.-f;
7.37
Repnnted from Molecdar and Celldar Mechanisms of Alcohol and Anesthetics'
Volume 625 of the Annais of rhe New York Academy of Sciences
June 28, 1991
Molecular Models of Anesthetic Action
on Sodium Channels, Including Those
from Human Braina
BERND W. URBAN,^^,^ CHRISTIAN FRENKEL,~,'
DANIEL S. D U C H , ~AND AUDREY B. KAUFF~
b~ePartrnentsof Anesthesiology
and Physiology
Comell University Medical College
New York, New York I0021
'Institute forAnaesthesiology
University of Bonn
Sigrnund-Freud-Strasse25
D 5300 Bonn I , Gennany
INTRODUCTION
As this contribution is part of the Haydon Memorial Symposium it may be
appropriate to outline in somewhat more detail the context in which the anesthesia
research originated by Denis Haydon was conducted and how it led to the investigations described in this report.
Even before the turn of this century it became clear that the hydrophobicity of
the anesthetic molecules played a crucial role in anesthetic action. The MeyerOverton correlation demonstrated that anesthetic potency and lipid partition coefficients were linearly related over many concentration decades.' Yet, while sharing a
common characteristic of hydrophobicity, anesthetic molecules were found to vary
tremendously in their chemical and physical properties. This then suggested a rather
nonspecific mode of action. It was thought that anesthetics dissolved in membranes
and thereby disrupted the membrane's structure. However, biological membranes
are highly complex and segregated. In order to evaluate the actions of anesthetics on
membranes, a simpler and better defined system was needed. Such a system became
available in the early 1960s, the lipid bilayer system. For the first time and 50 years
after Bernstein had proposed the membrane hypothesis in 1912, it became possible
to systematically characterize the physical properties of the bilayer aspect of memb r a n e ~Denis
,~
Haydon being one of the leading pioneers. Many subsequent reports
described how anesthetics perturbed membrane structure. An example was Denis
Haydon's demonstration of a correlation between the anesthetic potency of the
n-alkanes in whole animal anesthesia and their ability to dissolve in planar lipid
b i l a ~ e r sIt
. ~could be shown that higher homologues of the n-alkanes, which contrary
to expectations from the Meyer-Overton correlation had lost their anesthetic po"Supported by National Institutes of Health grants NS-22602 and GM-41102 (B.W.U.) and
postdoctoral fellowships frorn NATO 300-402-514-8 (C.F.) and National Research Service
NS-08146 (D.S.D.).
d ~ o r r e s p o n d e n c eand reprint requests should be addressed to the Institute for Anesthesiology, University of Bonn.
327
328
ANNALS NEW YORK ACADEMY OF SCIENCES
tency, were also excluded frorn dissolving in lipid bilayers, thus providing a possible
explanation for the anesthetic cutoff effect.'
Below the cutoff point n-alkanes were shown to thicken membranes. How could
such changes affect membrane excitability? Pure lipid bilayers which are perfect
insulators are rendered electrically excitable by the action of several classes of
specialized rnernbrane proteins. Since onset of anesthesia can be extremely rapid, ion
channels becarne obvious candidates for proteins being affected by the membrane
action of anesthetics. Ion channels constitute a class of rnernbrane proteins that
forrns the basis for membrane excitability, and this entire class is known to be
. ~the other possibility, that of anesthetics
susceptible to the actions of a n e s t h e t i c ~ If
dissolving in the hydrophobic Portions of the rnernbrane proteins themselves, is
ignored for a rnornent, a rnost important question rernains: how can changes
- in
membrane properties impair the function of membrane proteins?
Reconstitution studies, in which proteins are removed from their native mernbranes and inserted into lipid bilayer membranes of defined lipid cornposition, have
provided the best evidence so far that the lipid environment significantly affects the
properties of integral mernbrane proteins. Frorn these experiments, a large nurnber
of diverse proteins have been found to have altered functions depending on the
composition of the phospholipids with which they are in contact.' These studies have
shown that both specific properties of phospholipids such as head-group composition
and general properties of the hydrophobic bilayer such as microviscosity can have
dramatic effects on protein function. However, it remains to be demonstrated that
anesthetic-related changes in membrane properties actually do affect mernbrane
proteins. Although it has been argued that polar effects of anesthetics (such as
changes in surface charge) and hydrophobic effects (fluidity changes, mernbrane
thickening) occurred in the bilayer, sorne of these effects rnay actually happen at the
rnernbrane protein rnolecule itself. The lipid bilayer reconstitution technique allows
a direct test: if an anesthetic-induced change in a membrane property can cause a
change in membrane protein function, then the same change in rnernbrane properties brought about by an appropriate choice of membrane lipids in the absence of any
anesthetic should equally alter protein function.
This test was initially tried for antibiotic ion-channel-forrning molecules because
techniques were not yet available for incorporating biological ion channels into lipid
bilayers. In order to gain a rnolecular picture of drug action, the effects of hydrophobic compounds%nd alcohols7on grarnicidin A, an antibiotic that forms ion channels
in lipid bilayers,' were studied. A combination of rnernbrane thickness and mernbrane surface tension increases were found to account for decreases in channel
stability and lifetirnes which led to reduced membrane conductances in the presence
of hydrophobic anesthetics like the series of n-alkanes6 Changes in channel lifetimes
were later found to be comparable to those for bilayers in the absence of n-alkanes
when the lipid properties were changed instead.' Denis Haydon's thickness-tension
hypothesi?.' quantitatively accounted for the reduction in gramicidin ion channel
conductance by the n-alkanes and the hypothesis demonstrated a mechanisrn by
which purely hydrophobic substances through changes in rnembrane properties
could disrupt hydrogen bonds in a polypeptide or protein.
However, the gramicidin channel plays no role in naturally occurring excitable
rnernbranes while the voltage-dependent sodium channel is a ubiquitous ion channel
mediating fast propagated action potentials. Vital to nerve impulse transrnission and
cell communication, sodium channels play an important role in signal integration.I0 A
wide range of general as well as local anesthetic compounds suppress voltagedependent sodium ~hannels."-'~
Electrophysiologically, voltage-dependent sodium
channels have probably been studied more extensively than any other rnernbrane
URBAN et al.: MOLECULAR MODELS
329
channel and much is known about their biochemistry, pharmacology, and genetiCS.lU,16.17Ion channels can be grouped into farnilies of evolutionary and structurally
related gene products; for example, a 55% sequence homology exists between
sodium and calciurn channel~.''.'~
Because sodium channels have served as a model
for the description of almost all other voltage-dependent ion channels and, with
suitable rnodifications, even for chernically activated channels," they provide a good
starting point for the study of anesthetic interactions with ion channels.
Initially, the expertise to incorporate sodiurn channels into lipid bilayers did not
exist. Instead, capacity measurements were used to monitor anesthetic-induced
changes in the rnernbrane environment of sodiurn channels while simultaneously
recording the irnpact of these changes on sodiurn currents under voltage clamp
conditions. The initial choice for a sodium channel preparation fell on the squid giant
axon rnernbrane because the Hodgkin-Huxley formalism17 provided a detailed rnathematical description of its sodiurn currents that fitted the experimental data very
well. Though its parameters could not be thought of as reflecting specific rnolecular
events, one would expect changes in the Hodgkin-Huxley parameters, particularly in
their voltage dependence, if membrane properties such as thickness, surface poten' ~ . ' ~ experiments
.~~
t i a l ~or
, fluidity were altered. A many-year study f ~ l l o w e d . ~ ~ .These
systematically characterized the actions of different classes of anesthetics. The
classes were distinguished by the physicochernical properties of the anesthetic
compounds and involved (a) hydrophobic compounds, (b) polar and surface-active
substances such as alcohols, and (C) inhalation anesthetics. A combination of
separate effects on sodiurn conductance, activation, and inactivation was responsible
for the reduction in sodium currents.15 One of the intriguing results was the
observation that the slopes of the activation and inactivation curves as functions of
potential were generally reduced in the presence of anesthetics; they did not
increase. The total inhibitory anesthetic responses of sodiurn currents resulted from
the integration of separate inhibitory and excitatory anesthetic actions on distinct
sodium channel functions. Any one anesthetic class suppressed sodiurn currents by
more than one rnechanism, while different classes of anesthetics had distinct spectra
of anesthetic actions. Evidence concerning the underlying molecular rnechanisms
was indirect, but surface potentials, membrane thickness, surface tension, rnicroviscosity, and hydrogen bonding appeared to play a r ~ l e . ' ~The
. ' ~ sarne Pattern ernerged
again when anesthetic actions on potassiurn and grarnicidin channels were studied,
suggesting that a given anesthetic compound affected different ion channels in a
sirnilar manner. There were also differences. Thus, the quantitative correlation
between the shifts in the steady-state activation curves for sodium and potassiurn
currents was poor. This suggested that the lipid environrnent of the two types of ion
channel rnight have been different or that there was, at least in sorne instances, an
interaction of the anesthetics with the channel protein itself. While certain actions of
halogenated ethers correlated with their abilities to form hydrogen bonds, their
effects could have arisen frorn specific interactions with lipids or proteins other than
ion channels.
Except for gramicidin channels, definitive rnolecular interpretations of the
pararneter changes rernained elusive, rnostly because of the uncertainty as to the
immediate membrane environment of the ion channels involved. Biological rnernbranes are inhornogeneous. Different ion channels might be surrounded by
"preferred" lipids and specific proteins modulating their function. While overall
membrane Parameters may have been accessible to rneasurements, the properties
and cornposition of small segregated rnernbrane areas remained uncharacterized.
This basic problem, encountered with the classic squid giant axon preparation.
rernains the sarne with any biological preparation, even those that ernploy rnolecular
330
ANNALS NEW YORK ACADEMY OF SCIENCES
techniques such as patch clarnping o r genetic expression of ion channels in oocytes.
By rernoving a rnernbrane protein frorn its biological rnernbrane and inserting it into
artificial lipid bilayers, it rnay becorne possible to separate direct anesthetic actions
on rnernbrane proteins frorn those rnediated via the rnernbrane environrnent. It is
significant perhaps that one of the few quantifiable hypotheses of anesthetic action,
~
frorn work with lipid
Denis Haydon's thickness-tension h y p ~ t h e s i s ,originated
bilayers, strongly recornrnending this approach for rnechanistic anesthetic studies.
When Krueger, Worley, and French established a technique for studying the
steady-state behavior of voltage-dependent sodiurn channels in lipid bilayers," it
seemed a prornising tool for probing the rnolecular nature of anesthetic interactions.
URBAN et al.: MOLECULAR MODELS
and its on rate under our experimental conditions is very low. Therefore there was a
great probability that the sarne channels were obsewed throughout the experirnent
and that the nurnber of channels in the rnembrane would rernain small and constant.
Single channels were identified22.25as sodiurn channels by their conductance, fractional Open time (ix., the fraction of time a channel rernained in the Open state),
CONTROL
METHODS
When a planar bilayer is forrned across the hole of a thin teflon partition it
consists of a birnolecular leaflet of lipid rnolecules separating two aqueous cornpartrnents, which can be conveniently viewed as corresponding to the extra- and
intracellular cornpartments. Biological rnernbranes can be broken up in such a way
that the sodiurn-channel-containing rnernbrane fragrnents reseal to form srnall
rnernbrane vesicles. Alternatively, sodiurn channels are purified frorn these rnernbrane fragrnents and reconstituted into artificial lipid ve~icles.~'
Either one of these
vesicle types can be added to the vicinity of the lipid bilayer and, rnost likely through
the process of rnernbrane fusion, sodiurn channels are transferred frorn vesicles into
the planar lipid bilayer.
Bilayer procedures were as previously d e ~ c r i b e d . " ,All
~ ~ experirnents were conducted in syrnrnetrical 500 rnM NaCl (buffered at p H 7.4 with 10 rnM HEPES) at
roorn ternperature (22-25°C). Planar bilayers were forrned frorn synthetic, (4:l)
1-palrnitoyl-2-oleoyl-phosphatidylethanolarnine
and 1-palrnitoyl-2-oleoyl-phosphatidylcholine in decane. The sodiurn channel frorn Electrophotus electricus was purified
and reconstituted into vesicles as previously described." With the approval of the
Cornrnittee on Human Rights in Research at Cornell University Medical College,
diseased and healthy human brain cortical tissue sarnples (frontal and temporal
lobe) were obtained as surgical waste tissue frorn patients undergoing routine
craniotornies. No functional differences were observed in sodiurn channels frorn the
different tissues." Synaptosornal fractions were prepared as described." Synaptosomal fractions or reconstituted vesicles were added close to a preforrned bilayer
rnernbrane in the presence of 1 p M batrachotoxin (trans) or 50 p M veratridine
(syrnrnetrical), cornrnonly used sodiurn channel activators."." Channel orientation
was deterrnined by channel gating characteristics and/or by tetrodotoxin block,
allowing the use of the electrophysiological sign convention in the presentation of the
results.
RESULTS
Batrachotoxin-activated human brain sodiurn channels in lipid bilayers rernained
Open at rnernhrane potentials rnore positive than -60 rnV, except for brief closures
(see FIGURE1A). Batrachotoxin, which rernoved sodiurn channel inactivation,
perrnitted steady-state studies in which the sarne channels could be observed for
rnany hours, depending on the anesthetic used. Batrachotoxin, in addition, had the
function of a label. Its off rate has been estirnated to be of the order of four hours"
33 1
PENTOBARBITAL
PROPOFOL
D
MIDAZOLAM
FIGURE 1. Original current traces frorn batrachotoxin-activated single human sodium channels at -45 rnV rnernbrane potential under control conditions and when exposed to anesthetics.
Records were low-pass filtered with a 50 Hz corner frequency. 0 indicates the fully Open, C the
fully closed channel level.
steady-state activation characteristics, and sornetimes by their sodium selectivity and
block by tetrodotoxin, a sodium channel specific inhibitor.
Single channel behavior such as steady-state activation could change spontaneously with time. However, none of the observed channel properties showed any
systernatic dependence on channel or bilayer lifetirne. Therefore single sodiurn
332
ANNALS NEW YORK ACADEMY OF SCIENCES
channels were monitored for at least 30-40 minutes before any anesthetic was added
to the bilayer chambers. During the control period batrachotoxin-modified channels
showed the expected characteristics previously described." They remained Open
almost all of the time, as demonstrated by a fractional Open time of 0.94 2 0.04
[-Cstandard deviation (SD), n = 57 membranes] at potentials more positive than
-45 mV. Their single channel conductance was 26.0 2 0.6 pS (n = 68). The
midpoint of steady-state activation occurred at -84 2 10 mV (n = 71). The effective
1.7. The
gating charge (measure of the steepness of the activation curve) was 2.7 I
average number of channels per membrane was 2.2.
Immediately after the addition of the anesthetic agent at sufficiently high
concentrations, sodium channels spent more time in the closed state (FIGURE1B-E).
In the case of pentobarbital and ketamine, the current traces assumed a noisy
appearance as the transitions were too fast to resolve with the bilayer setup (filtered
at 50 Hz). This fast flicker was not the result of nonspecific membrane noise because
the traces were much less noisy when complete channel closures could be seen." In
URBAN et al.: MOLECULAR MODELS
PENTOBARBITAL
A
PROPOFOL
K~~ = 20 UM
Block,..:
28%
0 4
08
12
1.6
Penioborbilol conceniroiion IrnM)
MIDAZOLAM
Midazolam concentralion (mM)
20
Propolol concentration I p M )
C
Kelarnine concenlralion (rnt.4)
FIGURE 3. Time-averaged conductance block expressed as the fraction of the average
conductance reduction and control conductance plotted as function of anesthetic concentrations for human brain sodium channels. Computer-fitted concentration-response curves yielded
the values for the maximal block and the half-maximal block concentration, ED:,. Data for each
membrane and each anesthetic concentration have been pooled for membrane potential
between -45 mV and +45 mV. Error bars indicate SEM. The clinically relevant ranges are
indicated by hatching.
FIGURE 2. Time-averaged current-voltage curves for human sodium channels under control
conditions and when exposed to different concentrations of anesthetics. The slope conductances were obtained by linear regressions as indicated, the error bars indicate standard error
of the mean (SEM).
the case of propofol and midazolam, long closures as well as brief closures became
prominent (FIGURE1C-D).
Although there was no noticeable decrease in single channel amplitudes, the
current flowing across the membrane, averaged over time and many channel openings and closings, decreased because the channels spent less time in the Open state.
Relative to control, these time-averaged currents were suppressed. The current
reductions were the same at different potentials within the range of -45 mV and
+45 mV, provided the sampling period was sufficiently large (several minutes at each
potential). When time-averaged currents were plotted as a function of potential,
'
ANNALS NEW YORK ACADEMY OF SCIENCES
URBAN ei al.: MOLECULAR MODELS
linear relationships resulted just as under control conditions, except for a reduced
slope (FIGURE2). With increasing anesthetic concentrations the average currents
decreased further (FIGURE 2). Extracellular or intracellular additions of anesthetics
resulted in comparable suppressions.
The suppressions were quantitated by converting the time-averaged currents into
conductances and averaging them over the membrane potential range of -45 to +45
mV. Conductance block was calculated as the difference of the conductances before
and after the addition of the anesthetic and plotted as a function of anesthetic
concentration. The resulting dose-response curves could be approximated by rectangular hyperbolae (FEURE 3). The concentrations at which the currents were
reduced by 50% of the maximal suppression (ED„,) were obtained from weighted
least-square fits. For pentobarbital and propofol the ED„ values were much closer to
the clinically relevant concentration r a n g e ~ ~ "than
' ~ for ketamine and midazolam
which exceeded the clinically relevant do~es'"'~by more than an order of magnitude
(FIGURE4).
The voltage dependence of sodium channel openings and closing events was
maintained jn lipid bilayers, and the fractional Open time of human brain sodium
channels increased with membrane d e p ~ l a r i z a t i o n . ~Since
' ~ ~ " batrachotoxin removed
inactivation, sodium channels remained almost permanently Open at depolarized
potentials (more positive than -45 mV) with a fractional Open time of almost one,
while at very hyperpolarized potentials (more negative than - 120 mV) the fractional
Ketamine 0.1 mM
334
GlpS
Ketamine 0.5 mM
GlpS
25[
E
.----..-.----.
A
Propofol
A
X-----------
..................
X
Pentobarbital
Midazolarn
Ketarnine
FIGURE 5. Three successive steady-state activation responses at 0.1 m M ketamine (A-C) and
at 0.5 mM ketamine (E-G), respectively, for human brain sodium channels. Continuous lines
represent steady-state activation curves that have been obtained by fits to two-level Boltzmann
distributions. The fits at each ketamine concentration have been superimposed in the bottom
panels (D and H) to illustrate the variability.
multiples of clinical d o s e
FIGURE 4. The dose-response curves of FIGURE
3 normalized with respect to maximal
blocking effect and plotted as function of multiples of clinical doses. Clinical doses: pentobarbital 0.2 mM,2Lketamine 0.02 mM,27propofol0.055 mM,2%idazolam 0.01 mM."
Open time decreased to Zero as they remained permanently closed. In the intermediate potential range the channels opened and closed continuously, resulting in a
E To
fractional Open time that ranged somewhere between 0 and 1 ( F ~ G U R 5).
measure steady-state activation characteristics, membrane potentials were changed
starting from a holding potential of +SO mV with a decrementing potential sequence
ANNALS NEW YORK ACADEMY OF SCIENCES
URBAN et al.: MOLECULAR MODELS
337
were 2.1, 3.3, and 12.4, respectively (average 5.9), while the fit to the averaged data
points produced an effective gating charge of 2.4. This difference between the values
of 5.9 and 2.4 for gating charges averaged in different ways already indicates that the
value of the effective gating charge not only reflects the electrostatic component of
the conformational energy change but also represents an averaging over different
molecular states o r conformations.
When successive curves were averaged for several channels at 0.5 mM ketamine,
the resulting average activation curve became shallower with the higher anesthetic
concentrations (data not shown). This effect was observed for pentobarbital (FIGURE
6) and propofol (FIGURE7) as well. This variability made it necessary to record the
anesthetic response of the Same channel for as long as possible. Such observation was
usually terminated when a channel no longer opened, when additional channels
incorporated, o r when the membrane became noisy o r broke. Channels in membranes exposed to pentobarbital (163 min, n = 14 experiments) and ketamine (149
min, n = 14) could be observed much longer on average than in the presence of
propofol (39 min, n = 9) o r midazolam (36 min, n = 21). It is possible that these
latter anesthetics affected batrachotoxin binding as well.
In this context it became relevant to study the role that batrachotoxin plays in the
anesthetic response. Although it is already possible to observe unmodified purified
FIGURE 6. Six successive steady-state activation responses at 0.67 rnM pentobarbital for
human brain sodiurn channels. Continuous lines represent two-level Boltzrnann distributions.
The six steady-state activation responses were averaged and fit by another Boltzrnann distribution which is indicated as the dashed line in each of the six panels.
of - 10 mV steps until channels were fully closed. The membrane potential was then
returned in +10 mV steps back to the holding potential of +50 mV. Each potential
was held for 4 seconds. Channel fractional Open time (or time-averaged conductance) was calculated and plotted as a function of membrane potential. This
collection of data, referred to as the steady-state activation response, was fitted by a
two-level Boltzmann distribution function (FIGURE5) as already described." This
function, the steady-state activation curve, is characterized by the midpoint potential, Va, at which the channel spends half of the time in the closed state, and by the
effective gating charge, L,. The product z, V*is related to the free energy difference
between the voltage-gated Open and closed state of the channel, but z, is more
conveniently used as a measure of the steepness of the steady-state activation curve.
The curve becomes steeper with increasingz,.
After addition of anesthetic at a concentration around or exceeding the ED„, the
gating behavior of channels became much more variable. This is illustrated for
ketamine in FIGURE5, where three successive gating curves are shown at a concentration of ketamine well below (FIGURE5A-D) and closer to the ED„, (FIGURESE-H).
Successive steady-state activation curves recorded from the Same channel could
differ greatly in their midpoint potentials and slopes, particularly at the higher
anesthetic concentrations. This greater variability led to an apparent reduction in the
effective gating charge when the data from successive gating runs were averaged
before they were fit to a Boltzmann distribution. In this particular example (0.5 mM
ketamine, FIGURESE-H), the effective gating charges from the three individual runs
FIGURE 7. Six successive steady-state activation responses at 0.028 rnM propofol for human
brain sodium channels. Continuous lines represent two-level Boltzrnann distributions. The six
steady-state activation responses were averaged and fit by another Boltzmann distribution
which is indicated as the dashed line in each of the six panels.
control
Veratridine
control
1.34 rnM pentobarbital
FIGURE 8. Original current traces from batrachotoxin-activated and veratridine-activated single purified eel sodium channels at 60 mV membrane
potential. Records were low-pass filtered with a 50 Hz Corner frequency. 0 indicates the fully Open, C the fully closed channel level. Note that
veratridine-activated channels are about half the size of batrachotoxin-activated channels. While it suffices to show single traces for batrachotoxinactivated channels to demonstrate typical behavior, this is not true for veratridine-activated channels with their sometimes long Open and closed
intervals. The bottom trace for veratridinelcontrol represents a fully Open channel; for veratridinelpentobarbital the bottom trace shows a fully closed
channel.
$
L
m
z
0
m
tn
-0.5-
0.34mM pentobarbital
0.2 pS
slope = 9.9 I
-1.0-
0.67 mM pentobarbital
slope = 9.2 ? 0.2 pS
FIGURE 9. Current-voltage curves from single channel amplitudes of purified eel sodium channels under control conditions and when exposed to
different concentrations of pentobarbital. The dope conductances were obtained by linear regressions as indicated, the error bars indicate standard
deviations. A: Control, 10.4 I 0.1 PS. B: 0.335 mM, 9.9 I 0.2 PS. C: 0.67 mM, 9.2 0.2 PS. D: 9.3 I 0.2 PS.
W
W
W
340
ANNALS NEW YORK ACADEMY OF SCIENCES
sodium channels in lipid bilayers (vesicles3' or planar bilayersI2), the experimental
conditions are not yet suitable for pharmacological studies. In addition, as the
microenvironment for a sodium channel is known best for a purified channel, the
study of anesthetic effects on purified channels has to be considered in order to
dissect the components of anesthetic interactions. Human sodium channels have not
yet been purified. The cleanest purified preparation available for functional studies
appears to be that of the sodium channel from the electroplax of the electric ee1.22.'3
In biochemistry, the alkaloid toxin veratridine has been widely used to characterize
the functionality of purified sodium channels. Therefore the effects of pentobarbital
on veratridine-modified purified eel sodium channels were studied and the preliminary results are as follows.
URBAN et al.: MOLECULAR MODELS
341
Furthermore, the current traces during channel openings could become noisier
than the background current. In Support of this observation, current-voltage relationships constructed from transition histograms (current traces were filtered below the
noise frequency) showed a slight decrease at higher pentobarbital concentrations
(FIGURE9).
While the fractional Open time of batrachotoxin-modified channels was voltage
independent at potentials more positive than -45 mV, the fractional Open time of
veratridine-modified channels rose monotonically with increasing potential throughout this rangeI3 (FIGURE10). Therefore, in contrast to batrachotoxin-activated
sodium channels (FIGURE2), there was no potential range within which the
time-averaged current-voitage relationships were symmetrical and linear (FIGURE
10). However, the suppression by pentobarbital (FIGURE10) did not show much of a
voltage dependence. When averaged over the - 15 mV to +75 mV potential range,
the time-averaged conductance was suppressed to 54% of control by 0.67 mM
pentobarbital and to 29% of control by 1.34 mM pentobarbital.
DISCUSSION
Membrane Potential (mV)
FIGURE 10. Voltage dependence of veratridine-modified purified eel sodium channel conductances normalized to a single channel before and after addition of 0.67 mM and 1.34 mM
pentobarbital. All points are the average of 3 membranes. Each point was sampled a minimum
of three minutes per membrane.
Qualitatively, the pentobarbital-induced flicker of batrachotoxin-modified eel
sodium channels (FIGURE8) was similar to that observed with batrachotoxinmodified human brain sodium channels (FIGURE1). However, in contrast, the major
portion of the reduction in fractional Open time of veratridine-modified eel channels
did not arise from fast transitions but from changes in the long duration (seconds)
Open and closed transitions (FIGURE8). As the long Open and closed events in
FIGURE8 suggest, long observation times were required in order to measure the
pentobarbital-induced current suppression for a single channel. There was also
evidence of an increase in fast closed transitions during the long duration openings
(FEURE 8).
The results obtained in this study showed that all investigated general anesthetics
depressed at least two major functions of batrachotoxin-modified sodium channels
from human brain, leading to a voltage-independent reduction of the fractional
channel Open time and an interaction with the voltage-dependent steady-state
activation, respectively. At the same concentrations, the suppression of the timeaveraged conductances for batrachotoxin-modified human sodium channels and for
veratridine-modified purified eel electroplax sodium channels were very similar. The
effects of pentobarbital and propofol were detectable at concentrations that were
within the clinical range,"." while ketamine and midazolam showed similar effects
only at concentrations exceeding that range by more than an order of m a g n i t ~ d e . ~ ~ . ~ ~
The extent to which anesthetic actions are altered in the presence of alkaloid
toxins needs to be considered and explored in future. In the past, toxins have been
successfully used in the dissection of ion channel function.1° The alkaloid toxins are
also of interest as they may be probing yet unidentified regulatory sites of sodium
channels, similar to regulatory sites that have been found for other ion ~hannels.~""
It is possible that for sodium channels, anesthetic action and channel regulation may
be linked. However, the removal of normal inactivation gating and the high fractional
Open time of the Open channel by the alkaloid toxins may allow anesthetics greater
access to their interaction sites than in normal unmodified channels. If quantitative,
rather than order of magnitude, comparisons of anesthetic potency are desired, they
will have to be done with unmodified channels under physiological conditions.
O n the other hand, alkaloid toxins may simply be aiding in the detection of
conformational changes resulting from certain anesthetic interactions. A batrachotoxin-"labeled" channel can be observed electrophysiologically over many
hours, permitting the measurement of control behavior and several anesthetic doses
on the same channel. What has been striking when observing a single molecule for
hours is the random variability of its behavior, necessitating sufficiently long periods
of observation to obtain reproducible results. Anesthetics appear to substantially
increase this variability. It takes a stable system such as that of planar lipid bilayers to
demonstrate that this variability is indeed random and not due to a progressive
deterioration of the preparation.
Hydrophobic interactions of anesthetics need not be limited to the lipid bilayer.
Ion channels share common design features, including hydrophobic and other
342
ANNALS NEW YORK ACADEMY OF SCIENCES
segments of alpha-helical s t r ~ c t u r e . 'The
~ alpha helices are thought to be in close
contact with each other through their hydrophobic portions. Sodium channels
.'~
sites other than the bilayer where
possess at least 24 of these ~ e g m e n t s , ' ~offering
hydrophobic anesthetics might act. Anesthetics that manage to form a wedge
between hydrophobic helices might thus distort protein conformation and disrupt
protein function. Alpha-helical segments with hydrophobic as well as positively
charged domains are thought to be directly involved in channel gating. Because of its
capacity to undergo hydrophobic and perhaps additional polar interactions, the
anesthetic molecule might bind to such segments and lift the protein out of its stable
conformational energy minimum into a more unstable state. As a consequence, the
protein would cycle more readily between conformational states, as observed experimentally. This variability, for example, would lead to less-well-defined transitions
following activation by membrane potential, leading to shallower activation curves.
Hydrophobic interactions of anesthetics with alpha helices would thus be equally
compatible with the Meyer-Overton correlation. The additional potential for polar
interaction could explain the parallel shift of Meyer-Overton correlations for anesthetics with different capacities to form hydrogen bonds."
Qualitatively, the finding of more than one type of anesthetic action o n sodium
currents for anesthetics is in line with our studies of volatile and gaseous anesthetics
on peripheral nerve,''." where anesthetics were found to shift activation and inactivation curves as well as reduce the maximal conductance. The present studies also offer
an alternative interpretation of the reduction in slopes of activation and inactivation
curves observed in the sodium currents of squid axons. The d o p e reductions may
result from an averaging over a larger number of conformational states accessible in
the presence of anesthetics.
The sodium channel is not the only ion channel involved in the clinical actions of
anesthetics. Other membrane proteins should be incorporated into bilayers and
studied, including calcium and potassium channels and the y-aminobutyric acid
(GABA)/benzodiazepine and acetylcholine receptors. Eventually more complex
Systems could be built. For example, sodium channels might be incorporated into
bilayers together with opioid receptors to test the suggestion4' that opioids modulate
sodium channel function through an interaction between the stereospecific opioid
receptor and the intracellular aspects of the sodium channel. By building an excitable
membrane from scratch, it may thus become possible to identify the components of
membrane protein function that are significantly disrupted during anesthesia.
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