTA, and 10 HEPES (KMSint; pH 7.2) or 120 potassium glutamate, 1 EGTA, and 10 HEPES (KGint; pH 7.2). The electrode solution was 3 M KCl.Anesthetics
Solutions containing CHCl3 were prepared by dilution of a saturated (60 mM) solution in the external medium. Solutions containing the volatile anesthetics halothane and isoflurane were prepared by vaporizing the anesthetic in clinical vaporizers (Ohio) into external solution in an equilibrator (R. S. Weber and Associates, Westlake Village, CA) for 30 min. Equilibrated solutions were collected into gastight glass syringes. Solution exchange was done manually or via Teflon tubing in a gravity-fed perfusion system. Anesthetic concentration was determined by headspace sampling with a gas chromatograph (Hewlett-Packard HP 6890 Series with headspace sampler HP 7694) from an aliquot of the anesthetic-containing solution withdrawn directly from the equilibration chamber (halothane and isoflurane) or sealed vial (CHCl3) right before loading the perfusion syringes. The aliquot (200 µl) was rapidly placed at the bottom of a vial containing 5 ml of distilled water, and the vial was immediately sealed and kept for subsequent processing. The concentrations of the anesthetics used here are either higher than or equivalent to the minimum alveolar concentration (MAC) of each agent (MAC is 1 mM for CHCl3, 0.26 mM for halothane, and 0.4 mM for isoflurane). A clinically relevant concentration for CHCl3 is 1 mM (1 MAC). In some experiments, the saturated 60 mM CHCl3 solution was used. The highest concentrations of halothane and isoflurane used in this study correspond to 3.95 and 2.2 MAC, respectively. Clinically relevant concentration ranges are 0.5-2 MAC for halothane and 0.4-2 MAC for isoflurane.Electrophysiology
Ionic currents and gating currents were recorded with the cut-open oocyte Vaseline gap voltage-clamp technique, as described elsewhere (24, 25), using a commercial voltage clamp (Clampator, DAGAN Instruments). Briefly, the oocyte was placed in a three-compartment chamber. Currents were recorded from an area of the oocyte of one-fifth to one-fourth of the total surface of the oocyte that was exposed in the top compartment. The middle compartment served as a guard for leakage currents between the bottom (internal medium) and the top (external medium) compartments. The solutions in the top and middle compartments were identical and were exchanged at the same time. The bottom chamber contained internal solution, and contact between the solution and the oocyte cytoplasm was established by permeabilizing the bottom of the oocyte with saponin (0.3%). Permeabilization was monitored by following the change in the capacitive transient for a 10-mV pulse. The oocyte was impaled with a low-resistance (<1 MA custom-built acquisition system (hardware and software) was used to
elicit and record the data digitally. Data were acquired by sampling at
50-100 kHz for short pulses (10, 15, or 20 ms) and at 10-20
kHz for long pulses (45, 50, or 100 ms) and filtered at one-fifth of
the sampling frequency. When required, the capacitive transient was
subtracted online using P/4 protocols. Analysis was performed on
the digitally stored data with Analysis, custom software written to
complement the acquisition. Fits (single and double exponentials and
Boltzmann distributions) were done with NFIT.
Fractional conductance data were obtained from the instantaneous value
of the ionic currents upon repolarization. In practice, the peak tail
current was measured isochronally at a point close to the
repolarization time [see Fig.
5C; measurement made at
0.62 ms for the conductance-voltage
(G-V)
curve] or from the fit of the tail currents to exponential
functions extrapolated to time 0 (see
G-V
curves in Figs. 2D and
7C). The latter method was normally used to analyze
G-V data in experiments in which the anesthetic caused a change in kinetics.
G-V
curves were fitted to sequential models with Boltzmann distributions
based on the state sequence C0
C1
C2
O
Cf, where
the Ci
(i = 0, 1, 2) are closed states
leading to an open state (O). The blocked state (Cf) is populated from the open
state. Cf was proposed by Hoshi et al. (15) to account for
a fast flickery state observed at depolarized potentials in
single-channel data analysis of the IR mutant of the
ShakerB channel. The single-channel
data were analyzed with consideration of various possibilities for the
location of this Cf state; the
best fit to the data was given by models in which this state is
accessed from the open state. The macroscopic manifestation of this
state is a reduced probability of being open and a small 10% decline
in the peak current after a few milliseconds. The extent of occupancy
of the O
Cf transition
varies among oocytes (23). In the inactivating channel, inactivated
state(s) would stem in parallel from the open state (not shown) (15). Also, the first two transitions carry most of the gating charge, whereas C2
O and O
Cf are only slightly voltage
dependent, with only ~0.4 charges and <0.1 charges, respectively
(23). The equation used to fit the
G-V
data was of the form
![]() |
![]() |
![]() |
(1) |
![]() |
![]() |
(2) |
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To address the question of whether volatile anesthetics interact directly with the protein of a voltage-gated channel, the nature and specificity of the effects on the gating mechanisms of the Shaker K+ channel were studied by recording gating currents from oocytes expressing the nonconducting (W434F), noninactivating (IR) mutant. Gating charge movement on the order of 1 µA can be recorded from oocytes 1 day after injection (e.g., see Ref. 22). The effect of the anesthetics on the gating charge was evaluated by studying the anesthetic-induced changes in the magnitude and kinetics of the charge movement and in their voltage dependence.
Experiments in CHCl3
Noninactivating ShakerB channel.
GATING CURRENTS. Figure
1 shows the results obtained
by exposing W434F IR channels to 1 and 60 mM
CHCl3. In Fig.
1A are superimposed control and
experimental gating currents records.
CHCl3 (1 mM) had little or no
effect on either the magnitude or the time course of the "on"
gating charge movement
(Ig,on),
whereas it considerably accelerated the return of the gating charge
(Ig,off). The
CHCl3-induced faster relaxation of
Ig,off was absent
at 60 mV, developed at potentials more positive than
50
mV, and became clear at the more depolarized potentials. The
acceleration of
Ig,off produced by 60 mMCHCl3 was larger, as
illustrated with the superimposed traces shown in Fig.
1B,
inset. As at the lower concentration of CHCl3, the acceleration of the
return of the gating charge develops with depolarization becoming
evident at potentials at which the channels open (e.g., see
G-V
relation in Fig. 2D). This is
clearly seen in the plot shown in Fig.
1B. The time constants (
) of the
decay phases of
Ig,on and
Ig,off revealed
that control and treated on and off gating currents had similar time
courses at potentials at which the probability of opening is low, below
30 mV. At the potentials at which the channels are open,
Ig,off in
CHCl3 (60 mM) is fivefold faster
than the control (5.1 ± 0.3, mean ± SE, from
20 to +60
mV). Ig,on, on
the other hand, decayed with similar kinetics in control and
experimental data. There was an average 5 ± 2% reduction in the
of decay of
Ig,on between
10 and 60 mV. Acceleration of
Ig,off was seen
in 9 of 11 cells studied with 60, 10, or 1 mM
CHCl3; the effects were reversible in 8 of the 9 cells and ranged from 1.5- to 9-fold reduction in the
.
|
|
Inactivating ShakerB channel. GATING CURRENTS. In contrast to the results with the W434F IR mutant (Fig. 1), the effect of CHCl3 on the kinetics of Ig,off was prevented in channels with intact inactivation. This is shown in Fig. 3, inset. It is clear that in the inactivating W434F channel, Ig,off were mostly immobilized and had similar kinetics in CHCl3 and in the control. Ig,on, on the other hand, appeared to be slightly accelerated in CHCl3. The Q-V plot shown in Fig. 3 illustrates that the total charge moved (Qon, Qoff) at each potential remained the same in CHCl3. There is also no evidence of a change in the voltage dependence of the gating charge movement following CHCl3 treatment. Qoff was immobilized in CHCl3 to the same extent and with the same voltage dependence as the control, indicating that the anesthetic-induced acceleration of Ig,off seen in the absence of inactivation is prevented or not detectable for all voltages when inactivation takes place.
|
Experiments in Halothane
Gating currents. Halothane had no effect on the kinetics or magnitude of the gating charge movement of the W434F IR channel, as illustrated in Fig. 4A. The traces at the top are currents recorded with 1 mM CHCl3. After washout of the CHCl3, the oocyte was subjected to 2.5 mM halothane. The H4 W434F IR clearly responded to the CHCl3 but not to the halothane applied immediately afterward. Halothane had no effect on gating currents in four of four oocytes tested. In two of these, CHCl3 produced acceleration of Ig,off. This same high concentration of halothane was used in an oocyte expressing wild-type H4 W434F channels (Fig. 4B). As in the case of the W434F IR (Fig. 4A), even at this high concentration, halothane had only minimal effects on the kinetics of the gating charge. The Q-V curves for Qon and Qoff in control and halothane-treated channels superimposed (data not shown). Ionic currents through the inactivating and noninactivating conducting channels were expected to be unaffected by halothane.
|
Ionic currents.
Halothane had no effect on channel kinetics (Fig.
5). There was no effect on the rates of
activation or deactivation of H4 IR channels, as evidenced by the lack
of effect on the time course of the macroscopic ionic currents (Fig.
5A). In the presence of a relatively
high concentration of halothane (2.5 mM for the data shown in Fig. 5),
ionic currents were depressed by 10-20% [12% in the
current-voltage
(I-V)
plots in Fig. 5B]. There was a
block of the conductance (28% in the
G-V
in Fig. 5C and Table 1), which became
constant for voltages positive to 10 mV. The fractional conductance curves of control and halothane-treated currents
superimposed (Fig. 5C,
inset), indicating no change in the
voltage dependence of the conductance. This result differs from that
with CHCl3 in that halothane, as
expected from the gating current experiments, did not produce changes
in the kinetics of the ionic conductance, suggesting that it may mostly
affect the single-channel conductance. The blocking effect of halothane
on the ionic currents was observed in five of five oocytes; the average
block of 14.8 ± 2.8% was reversible after washout.
|
Experiments in Isoflurane
Gating currents.
Isoflurane had a marked effect on the kinetics of the gating charge
movement in W434F IR channels. The most dramatic effect was a
considerable slowing down of
Ig,off, as shown
in the superimposed traces in Fig.
6A. Again,
the effect was more pronounced at higher depolarizations. The time
constants of the decay of
Ig,on and Ig,off are shown
in Fig. 6B,
top and
bottom, respectively. Isoflurane induced a small increase in both
f and
s values for
Ig,on. In contrast, isoflurane doubled the time constant of the off gating currents for pulses more positive than
40 mV. At potentials at which the probability of opening is small, isoflurane had only a small
effect. This was observed in two of three oocytes tested; the effect
was reversible and amounted to 2.4- and 3.4-fold increases in the time
constant of decay of
Ig,off. The
effect of isoflurane, although opposite, was similar to that of
CHCl3 and halothane in that it
became evident as channels opened and in that there was no effect on
the voltage dependence of the gating process (there was only a small
depolarizing shift in the Qon vs. V
relation; not shown). The consequence of the kinetic change induced by
isoflurane near the open state of the channel on channel conductance
was explored in the conducting H4 IR channel.
|
Ionic currents.
Currents through the noninactivating mutant H4 IR (Fig.
7) were dramatically affected by
isoflurane. The anesthetic induced an increase in the current elicited
by test depolarizations
(I-V plot in Fig. 7A and Fig.
7A,
inset); in the example, the increase was 30%. Isoflurane clearly caused a decreased rate of deactivation, as the tail currents appear to be larger and more prolonged (Fig. 7A,
inset). The change in the time
course of the tail currents induced by isoflurane is illustrated in the
semi-logarithmic plot in Fig. 7B. The
time constants of both fast and slow components are increased in
isoflurane compared with the control and the wash. Isoflurane produced
an increase in the time constants (>4-fold for
s at
50 mV) that is
consistent with the reduction in the rate of return of the gating
charge in the nonconducting H4 W434F IR mutant. A pronounced increase
in the conductance was obtained from the
G-V
relations (Fig. 7C); it was higher
at the foot of the
G-V
curve (e.g., 6-fold at
30 mV) than at more depolarized potentials (e.g., 1.5-fold at +60 mV). This is also evident from the
fractional conductance curves (Fig.
7C,
inset), from which it is apparent
that the conductance started developing at more negative potentials in
isoflurane than in the control. This was quantified further by fitting
the data to the five-state model, C0
C1
C2
O
Cf, described in
MATERIALS AND METHODS (Fig. 7C). In isoflurane there is a
leftward shift in the voltage of activation of the channels (3 mV for
V1), a fourfold
decrease in A, and a strong eightfold
increase in B (Table 1). The change in
A was expected from the slower return
of Ig,off and
tail currents when in isoflurane. The change in
B indicates that it is much less
likely to find the channel in the blocked state. Both contribute to an
increase in the conductance (see Molecular Mechanisms
below). Isoflurane induced an increase in the ionic
current in 9 of 12 oocytes (ratio of isoflurane to control: peak ionic
current, 1.35 ± 0.15; peak tail current, 1.45 ± 0.06) and
slowed down tail current kinetics in 11 of 12 cells (ratio of
isoflurane to control:
f, 1.5 ± 0.1;
s, 2.9 ± 1.1);
the effects were reversible after washout in 9 of the 12 cells.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The ShakerB K+ channel was used as a model voltage-dependent ion channel to study the effects of three volatile anesthetics on its gating properties and on the voltage-dependent characteristics of the ionic conductance. The results can be summarized as follows. 1) The volatile anesthetics differentially affected the kinetics of the return of the gating charge in the noninactivating channel: CHCl3 accelerated it, halothane had no effect, and isoflurane slowed it considerably. 2) The effect on Ig,on was small, and there was no effect in the total amount of charge moved. 3) In channels with intact inactivation, CHCl3 and halothane had little effect on the magnitude or kinetics of the gating charge movement. 4) Halothane and CHCl3 produced a block of the ionic conductance, whereas isoflurane increased it. 5) Analysis of the G-V data indicated that none of the anesthetics produced changes in the voltage dependence of the conductances (there were no changes in zi). 6) CHCl3 induced a hyperpolarizing shift in the G-V and a destabilization of the open state by increasing the rates of exit from the open state to the last closed state and the blocked state. Halothane produced a small depolarizing shift and a reduction of the maximal conductance. Isoflurane produced a small hyperpolarizing shift and a stabilization of the open state; in isoflurane, there was strong destabilization of the blocked state. 7) The effects of the three anesthetics were seen as channels opened.
Direct Effects on the Channel Protein
The data presented in this paper suggest that inhalation anesthetics interact directly with the channel protein of voltage-gated ShakerB K+ channels and not through an indirect effect on bilayer properties. The effect is selective in that the three anesthetics tested had different effects on the function of the channels. The data suggest that the gating machinery involved in channel activation does not respond to the presence of the anesthetics until transitions close to channel opening. If it is assumed that the anesthetics reach and change the environment of hydrophobic regions within the protein, the small effects on Ig,on imply that the main charge-contributing transitions occur independently of changes in the hydrophobic environment close to where these gating charges are moving. These data do not rule out, however, possible effects on uncharged steps or on transitions carrying less charge and moving at early stages in the activation pathway. Effects of the anesthetics on such steps would have been detected had they introduced a rate-limiting step at earlier stages in channel activation. Although the effects of CHCl3, halothane, and isoflurane on the kinetics of Ig,on of the Shaker channels were small, the effects on the return of the gating charge and channel closing were considerable and were very much dependent on the anesthetic. Although the nature of the effects, however small, on Ig,on could be related to anesthetic-induced changes in the properties of the bilayer, it is hard to envision a common mechanism of action mediated by anesthetic effects on bilayer properties to explain the differential effect of CHCl3, halothane, and isoflurane on Ig,off and tail kinetics.The effects of CHCl3 on Ig,on of the Shaker channels differ from those previously reported for the Na+ channel of the squid giant axon by Fernandez et al. (10). They observed that CHCl3, at high concentrations (60 mM), produced a block of the fast component of Ig,on with a concomitant full block of the Na+ conductance. The slow component of the Ig,on was not affected by the CHCl3. In contrast, the kinetics of the movement of dipicrylamine (a lipophilic ion) were dramatically accelerated by the same concentration of the anesthetic. The results were interpreted to imply that the effect of the anesthetic on the gating charge movement was not merely an effect of the physical properties of the bilayer but a direct effect on the gating machinery of the channel. With 60 mM CHCl3, the conductance of the Shaker channel was also fully blocked (data not shown), but this occurred without a block of any of the components of Ig,on and with a small, but variable, change in kinetics. The difference in the effects of CHCl3 on the two channels (Na+ and K+) gives further support to specific interactions of the anesthetics with the channel protein.
Molecular Mechanisms
The results indicate a clear qualitative difference in the modulation of K+ channels produced by the different anesthetics. To gain more insight into the mechanisms by which the anesthetics may be affecting the noninactivating channels, we have modeled the effects of the anesthetics on the G-V relations of the conducting channels. Several models for the gating of ShakerB K+ channels have been proposed (2, 15, 23). The simplified version of one such model, after Rodriguez and Bezanilla (23), used in this study is shown in the scheme
![]() |
With halothane, there was no clear evidence of kinetic changes, therefore, most of the effect was expected to be in the conductance proper. Indeed the model predicted a reduced Gmax for halothane. This could be the result of single-channel conductance reduction or an entry to a blocked state.
The inactivation particle precluded the effect of CHCl3 on the return of the gating charge and on channel deactivation. This observation suggests that the site with which CHCl3 interacts is not exposed to the anesthetic when the inactivating particle is tethered to the channel. Alternatively, the binding of the inactivation particle to its docking site does not allow the CHCl3 effect to reach completion. Because hydrophobic interactions play an important role in the docking of the inactivation particle at its site (20), it is tempting to speculate that CHCl3 could interact with that site to produce a change that eases the return of the gating charge upon repolarization but with a much decreased affinity than the inactivating particle itself. The data on the wild-type Shaker channel suggest, however, that the influence of the inactivating particle on the action of 1 mM CHCl3 is not state dependent, because the anesthetic does not affect the closing rate of the fraction of channels that have not inactivated even at short depolarizations (data not shown).
Relevance to K+ Channel Function in General Anesthesia
K+ currents contribute to the control of neuronal excitability and are, therefore, relevant to general anesthesia. For example, A-type currents play a critical role in determining the output of CA1 pyramidal neurons by dampening dendritic excitability (14). Propagation of the action potential is controlled by an A-type conductance in hippocampus (5). Also, K+ currents have been implicated in the development of anesthetic sensitivity in Drosophila (27). In those studies, impairment of A-type currents resulted in behavioral insensitivity to isoflurane. It is important, therefore, to understand which channels are involved in the response to general anesthetics and what are the molecular mechanisms responsible for their action.During an action potential, the changes in membrane potential drive the opening and closing of channels as determined by the voltage dependence and rates of the gating processes. Delayed rectifier K+ currents are important for membrane repolarization during an action potential, and the resting K+ conductance determines the membrane potential and, consequently, excitability. The H4 IR mutant studied here has characteristics of a delayed rectifier channel. An acceleration of channel closing alongside a reduction of the conductance to K+, like that seen in CHCl3, would make the repolarizing phase of the action potential slower, and the action potential would broaden. A similar result would be expected with halothane, which reduces the conductance proper. Conversely, the membrane would repolarize at a much faster rate with an increased conductance and a reduction of the rate at which channels close, as would occur in the case of the noninactivating channels exposed to isoflurane. Changes in the K+ conductance also affect the onset of the action potential; the firing threshold is reached earlier when the conductance is decreased and is delayed when it is increased. All of the above effectively change the firing frequency and conduction velocity of the cell. As mentioned above, A-type currents also play important roles in excitability and action potential propagation. Depending on the resting potential of a neuron, A-type currents will influence the duration and repolarization rate of the action potentials or influence the latency between action potentials and, consequently, the firing frequency. In the latter scenario, which occurs in neurons with depolarized resting potentials, a higher conductance and longer times in the open state would lengthen the period between action potentials, effectively reducing the firing frequency. A reduced conductance or shorter openings, on the other hand, would speed this process. The rates at which these changes occur and the voltage dependence of the processes involved are governed by the gating mechanisms of the channels, and, as is clearly illustrated in this study, general anesthetics can have a dramatic influence on the gating process of K+ channels.
Although there is a large body of evidence for volatile anesthetic block of K+ channels in native cells as well as in expressed recombinant channels, most of the reports concern anesthetic effects on other types of K+ channels (9, 17, 29). In general, CHCl3, halothane, and isoflurane depress currents through most K+ channels. However, it is also known that at low concentrations volatile anesthetics can produce hyperexcitability, whereas at higher concentrations, still within the clinically relevant range, there is depression (8, 18, 29). This type of behavior could be explained by differential modulation of the various types of K+ channels in cell bodies, dendrites, and axons (8). There is, however, precedent for activation of K+ currents induced by volatile anesthetics. In Aplysia neurons, Winegar et al. (31) found that halothane and isoflurane induced an increase in the probability of opening of a resting K+ channel, the S channel, which hyperpolarized the resting membrane potential, producing a loss of excitability. The inhibition by CHCl3 and halothane and the stimulation by isoflurane seen in the present study are, therefore, consistent with previous observations in isolated cells and axons.
In a comparative study with different clones of K+ channels, Zorn et al. (32) reported block by halothane of ShakerB (ShB1) currents expressed in Xenopus oocytes at concentrations similar to those used in the present study. The peak current was reduced, and no change in channel inactivation was observed. In another study with recombinant channels, Vener et al. (30) reported block of peak and steady-state conductance and increased rates of activation and inactivation of the Shaker analog RCK2 (Kv1.6) treated with halothane (0.5-8%). The results presented in this paper are in line with the inhibitory effect of halothane on channel conductance seen in the ShB1 (32) and in the RCK2 (30), but, in contrast to the RCK2, halothane had little or no influence on channel kinetics of any of the clones studied, as in the ShB1.
In conclusion, Shaker-like K+ channels are differentially modified by the action of general anesthetics. The evidence points toward a direct interaction of the anesthetics with the channel protein as opposed to an indirect effect due to perturbation of the bilayer milieu. The action of the anesthetics tested involves modification of the channel at transitions near the open state, affecting kinetics, mostly of closing, and channel conductance. On the basis of the data presented here, neural function dependent on the activity of noninactivating Shaker-like delayed rectifying channels would differ substantially depending on the anesthetic. One could predict that halothane and CHCl3 would produce different firing patterns and repolarization rates than isoflurane. Although there are differences that could be attributed to the nature of the K+ channels studied, the data presented in this paper are consistent with that obtained in similar studies in this and other systems at clinically relevant concentrations of volatile anesthetics.
![]() |
ACKNOWLEDGEMENTS |
---|
I am indebted to Drs. D. Papazian and L. Toro for the generous gift of Shaker clones, to R. Katayama for excellent technical assistance, to Y. Jin and D. Grenet for oocyte isolation and sample injection, to A. Hui and J. Angione for hardware and software support, to Drs. F. Bezanilla, M. S. Gold, and R. Latorre for reviewing the manuscript and for helpful discussion, and to Drs. F. Bezanilla and E. Stefani for helpful input and continuing support.
![]() |
FOOTNOTES |
---|
This project was supported by National Institute of General Medical Sciences Grant GM-53781 and by the Dept. of Anesthesiology, University of California, Los Angeles.
Preliminary reports of these data were published in abstract form (4) and presented at the Fifth International Conference on Molecular and Cellular Mechanisms of Anaesthesia (University of Calgary, Calgary, Alberta, Canada, 1997).
Address for reprint requests: A. M. Correa, UCLA Medical Receiving/Anesthesiology, 650 Circle Dr. South, BH-612 CHS/Correa, Los Angeles, CA 90095-1778.
Received 5 November 1997; accepted in final form 11 June 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bean, B. P.,
P. Shrager,
and
A. D. Goldstein.
Modification of sodium and potassium channel gating kinetics by ether and halothane.
J. Gen. Physiol.
77:
233-253,
1981[Abstract].
2.
Bezanilla, F.,
E. Perozo,
and
E. Stefani.
Gating of Shaker K+ channels. II. The components of gating currents and a model of channel activation.
Biophys. J.
66:
1011-1021,
1994[Abstract].
3.
Charlesworth, P.,
G. Pocock,
and
C. D. Richards.
Calcium channel currents in bovine adrenal chromaffin cells and their modulation by anaesthetics.
J. Physiol. (Lond.)
481:
543-553,
1994[Abstract].
4.
Correa, A. M. Gating and ionic currents of
voltage-gated ionic channels exposed to general anesthetics (Abstract).
Biophys. J. A439, 1994.
5.
Debanne, D.,
N. C. Guérineau,
B. H. Gähwiler,
and
S. M. Thompson.
Action-potential propagation gated by an axonal IA-like K+ conductance in hippocampus.
Nature
389:
286-289,
1997[Medline].
7.
Dilger, J. P.
Basic pharmacology of inhalational anesthetic agents.
In: The Pharmacological Basis of Anesthesiology, edited by T. A. Bowdle,
A. Horita,
and E. D. Kharasch. New York: Churchill Livingstone, 1994, chapt. 23, p. 497-521.
8.
Elliot, J. R.,
and
B. W. Urban.
Integrative effects of general anesthetics: why nerve axons should not be ignored.
Eur. J. Anaesthesiol.
12:
41-50,
1995[Medline].
9.
Eskinder, H.,
D. Gebremedhin,
J. G. Lee,
N. Rusch,
F. D. Supan,
J. P. Kampine,
and
Z. J. Bosnjak.
Halothane and isoflurane decrease the open state probability of K+ channels in dog cerebral arterial muscle cells.
Anesthesiology
82:
479-490,
1995[Medline].
10.
Fernandez, J. M.,
F. Bezanilla,
and
R. E. Taylor.
Effect of chloroform on charge movement in the nerve membrane.
Nature
297:
150-152,
1982[Medline].
11.
Franks, N. P.,
and
W. R. Lieb.
Molecular and cellular mechanisms of general anaesthesia.
Nature
367:
607-614,
1994[Medline].
12.
Goldin, A. L.
Maintenance of Xenopus laevis and oocyte injection.
Methods Enzymol.
207:
266-279,
1992[Medline].
13.
Herrington, J.,
R. C. Stern,
A. S. Evers,
and
C. J. Lingle.
Halothane inhibits two components of calcium current in clonal (GH3) pituitary cells.
J. Neurosci.
11:
2226-2240,
1991[Abstract].
14.
Hoffman, D. A.,
J. C. Magee,
C. M. Colbert,
and
D. Johnston.
K+ channel regulation of signal propagation in dendrites of hippocampal pyramidal neurons.
Nature
387:
869-875,
1997[Medline].
15.
Hoshi, T.,
W. N. Zagotta,
and
R. W. Aldrich.
Shaker potassium channel gating. I. Transitions near the open state.
J. Gen. Physiol.
103:
249-278,
1994[Abstract].
16.
Janoff, A. S.,
M. J. Pringle,
and
K. W. Miller.
Correlation of general anesthetic potency with solubility in membranes.
Biochim. Biophys. Acta
649:
125-128,
1981[Medline].
17.
Kulkarni, R. S.,
L. J. Zorn,
V. Anantharam,
H. Bayley,
and
S. N. Treistman.
Inhibitory effects of ketamine and halothane on recombinant potassium channels from mammalian brain.
Anesthesiology
84:
900-909,
1996[Medline].
18.
MacIver, M. B.,
and
D. L. Tanelian.
Volatile anesthetics excite mammalian nociceptor afferents recorded in vitro.
Anesthesiology
72:
1022-1030,
1990[Medline].
19.
Mihic, S. J.,
Q. Ye,
M. J. Wick,
V. V. Koltchine,
M. D. Krasowski,
S. E. Finn,
M. P. Mascia,
C. F. Valenzuela,
K. K. Hanson,
E. P. Greenblatt,
R. A. Harris,
and
N. L. Harrison.
Sites of alcohol and volatile anaesthetic action on GABAA and glycine receptors.
Nature
389:
385-389,
1997[Medline].
20.
Murrell-Lagnado, R. D.,
and
R. W. Aldrich.
Interactions of amino terminal domains of Shaker K+ channels with a pore blocking site studied with synthetic peptides.
J. Gen. Physiol.
102:
949-975,
1993[Abstract].
21.
Pancrazio, J. J.
Halothane and isoflurane preferentially depress a slowly inactivating component of Ca2+ channel current in guinea-pig myocytes.
J. Physiol. (Lond.)
494:
91-103,
1996[Abstract].
22.
Perozo, E.,
R. MacKinnon,
F. Bezanilla,
and
E. Stefani.
Gating currents from a nonconducting mutant reveal open-closed configurations in Shaker K+ channels.
Neuron
11:
353-358,
1993[Medline].
23.
Rodriguez, B. M.,
and
F. Bezanilla.
Transitions near the open state in Shaker K+ channel: probing with temperature.
Neuropharmacology
35:
775-785,
1996[Medline].
24.
Stefani, E.,
and
F. Bezanilla.
The cut open oocyte voltage clamp technique.
Methods Enzymol.
293:
300-318,
1998[Medline].
25.
Stefani, E.,
L. Toro,
E. Perozo,
and
F. Bezanilla.
Gating of Shaker K+ channels. I. Ionic and gating currents.
Biophys. J.
66:
996-1010,
1994[Abstract].
26.
Study, R. E.
Isoflurane inhibits multiple voltage-gated calcium currents in hippocampal pyramidal neurons.
Anesthesiology
81:
104-116,
1994[Medline].
27.
Tinklenberg, J. A.,
I. S. Segal,
G. Tianzhi,
and
M. Maze.
Analysis of anesthetic action on potassium channels of the Shaker mutant of Drosophila.
Ann. NY Acad. Sci.
625:
532-539,
1991[Abstract].
28.
Urban, B. W.
Differential effects of gaseous and volatile anaesthetics on sodium and potassium channels.
Br. J. Anaesth.
71:
25-38,
1993[Medline].
29.
Urban, B. W.,
and
D. A. Haydon.
The actions of halogenated ethers on the ionic currents of the squid giant axon.
Proc. R. Soc. Lond. B Biol. Sci.
231:
13-26,
1987[Medline].
30.
Vener, D.,
T. Johnson,
T. Woodward,
and
G. Kirsch.
Halothane interaction with cloned rat brain K+ channels (Abstract).
Anesthesiology
79:
A383,
1993.
31.
Winegar, B. D.,
D. F. Owen,
C. S. Yost,
J. R. Forsayeth,
and
E. Mayeri.
Volatile general anesthetics produce hyperpolarization of Aplysia neurons by activation of a discrete population of baseline potassium channels.
Anesthesiology
85:
889-900,
1996[Medline].
32.
Zorn, L.,
R. Kulkarni,
V. Anantharam,
H. Bayley,
and
S. N. Treistman.
Halothane acts on many potassium channels, including a minimal potassium channel.
Neurosci. Lett.
161:
81-84,
1993[Medline].