(Received for publication, April 20, 1995; and in revised form, June 12, 1995)
From the
The molecular basis of general anesthetic action on membrane
proteins that control ion transport is not yet understood. In a
previous report (Covarrubias, M., and Rubin, E.(1993) Proc. Natl.
Acad. Sci. 90, 6957-6960), we found that low concentrations
of ethanol (17-170 mM) selectively inhibited a
noninactivating cloned K channel encoded by Drosophila Shaw2. Here, we have conducted equilibrium
dose-inhibition experiments, single channel recording, and mutagenesis in vitro to study the mechanism underlying the inhibition of
Shaw2 K
channels by a homologous series of n-alkanols (ethanol to 1-hexanol). The results showed that:
(i) these alcohols inhibited Shaw2 whole-cell currents, the equilibrium
dose-inhibition relations were hyperbolic, and competition experiments
revealed the presence of a discrete site of action, possibly a
hydrophobic pocket; (ii) this pocket may be part of the protein because n-alkanol sensitivity can be transferred to novel hybrid
K
channels composed of Shaw2 subunits and homologous
ethanol-insensitive subunits; (iii) moreover, a hydrophobic point
mutation within a cytoplasmic loop of an ethanol-insensitive
K
channel (human Kv3.4) was sufficient to allow
significant inhibition by n-alkanols, with a dose-inhibition
relation that closely resembled that of wild-type Shaw2 channels; and
(iv) 1-butanol selectively inhibited long duration single channel
openings in a manner consistent with a direct effect on channel gating.
These results strongly suggest that a discrete site within the ion
channel protein is the primary locus of alcohol and general anesthetic
action.
Aliphatic alcohols (n-alkanols) and volatile
anesthetics are lipophilic agents that can affect the transport of ions
across the cell membrane. Therefore, this is thought to be the basis
for the biological actions of ethanol and other general anesthetics in
the nervous system. According to the Meyer-Overton rule the ability of
these agents to partition in lipid-like environments correlates with
their general anesthetic potency in
vivo(1, 2) . Thus, the core of the lipid bilayer
was an attractive locus of action (``lipid hypothesis'').
This hypothesis predicted that membrane proteins, and especially those
involved in ion transport, will be indirectly affected by lipophilic
anesthetics as the result of a physical perturbation of the lipid
bilayer(3, 4, 5) . However, a number of
observations have challenged this hypothesis(2, 5) :
(i) membrane disordering caused by anesthetic concentrations of n-alkanols can be mimicked by a temperature change of 1
°C(2) ; (ii) certain enzymes are affected by n-alkanols and volatile anesthetics in the absence of
phospholipids(6, 7, 8) ; (iii) long chain n-alkanol derivatives exhibit a cut-off effect in their
anesthetic potency(2, 7, 9, 10) ,
and certain volatile anesthetics display
stereoselectivity(11) ; and (iv) only a limited number of ion
channels with diverse functional properties are affected by anesthetic
concentrations of n-alkanols and/or other general anesthetics.
Such targets include a subset of neurotransmitter-gated ion channels,
namely
-aminobutyric acid receptors and N-methyl-D-aspartate
receptors(2, 4, 12) , high voltage and low
voltage Ca
channels(13) , and certain
K
channels(11, 14, 15) .
Although many of these targets are similarly affected by n-alkanols and volatile anesthetics, some show differential
sensitivity(16) , thereby suggesting additional specificity. To
explain these observations an alternative hypothesis of general
anesthesia and alcohol action proposes that lipophilic anesthetics may
act directly on membrane proteins (``protein hypothesis'')
through an interaction with relatively specific hydrophobic clefts or
pockets(2) . Proteins that are sensitive to these agents may
have common structural motifs, which are located in or associated with
a region of the molecule that is critical to its function. Low affinity
bulk sites and high affinity discrete sites may actually coexist in the
membrane, but only the latter would be relevant to the anesthetic
effect. Low affinity sites could involve the core of the lipid bilayer,
the protein-lipid interface, or putative membrane-spanning segments of
the polypeptide. In absolute terms, however, the binding affinity of
putative general anesthetic and n-alkanol binding sites seems
to be in the high micromolar to millimolar range, which suggests weak
and unconstrained interactions(4) . Thus, the term high
affinity is used here in a relative manner when compared with that of
low affinity sites like those mentioned above.
Although valuable
studies have been made to demonstrate the protein hypothesis of alcohol
and general anesthetic
action(2, 10, 17, 18, 19, 20) most of the evidence is still indirect, and the molecular
bases remain unknown. In a previous study we found that a cloned
K channel encoded by Drosophila Shaw2 is
selectively inhibited by physiologically relevant levels of ethanol in
a concentration-dependent manner(14) . Such inhibition was
found to be rapid, reversible, and voltage-independent. By contrast, 12
K
channels homologous to Shaw2 (all members of the
Shaker superfamily) required >200 mM to exhibit significant
inhibition(14, 21) . Since the same cell type (Xenopus oocytes) was used in these studies and, therefore,
the membrane environment was constant, we proposed that the unique
sensitivity of the Shaw2 K
channel to ethanol was
specified by its protein moiety. Compared to other members of the
Shaker superfamily, Shaw2 K
channels also bear unique
biophysical properties(14, 22) , such as lack of
inactivation, activation with no apparent delay, low voltage
sensitivity, and low open probability. Thus, they are unlikely to
control spike repolarization (typically the role of delayed rectifier
K
channels) but may help to determine the passive
electrical properties of the membrane. Native channels with functional
properties analogous to those of Shaw2 have been recorded from nerve
and muscle cells(23, 24, 25) . To investigate
the molecular mechanism underlying inhibition of Shaw2 K
channels by ethanol and its relationship to the bases of general
anesthetic action, we studied the interactions between wild-type and
mutant channels with members of the homologous series of n-alkanols. These experiments demonstrate the presence of a
discrete hydrophobic site of action probably located in the Shaw2
polypeptide. In addition, we show that channel inhibition results from
a discrete effect on channel gating.
Figure 1:
Equilibrium dose-inhibition
curves. A, whole-oocytes Shaw2 K currents evoked by a
450-ms step depolarization from -100 to +40 mV. Currents in
the absence and presence of 1-butanol are shown superimposed. Top to bottom (in mM), 0, 4.4, 5.5, 11, 22,
33, 44, and 55. B, normalized current amplitude
(I/I
) as a function of 1-butanol concentration. Symbols represent three separate measurements. Lines represent
minimized fits to a Langmuir adsorption isotherm of the following form:
I/I
= 1/(1 +
([A]/K)
), where [A]
is the alcohol concentration, K is the apparent equilibrium
constant, and n is the Hill coefficient. Solidline, best fit parameters (K = 16
mM, n = 1.1); dottedline,
best fit parameters (K = 14 mM, n = 1.2) assuming a background level of 5%. C,
logarithmic dose-inhibition curves for the indicated alcohols. Lines were generated as described above. In general,
the background level was estimated to be <10%. However, because the
absolute saturating level cannot be reached, for consistency no
background level was assumed to obtain the best fit. Table 1summarizes the best fit parameters. D,
competition dose-inhibition curve. Lineacrosssolidsymbols (1-hexanol alone, control) is a
minimized fit obtained as described above (K = 1 mM, n = 0.7). Lineacrosscircles (1-hexanol concentration varied in the presence of ethanol at 170
mM) was generated according to the following equation:
I/I
= 1/(1 + [C2]/K
+ [C6]/K
), where
[C2] and [C6] are the concentrations of ethanol and
1-hexanol, respectively, and K
and K
are the corresponding equilibrium constants
(their values were obtained from Table 1). This equation
describes direct competition between two alcohols for a common binding
site. Symbols in C and D represent the mean
± S.D. of 3-6 determinations.
Figure 2: Inhibition of Shaw2 currents by n-alkanols is proportional to alkyl chain length. A, apparent equilibrium constants as a function of number of methylene groups in the alkyl chain. Line represents a linear regression (r= 0.99). B, standard free energy of binding as a function of the number of methylene groups in the alkyl chain. Here, K is expressed in unitary mole fraction units. Line represents a linear regression (r= 0.99) with a slope of -2.93 kJ/mol. Circles and opentriangles represent measurements for hKv3.4-Shaw2 tandem dimer and hKv3.4/G371I ( Fig. 3and Fig. 9).
Figure 3:
Properties of a hybrid human-fly
K channel: current kinetics. A, diagram of a
human-fly tandem dimer. Linelength and boxwidth are proportional to polypeptide length. Boxes represent putative transmembrane segments (S1-S6); J indicates the junction between hKv3.4 and Shaw2 polypeptides (see
``Materials and Methods''). B, diagram of a putative
configuration of a tetrameric channel made of tandem dimers (HFHF). An alternative configuration (HHFF) cannot be ruled
out because the entire linker region between the cores of both subunits
is sufficiently long(37, 38) . C, D,
and E, whole-oocyte outward K
currents
expressed by Shaw2, hKv3.4, and human-fly tandem dimer, respectively.
Currents were evoked by 900-ms step depolarizations from -100 to
+50 mV.
Figure 9:
Effect of 1-hexanol on wild-type and
mutant hKv3.4 K channels. A, whole-oocyte
outward K
currents evoked by 125-ms step
depolarizations from -100 to +40 mV. Currents were recorded
before and after exposing oocyte to 8 mM 1-hexanol. Currents
in the presence of 1-hexanol were taken after the inhibitory effect had
equilibrated (30-60 s). This effect was reversible. B,
dose-inhibition relation for wild type and G371I. The line represents a minimized fit obtained as described in the legend to Fig. 1. Best fit parameters were K = 3.3 mM and n = 1. Dashedline represents the control level. Symbols represent the mean
of 3 and 2 determinations from wild type and G371I,
respectively.
Figure 4:
Properties of a hybrid human-fly
K channel: inhibition by n-alkanols.
Dose-inhibition curves for ethanol (A) and 1-hexanol (B). Curves were analyzed as described in the legend
to Fig. 1. Shaw2 best fit parameters were as follows: K = 168 mM (n = 1) and K = 1 mM (n = 0.7) for ethanol and
1-hexanol, respectively. Tandem dimer best fit parameters were as
follows: K = 702 mM (n = 0.8)
and K = 5.5 mM (n = 1) for ethanol and
1-hexanol, respectively. Since hKv3.4 exhibits an apparent threshold
effect with both ethanol and 1-hexanol, we did not attempt to explain
these data quantitatively (see ``Discussion''). Dashedline represents the control level. Symbols represent the mean ± S.D. of two to four
determinations.
Figure 5: Inhibition of Shaw2 single channel currents by 1-butanol. Consecutive single channel currents recorded at 0 mV from an inside out patch under control conditions (left), exposing the cytoplasmic side to 88 mM 1-butanol (center), and after washout (right). Currents shown were low-pass filtered at 2.5 kHz (-3 db, 8-pole) and digitized at 20 kHz. Line across the traces is the zero current level. Analysis of the complete set of records is shown in Fig. 6B and 7.
Figure 6:
Effect of 1-butanol on Shaw2 single
channel conductance and open time. A, single channel
current-voltage relation in the absence () and presence of 44
mM 1-butanol (circo). Mean current amplitudes at each membrane
potential were estimated from Gaussian fits to amplitude histograms.
The calculated slope conductances (solidlines) were
23 and 25 pS for control and 1-butanol, respectively. B,
cumulative histograms of open times. This represents the probability
that the open time is equal or shorter than the time on the abscissa.
Figure 7: Effect of 1-butanol on the distributions of closed times and open times. Logarithmic histograms of closed times (A) and open times (B) from single channel records in the absence of alcohol (control), in the presence of 1-butanol, and after washout. The number of binned events in these histograms were (from top to bottom): 1377, 368, and 1738. Solidthicklines represent best fits to a single exponential (closedtime) or a sum of two exponential terms (opentime). Each component of the sum is shown separately by thinsolidlines. The best fit parameters are shown in Table 2(Experiment 9482405/16).
Figure 8:
The S4-S5 linker of 11 members of the
Shaker superfamily of voltage-gated K channels. The
letters d, h, m, and r stand for Drosophila, human, mouse, and rat, respectively. Bold and underlinedcharacters represent identity or
similarity relative to Shaw2 (assuming S
T and K
R). Asterisks mark positions that form part of the leucine-heptad
repeat(44) . Column on the right indicates
whether a channel was significantly inhibited by <200 mM ethanol(14, 21) .
Figure 10:
Effect of ethanol on wild-type and mutant
Shaw2 K channels. A, whole-oocyte outward
K
currents evoked by 450-ms step depolarizations from
-100 to +40 mV. Currents were recorded before and after
exposing oocyte to 170 mM ethanol. Currents in the presence of
ethanol were taken after the inhibitory effect had equilibrated
(30-60 s). This effect was reversible. B, whole-oocyte
Shaw2 K
currents evoked by a 9000-ms step
depolarization from -100 to +20 and +10 mV (wild type
and I319G, respectively). For I319G, current decay at +10 and
+60 mV did not seem significantly
different.
We have studied the interaction of various members of the
homologous series of n-alkanols with a cloned K channel encoded by Drosophila Shaw2. These compounds
cause depression of the nervous system in vivo and have been
commonly used to study the biological bases of general anesthesia and
alcohol action(2, 3, 9) . Since Shaw2
membrane currents are selectively inhibited by n-alkanols at
low concentrations, we investigated the functional and molecular bases
of this action. The results led to the following main conclusions: (i)
current inhibition results from a selective reduction in the
probability that channels enter a long duration open state; and (ii)
the site of n-alkanol action is a discrete hydrophobic cleft
or pocket, most likely located in the channel polypeptide.
Inhibition of a long duration open state by n-alkanols can
be interpreted according to Fig. SI, where C represents a single
closed state in the activation pathway or a very rapid equilibrium
between several closed states that precede channel opening and O and O
represent brief duration and long duration open
states, respectively. The rates that control channel opening and
closing are represented as
and
, respectively. The following
arguments suggest that Fig. SIis a minimal mechanism that
qualitatively explains gating of Shaw2 channels. (i) The rising phase
of Shaw2 currents shows no delay, even at negative voltages, and gating
is weakly voltage-dependent(48) . Closed time histograms
suggested only one closed state directly in equilibrium with open
channels (Fig. 7A, Table 2). (ii) The presence of
two open states (O
and O
) was suggested from
the analysis of open time histograms, which is best described by the
sum of two exponential components (Fig. 7B). (iii)
Direct transitions O
O
do not seem
likely because 1-butanol selectively affects the frequency of long
duration openings with little effect on the time constants of long and
brief duration openings (Table 2). Accordingly, parameters
extracted from the analysis of open and closed time histograms can be
related to rate constants in Fig. SI: long mean open time
(
) = 1/
; brief mean open time
(
) = 1/
; mean closed time
(
) = 1/(
+
); the fractional contribution of long duration
openings to the the distribution of open times (f
) equals
the probability of C
O
(Table 2). Then, P(C
O
) =
/(
+
) and therefore,
= f
/
. Similarly,
= f
/
, where f
represents the fractional contribution of brief duration openings
to the distribution of open times (Table 2). Our data suggest
that mainly
is reduced by the action of n-alkanols. However, an accurate estimation of the opening
rates is presently limited because we do not know whether the analyzed
records represent the activity of a single channel, even in the absence
of overlapped openings (owing to a low open probability). Also,
is overestimated because of missed brief openings (Fig. 7B and Table 2). Nevertheless, as an
approximation, we find from the inside out patch data (Table 2)
that
is reduced from 8 to 0.5 s
(16-fold), whereas
is reduced only from 9 to 4
s
(
2-fold). If the proposed mechanism can
account for inhibition of the macroscopic membrane current by n-alkanols, we can predict that at a given concentration
f
(+)/f
(-) should equal the remaining
fraction of the membrane current (see below). f
(-)
and f
(+) are the fractional contributions of long
duration openings in the absence and presence of 1-butanol,
respectively. In addition, since there is little effect on brief
openings and the time constants of long and brief open times may differ
5-10-fold (Table 2), the proposed mechanism also predicts
that at high concentrations of n-alkanol a small proportion of
the membrane current (contributed by brief openings only) should be
relatively resistant to inhibition. The dottedline in Fig. 1B describes the data, assuming a simple
binding isotherm plus a constant background level representing 5% of
total current. Thus, at 88 mM 1-butanol (Fig. 1B and Table 2), (I/I
) - 0.05 = 0.1
and f
(+)/f
(-) = 0.2. The
close agreement between these values (within 10% of total current)
suggests that inhibition of
can explain the effect of n-alkanols on the macroscopic current. As suggested earlier,
an additional effect on the number of active channels(N) seems,
therefore, unlikely.
Figure SI:
A novel hybrid channel composed of two Shaw2
subunits and two hKv3.4 subunits expressed currents with a sensitivity
to n-alkanols greater than that of hKv3.4 alone. We therefore
concluded that such a phenotype is determined by the Shaw2 moiety. A
similar observation has been made with mutant Shaker channels and
tetraethylammonium(37, 38, 49) .
Tetraethylammonium is a K channel blocker that is
known to interact with specific amino acids near the outer and inner
mouths of the pore(50, 51) . Especially interesting in
our study was the finding that the apparent threshold effect observed
with hKv3.4 (resembling a sigmoidal dose-inhibition relation) is
eliminated in the case of hKv3.4-Shaw2 hybrids. In fact, ethanol and
1-hexanol dose-inhibition relations for hybrid channels were hyperbolic
with Hill coefficients of about 1.0 (Fig. 4). Thus, it seems
that interaction with a high affinity site dominates in the hybrid
channel, and such a site might be located in the Shaw2 moiety. In
hKv3.4 (and probably in other related channels insensitive to alcohols)
there may be multiple low affinity sites, and higher occupancy is
required to cause inhibition (resembling positive cooperativity). For
instance, multiple low affinity sites may be located in the
protein-lipid interface surrounding the channel oligomer. In Shaw2
however, there is a single high affinity site possibly located at the
protein-water interface. Consistent with the latter suggestion, we
found a standard free energy change per methylene group on the order of
-3 kJ/mol (Fig. 2). This value corresponds with the free
energy change estimated for the transfer of n-alkanols from
water to alkanes(31) . Moreover, we showed that a hydrophobic
point mutation in a putative cytoplasmic loop of hKv3.4 (G371I) was
sufficient to eliminate apparent sigmoidicity of the dose-inhibition
relation and introduce higher affinity (Fig. 9B). The
dose-inhibition relation for hKv3.4/G371I resembled that of Shaw2
(albeit with somewhat lower affinity; see below). These data suggest
that the effect of G371I in hKv3.4 is unlikely to result from a random
structural change. G371I may have created a hydrophobic cleft, and
binding of n-alkanols to this site allows inhibition of hKv3.4
with higher affinity. Alternatively, we cannot rule out that G371I
causes an allosteric shift between low and high affinity conformations
of the channel(2, 7) .
If the corresponding site in Shaw2 (I319) were a critical determinant of the native n-alkanol binding site, it seemed that I319G would reduce n-alkanol-dependent inhibition of Shaw2 currents. However, this was not the case, and the main apparent change caused by I319G was the presence of slow current decay (Fig. 10B). We propose the following explanation for this observation; most likely, various residues are important determinants of the binding site. This concept is supported by the fact that the sensitivity of hKv3.4/G371I was intermediate between that of Shaw2 and that of wild-type hKv3.4. If the putative hydrophobic pocket in Shaw2 is structurally critical, it is conceivable that, to preserve a stable conformation, compensatory changes have taken place in Shaw2/I319G. For instance, in I319G another hydrophobic amino acid from a nearby or distant region may play the role of I319. Similar compensatory changes have been proposed to occur in mutants of cystic fibrosis transmembrane conductance regulator(52) . Thus, the determinants of the n-alkanol binding site in Shaw2 might be redundant, and further mutagenesis experiments will be necessary to identify all critical residues.
Additional evidence suggests that the site of n-alkanol action in Shaw2 might be relatively specific.
Several functionally important hydrophobic pockets have been located in
voltage-gated Na channels and Shaker K
channels, mainly in the S4-S5 loop and within S6, where they form
part of the inactivation gate receptor and the binding sites of local
anesthetics and alkyl quaternary ammonium
derivatives(53, 54, 55) . However, n-alkanols and volatile anesthetics affect these channels with
very low affinity(14, 21, 32, 33) .
Clearly, the presence of functionally important hydrophobic pockets is
not necessarily equivalent to general anesthetic action with high
affinity. Thus, although the Shaw2 K
channel bears
significant similarity to those channels, its unique sensitivity to n-alkanols indicates the presence of a relatively specific
site of action, which can be partially engineered in a homologous
channel by a single amino acid substitution in the S4-S5 loop. It is
conceivable that both lipid and protein may form part of the n-alkanol binding site. Recent reports suggest however, that
the S4-S5 loop forms part of the inner mouth of the
channel(53, 56) , and it is not known how this region
may interact with membrane lipids surrounding the protein.
Overall
the data provide compelling evidence that favors the presence of a
discrete site of n-alkanol action in a cloned K channel and, therefore, support the protein hypothesis of general
anesthetic and alcohol action. Evidence of n-alkanol sites has
also been reported for ligand-gated ion channels, including N-methyl-D-aspartate receptors, nicotinic
acetylcholine receptors and ATP-gated ion
channels(5, 10, 19, 20) . However,
there is little information available about the nature of the binding
sites and their determinants. Nevertheless, n-alkanol binding
sites are probably diverse, and future structure-function studies
should help to reveal their differences and similarities.
Given the
unique biophysical properties of Shaw2, we can relate inhibition of
this channel by n-alkanols with a possible anesthetic effect.
Shaw2-like K channels have been recorded in nerve and
muscle cells(23, 24, 25) , Mainly because of
their weak voltage sensitivity and lack of inactivation, they may help
to determine the resting membrane potential. Inhibition of Shaw2 by
anesthetics would increase membrane resistance. This can cause
steady-state depolarization (57) and, in addition, may allow a
larger depolarization in response to a given excitatory stimulus.
Although these changes may initially cause excitation, their long term
effect could lead to inactivation of voltage-gated Na
channels and, therefore, inhibition of membrane
excitability(58, 59) .