(Received for publication, June 9, 1995; and in revised form, August 25, 1995)
From the
Volatile anesthetics at concentrations that are used in clinical
practice to induce anesthesia selectively inhibit activity of the
plasma membrane Ca-transport ATPase (Kosk-Kosicka,
D., and Roszczynska, G.(1993) Anesthesiology 79,
774-780). We have investigated the mechanism of the inhibitory
action of several anesthetics on the purified erythrocyte
Ca
-ATPase by employing fluorescence spectroscopy
measurements that report changes in the environment of intrinsic
tryptophans and of an extrinsic probe attached in the active site of
the enzyme. We have shown that the observed inhibition of the
Ca
-dependent activation of the enzyme correlates well
with the elimination of the Ca
-induced conformational
change that is important for the proper function of the enzyme.
Analysis of the anesthetics effects on the total tryptophan
fluorescence indicates a significant effect on enzyme conformation.
Similar changes have been observed in the sarcoplasmic reticulum
Ca
-ATPase. We propose that volatile anesthetics
inhibit Ca
-ATPase by interacting with nonpolar sites
in protein interior, in analogy to the binding demonstrated for
myoglobin, hemoglobin, and adenylate kinase (Schoenborn, B. P., and
Featherstone, R. M.(1967) Adv. Pharmacol. 5, 1-17;
Tilton, R. F., Kuntz, I. D., and Petsko, G. A.(1984) Biochemistry 23, 2849-2857). Such binding is expected to modify
conformational substate(s) of the enzyme and perturb its function. We
view this process as an example of a general phenomena of interaction
of small molecules with internal sites in proteins.
The molecular site and mechanism of action of inhaled volatile
anesthetics is not known despite intensive studies of over 30 years.
Various hypothesis have been developed ranging from lipids to proteins
as molecular targets and unspecific to specific sites of action (for
review, see Miller(1985), Koblin(1990), and Franks and Lieb(1994)). In
recent years the focus has shifted to membrane proteins, including ion
channels and receptors for which different anesthetic effects have been
reported, however, neither a specific nor an unifying mechanism of
action has been described. We have previously identified another
intrinsic membrane protein, the plasma membrane Ca pump, as a potential target for anesthetic action (Kosk-Kosicka
and Roszczynska, 1993; Kosk-Kosicka, 1994). Its
Ca
-ATPase activity and Ca
transport
are inhibited by a variety of inhaled anesthetics, both clinically used
and experimental ones (KoskKosicka and Roszczynska, 1993; Franks et
al., 1995). (
)We have described the effects of four
halogenated volatile anesthetics that are used in clinical anesthesia
on the normal process of enzyme activation by either calmodulin binding
or dimerization (Kosk-Kosicka and Roszczynska, 1993). All four drugs
inhibited these two activation pathways in a dose-dependent manner and
with a similar I
. The inhibition was observed at their
clinical concentrations suggesting that Ca
-ATPase was
not only a good model target of an intrinsic membrane protein but could
also be a pharmacological in vivo target for this group of
general anesthetics. In subsequent studies we have demonstrated that
the inhibition was selective as judged by the following criteria.
First, two other groups of general anesthetics, barbiturates and
alkanols, did not inhibit the enzyme at their anesthetic concentrations
(Kosk-Kosicka et al., 1992).
Second, several other
ATPases including Mg
-ATPase and
Na
,K
-ATPase showed significantly
lower sensitivity to the volatile anesthetics than did the
Ca
-ATPase. (
)Third, the above phenomena
were observed in three distinct plasma membranes, ranging from
erythrocytes to neuronal and endothelial cell types (Kosk-Kosicka et al., 1995b). As the site and mechanism of anesthetic action
have not been explained and only a few proteins among the several
reported to be targeted by anesthetics in vitro are affected
at clinically relevant concentrations the observed inhibition of the
Ca
-ATPase activity deserves a close attention.
In
the present study we have investigated the mechanism of enzyme
inhibition by volatile anesthetics by two experimental approaches:
Ca-ATPase activity assay and fluorescence
spectroscopy measurements. We have used dimeric
Ca
-ATPase whose activation is independent of a
modulatory protein calmodulin since we have previously established that
both normal modes of enzyme activation (by calmodulin binding to enzyme
monomers and by the self-association of monomers to dimers) are equally
sensitive to this group of general anesthetics (Kosk-Kosicka and
Roszczynska, 1993; Kosk-Kosicka and Bzdega, 1988; Kosk-Kosicka et
al., 1990). We have assessed the effects of the anesthetics on the
Ca
-dependent conformational changes of the enzyme by
monitoring the Ca
-dependent changes in fluorescence
intensity of two probes: 1) intrinsic tryptophan(s) that reflect a
conformational change which the Ca
-ATPase undergoes
upon binding the substrate Ca
in the initial step of
enzymatic cycle, and 2) an external probe, fluorescein
5`-isothiocyanate (FITC) (
)attached to lysine 601 in the
active site that normally binds ATP (Dupont, 1976; Inesi et
al., 1980; Kosk-Kosicka and Inesi, 1985; Kosk-Kosicka et
al., 1989). We demonstrate that the anesthetics inhibit both
measures in a dose-dependent manner and there is a good correlation
between the attenuation of the Ca
-dependent
conformational changes and the inhibition of the
Ca
-ATPase activity. In addition, the observed changes
in the total tryptophan fluorescence also suggest that the anesthetics
affect enzyme conformation. We analyze these findings with respect to
the demonstrated binding of small ligands such as xenon (a very potent
experimental inhaled anesthetic) in interior spaces of metmyoglobin
which affects the internal motions and substates of the protein
(Schoenborn and Featherstone, 1967; Tilton et al., 1984). As
proteins undergo motions small molecules enter and interact with
nonpolar sites in the protein interior (Englander et al.,
1972; Lakowicz and Weber, 1973; Eftink and Ghiron, 1976; Cooper, 1976;
Cohen et al., 1977; Lim and Sauer, 1991; Eriksson et
al., 1992; Lim et al., 1994). We postulate that
interaction of anesthetic molecules with nonpolar sites in the
Ca
-ATPase molecule modifies conformational
substate(s) of the protein which results in impairment of its enzymatic
function. We consider binding of small molecules in nonpolar internal
protein spaces a general phenomena whose occurrence, however, requires
compatibility between the nonpolar sites available in the protein and
the invading molecule. In the case of gaseous anesthetic such an
interaction may or may not have functional consequences at clinical
anesthetic concentrations depending on the structure (flexibility) and
function of a given protein, as it does for the
Ca
-ATPases but apparently not for myoglobin or
hemoglobin. The lack of a significant functional effect of the
demonstrated anesthetic binding in myoglobin or hemoglobin and their
relationship to anesthesia apparently eliminated it from consideration
as a mechanism of anesthetic action. In contrast, both function and
conformation of the Ca
-ATPase are significantly
disturbed by the anesthetics and their action on this intrinsic
membrane protein which controls intracellular Ca
homeostasis could certainly contribute to the pharmacological
anesthetic effects. Our model needs to be treated as hypothetical until
future advances in NMR or x-ray crystallography allow for its
verification.
Egg yolk phosphatidylcholine (P5763) and CNBr-activated
Sepharose 4B were purchased from Sigma; octaethylene glycol
mono-n-dodecylether (CE
) was obtained
from Nikko (Tokyo, Japan). Coupling of bovine calmodulin to Sepharose
was performed in accordance with Pharmacia Biotech Inc. instructions as
described earlier (Kosk-Kosicka and Bzdega, 1988). Enflurane and
isoflurane were obtained from Anaquest (Liberty Corner, NJ);
methoxyflurane was from Abbott Hospital Products; and thymol-free
halothane from Halocarbon Laboratories (River Edge, NJ). Halothane is a
two-carbon alkane halogenated derivative, the other three anesthetics
belong to halogenated methyl ethyl ether derivatives as shown in Fig. 1.
Figure 1: Chemical structures of volatile anesthetics used in the study.
The methods used for preparation of erythrocyte ghost
membranes and sarcoplasmic reticulum (SR), purification of the
Ca-ATPase from erythrocyte membranes, determination
of protein, and Ca
concentration were as described
previously (Kosk-Kosicka et al., 1983, 1986; Kosk-Kosicka and
Bzdega, 1988). Free Ca
concentrations were calculated
(Fabiato and Fabiato, 1979) from total calcium and EGTA concentrations,
based on the constants given by Schwartzenbach et al.(1957).
Total calcium was measured by atomic absorption. Sarcoplasmic reticulum
was prepared from rabbit skeletal muscle in the laboratory of Dr. Inesi
(Eletr and Inesi, 1972).
On-line formulae not verified for accuracy
where is the pre-exponential factor and
is the lifetime. The fractional intensity of each
component in the decay is given
by:
On-line formulae not verified for accuracy
the best fit between the data and the calculated value is
indicated by a minimum value for the goodness-of-fit parameter
:
On-line formulae not verified for accuracy
where is the number of degrees of freedom and
and
m are uncertainties in the phase and modulation values,
respectively.
Data are expressed as the mean ± S.E. of three to six independent experiments performed in duplicates. The data points were fitted by the nonlinear regression method.
We have demonstrated a strong correlation between the general
anesthetic potency (as expressed in MAC values) of several halogenated
volatile anesthetics and their ability to inhibit the
Ca-ATPase activity (Kosk-Kosicka and Roszczynska,
1993). The anesthetic concentrations in our activity assays were
expressed in volume % at 37 °C for easy comparison with their
anesthetic potency. At present we have recalculated the values into
millimolar concentrations (in addition, original data for
methoxyflurane have been included) and compared the concentrations of
the anesthetics in the reaction mixture that half-maximally inhibit the
purified erythrocyte Ca
-ATPase with their
concentrations in blood to which the enzyme may be exposed when 1 MAC
of a given anesthetic is administered in clinical anesthesia. Fig. 2shows that the concentrations of all four agents that the
enzyme may encounter during anesthesia inhibit enzyme function.
Figure 2:
Correlation between anesthetic and
inhibitory potencies of four inhalation anesthetics: isoflurane
(), methoxyflurane (
), enflurane (
), and halothane
(*) at 37 °C. The anesthetic potencies, expressed in millimolar
concentrations, in blood were calculated from published values of MAC
taking into consideration the appropriate blood/gas partition
coefficients (Eger, 1974; Koblin, 1990; Kosk-Kosicka and Roszczynska,
1993). Inhibitory potencies are expressed in millimolar concentrations
(in the reaction mixture) that half-maximally inhibit
Ca
-ATPase activity of the purified dimeric enzyme.
Both sets of values were obtained at 37 °C, i.e. human
body temperature.
The
anesthetics decrease both V and Ca
affinity of the Ca
-ATPase activity (Fig. 3). As shown in Fig. 3isoflurane at a
concentration that half-maximally inhibits the enzyme causes a small
yet statistically significant shift in the apparent K
for calcium from pCa 7.05 ± 0.05 to 6.9 ±
0.04. Similar results were obtained with other anesthetics (data not
shown).
Figure 3:
Calcium dependence of
Ca-ATPase activity in the presence (
) and
absence of isoflurane (
). The Ca
-ATPase activity
was assayed as described under ``Materials and Methods.'' The
reaction mixture contained 50 mM Tris maleate, pH 7.4, 120
mM KCl, 8 mM MgCl
, 150 µM
C
E
, 1 mM EGTA, and 3 mM ATP.
Various amounts of CaCl
were added to obtain the calcium
concentrations specified in the horizontal axis. Enzyme
concentration was 70 nM. The assays were performed at 37
°C. Inset shows normalized data of one typical experiment,
where 100% is the maximal activity either with or without isoflurane.
Six independent experiments were performed in duplicates. The mean K
for the enzyme in the presence of isoflurane
was significantly different from the control (p <
0.04).
The effects of volatile anesthetics on the enzyme were
subsequently examined at 25 °C by two experimental approaches:
Ca-ATPase activity assay and fluorescence
spectroscopy measurements. Fig. 4shows that at this temperature
the activity is also inhibited in a dose-dependent manner, similar to
the patterns observed at 37 °C except that higher concentrations
are required for a comparable extent of inhibition. This temperature
dependence is in agreement with nonpolar interactions between each
anesthetic and the enzyme (Collins and Washabaugh, 1985; Makhatadze and
Privalov, 1995).
Figure 4:
Inhibition of the
Ca-ATPase activity by isoflurane (
), enflurane
(
), methoxyflurane (
), and halothane (*). The
Ca
-ATPase activity was assayed as described under
``Materials and Methods.'' The reaction mixture contained 50
mM Tris maleate, pH 7.4, 120 mM KCl, 8 mM
MgCl
, 150 µM C
E
, 1
mM EGTA, 17.5 µM free Ca
, and 3
mM ATP. Ca
-ATPase concentration was 70
nM. The assays were performed at 25 °C. The specific
activity (100% activity) was 220 ± 9 µmol of
P
/mg of protein/h.
The effect of anesthetics on the functionally
important enzyme conformation has been assessed by measuring the
calcium-dependent increase in intrinsic tryptophan fluorescence induced
by substrate Ca binding and in addition, by the
decrease of the fluorescence of an external probe, FITC, bound in the
active site of the enzyme. As shown in Fig. 5the
Ca
-induced increase in tryptophan fluorescence is
attenuated by all three anesthetics in a similar dose-dependent manner.
Halothane effect could not be determined accurately due to the high
extent of quenching of tryptophan fluorescence by this compound. The
half-maximal reduction is observed at
0.16-0.26 mM concentrations of the agents in the order of efficiency shown in Fig. 5; the difference between their F
values may
be not statistically significant. The Ca
-induced
fluorescence increase is readily reversed by addition of EGTA both in
the presence and absence of anesthetics. Similar dose dependence is
observed in the attenuation of the Ca
-dependent
decrease in FITC fluorescence, even though the
F/
F
does not proceed to 0 (Fig. 6). Thus, there is a good
correlation between the inhibitory action of the anesthetics on the
Ca
-ATPase activity (I
=
0.20-0.27 mM) and the Ca
-induced
conformational change assessed by either tryptophan or FITC
fluorescence measurements (F
= 0.16-0.23
mM).
Figure 5:
Concentration dependence of the
suppressing effect of volatile anesthetics on the
Ca-dependent increase in the intrinsic tryptophan
fluorescence. The fluorescence measurements were performed at 25 °C
as described under ``Materials and Methods.'' Enzyme
concentration was 70 nM. Fluorescence increase recorded at 330
nm was induced upon addition of 1.2 µM free
Ca
. The Ca
-dependent change in
tryptophan fluorescence in the control in the absence of anesthetics
(1.2-2% of the total tryptophan fluorescence) is shown as 100%.
The
F/
F
at each anesthetic concentration is
expressed in percent of the Ca
-dependent fluorescence
increase in the presence of anesthetic as compared to the control
experiment. The increase was reversible upon addition of EGTA both in
the absence and presence of anesthetics. Symbols used for
anesthetics are as described in the legend to Fig. 4.
Figure 6:
Concentration dependence of the
suppressing effect of volatile anesthetics on the
Ca-dependent decrease of FITC fluorescence. Labeling
of the Ca
-ATPase with FITC and fluorescence
spectroscopy measurements were performed as described under
``Materials and Methods.'' Fluorescence decrease was induced
upon addition of 1.2 µM free Ca
to the
reaction mixture containing 70 nM FITC-labeled enzyme. The
Ca
dependent change in FITC fluorescence of the
control in the absence of anesthetics (1-2% of the total
fluorescence) is shown as 100%. The
F/
F
at
each anesthetic concentration is expressed in percent of the
Ca
-dependent fluorescence decrease in the presence of
anesthetic as compared to the control experiment. Symbols used
for anesthetics are as described in the legend to Fig. 5.
We have also analyzed the anesthetics effects on the
total tryptophan fluorescence intensity of Ca-ATPase.
All four studied compounds decrease the fluorescence in a
dose-dependent linear fashion (Fig. 7). In order to distinguish
which type of quenching process is taking place, lifetime fluorescence
intensity measurements were performed in the presence and absence of
isoflurane. As shown in Table 1intensity decays are very
heterogenous and three exponentials are needed to fit the data. This is
expected for a protein with several tryptophans and was observed
previously for the SR Ca
-ATPase (Lakowicz et
al., 1986; Gryczynski et al., 1989; Wang et al.,
1992). In the presence of isoflurane the lifetimes decrease (compare
exponential fits
and <
> in Table 1). These changes in lifetimes are characteristic of
collisional quenching (Pesce et al., 1971; Lakowicz, 1991). On
the other hand the apparent quenching constants, K
, calculated from the decrease in fluorescence
intensity (in Fig. 7and Table 2) are larger than those
possible if collisional quenching were entirely responsible for the
observed fluorescence decrease (for example, for other multitryptophan
proteins
-trypsin and pepsin the K
are 2.4 M
and 9.5 M
),
indicating contributions from other processes such as binding of the
quencher and conformational changes (Sellers and Ghiron, 1973; Eftink
and Ghiron, 1976; Lakowicz, 1991). Accordingly, steady-state
fluorescence intensity spectra recorded for the enzyme in the absence
and presence of increasing concentrations of isoflurane show a
progressive decrease of intensity and a red shift of up to 6 nm at 0.52
mM isoflurane concentration (Fig. 8), suggesting an
increased polarity of the tryptophan environment that could result from
a conformational change caused by the interacting anesthetic. It has
been shown that conformational changes caused by partial denaturation
of protein result in fluorescence intensity and lifetime decreases and
a red shift of the spectrum (Gryczynski et al., 1988). To
assess the contribution of a direct quenching of tryptophans by the
anesthetics we have tested the effect of anesthetics on the
fluorescence of the heat-denaturated enzyme and on free tryptophan in
the same reaction mixture, including detergent. The free tryptophan
fluorescence was not affected while there was a decrease in the
fluorescence of the denatured enzyme. In addition, no fluorescence
resonance energy transfer was observed between tryptophan and
anesthetics (no overlap of absorption and emission spectra). All these
findings point to a substantial contribution of conformational change
of the enzyme in the anesthetic effect on the total tryptophan
fluorescence.
Figure 7:
Concentration dependence of the total
intrinsic tryptophan fluorescence intensity of the
Ca-ATPase in the presence of halothane (*),
methoxyflurane (
), isoflurane (
), and enflurane (
).
F
is the total fluorescence intensity recorded at
330 nm in the absence of a volatile anesthetic; F
is the total fluorescence intensity at each concentration of
the anesthetic (both at time zero). The experiment was performed at 25
°C in 100 mM Tris-HCl, pH 7.4, 8 mM
MgCl
, 150 µM C
E
, 130
mM KCl, and 1 mM EGTA.
Figure 8:
Steady-state fluorescence emission spectra
of erythrocyte Ca-ATPase in the absence (upper) and presence (lower) of 0.52 mM isoflurane. Excitation wavelength was 290 nm. Spectra were
recorded at 25 °C in 100 mM Tris-HCl, pH 7.4, 8
mM MgCl
, 150 µM
C
E
, 130 mM KCl, and 1 mM
EGTA.
To examine the contribution of lipids in the decrease
of total tryptophan fluorescence due to anesthetics we investigated
their action on the sarcoplasmic reticulum Ca-ATPase.
This enzyme could be studied in the native membrane because it
constitutes
80% of the total membrane protein as compared to
0.01% of the membrane protein represented by the
Ca
-ATPase in the plasma membrane. Tryptophan
fluorescence of the SR enzyme is reduced by the four anesthetics with
the same order of efficiency as observed for the purified plasma
membrane Ca
-ATPase (Table 2). Also similar to
the plasma membrane enzyme, the apparent quenching constants are too
large for an exclusively diffusion controlled process. They are
approximately two times lower than for the purified plasma membrane
enzyme. The difference could arise from either an interference of the
surrounding lipids or differences in the enzyme protein molecules such
as existence of fewer sites for interaction with the anesthetics in the
SR enzyme or their lower affinity than in the plasma membrane enzyme.
The fact that about 2-fold lower anesthetic concentrations are required
for inhibition of the purified plasma membrane
Ca
-ATPase activity as compared to the enzyme in the
erythrocyte ghost membrane (Fig. 9: I
= 0.26
and 0.50 mM halothane) suggests that the lower apparent
quenching constants for the enzyme in SR may be caused by a decrease of
tryptophans accessibility to the anesthetics by the lipid bilayer. A
similar difference in halothane sensitivity of the
Ca
-ATPase in ghost membrane versus purified
was observed previously at 37 °C (Kosk-Kosicka and Roszczynska,
1993). As shown in Fig. 9, the Ca
-ATPase in SR
requires a 40-50% higher halothane concentration for half-maximal
inhibition than the enzyme in erythrocyte membrane. This result and the
fact that at concentrations up to 0.2 mM halothane the enzyme
in SR is activated, in contrast to the plasma membrane
Ca
-ATPase (Fig. 9), indicate that in addition
to the lipid effect, the nonpolar sites in the SR enzyme may have a
lower affinity for the anesthetics than comparable sites in the plasma
membrane enzyme.
Figure 9:
Comparison of the effect of halothane on
the Ca-ATPase activity in the purified
Ca
-ATPase (*), Ca
-ATPase in
erythrocyte ghost membranes (
), and Ca
-ATPase in
sarcoplasmic reticulum (
). The activity assay was performed at
25 °C as described under ``Materials and Methods.'' The
reaction mixture for sarcoplasmic reticulum contained the divalent
cation ionophore A23187 (10 µM) and the reaction time was
15 min as compared to 30 min for the plasma membrane
Ca
-ATPase. For both membranes the
Ca
-ATPase activity was calculated as the difference
between the activity detected in the presence of both Mg
and Ca
ions and absence of Ca
ions. The specific Ca
-ATPase activities (in
µmol of P
/mg of protein/h) were 100 in SR and 0.35 in
erythrocyte ghosts.
We have shown that volatile anesthetics affect both the
conformational state and activity of the Ca-ATPase.
There is a strong correlation between the attenuation of the
Ca
-induced functionally important conformational
change of the protein and impairment of its enzymatic function by
anesthetics. Also our data on anesthetic effects on the total
tryptophan fluorescence, including the decrease of fluorescence
intensity, the red shift, and the decrease in lifetimes, indicate that
anesthetics produce conformational changes in the enzyme molecule. To
explain these effects we suggest that the anesthetics enter the protein
and interact with nonpolar amino acids in its interior. To accommodate
the intruding anesthetic the protein molecule undergoes structural
rearrangement that results in the loss of its
Ca
-ATPase activity. In proposing this mechanism of
anesthetic action on the Ca
-ATPase we are considering
multiple x-ray diffraction and NMR data that have demonstrated binding
of gaseous anesthetics and other molecules, including urea and
steroids, in hydrophobic ``cavities'' in the interior of
several proteins (Schoenborn, 1965; Schoenborn et al., 1965;
Schoenborn and Featherstone, 1967; Nunes and Schoenborn, 1973; Brown et al., 1976; Sachsenheimer et al., 1977; Hibbard and
Tulinsky, 1978; Tilton and Kuntz, 1982; Tilton et al., 1984;
Otting et al., 1991; Arevalo et al., 1994; Jain et al., 1994; Williams et al., 1994; Hubbard et
al., 1994).
The Ca-ATPase seems to have a
fitting nonpolar site(s) available to all five volatile anesthetics
studied in our laboratory (present paper and in Kosk-Kosicka and
Roszczynska, 1993) and to xenon (Franks et al., 1995) as
judged by their inhibitory effects. Temperature effects (on the
Ca
-ATPase activity) support a nonpolar nature of
interactions between the anesthetics and the amino acids in the
Ca
-ATPase. It would be worthwhile to investigate
effects of bigger or/and less polarizable anesthetic molecules on the
enzyme to further test our hypothesis.
If volatile anesthetics
invade the interior of the Ca-ATPase molecule their
binding is expected to affect internal motions and conformational
substates of the enzyme (Schoenborn, 1965; Brown et al., 1976;
Hibbard and Tulinsky, 1978; Tilton et al., 1984; Lim et
al., 1994). The observed effects on total tryptophan fluorescence
might then reflect the invasion that results in subtle conformational
changes including tryptophan environment. The lack of the
Ca
-sensitive increase in tryptophan fluorescence in
the presence of anesthetics would reflect the increased stability of
the substate characteristic of a nonactive enzyme: enzyme with bound
anesthetic cannot attain the conformation necessary for its normal
activation. As a result no Ca
-ATPase activity can be
detected. Recent studies, including investigation of the effects of
amino acid mutations in the hydrophobic core of
-repressor on
internal packing interactions, have demonstrated that the protein
accommodates the potentially disruptive residues with shifts in its
-helical arrangement and becomes more stable since its packing is
improved. However, the rearrangements cause repositioning of functional
residues, which results in reduced function of the protein (Lim and
Sauer, 1991; Lim et al., 1994). By analogy, a distortion of
amino acid side chain(s) (resulting from anesthetic binding to the
Ca
-ATPase) that normally coordinate to Ca
may cause a change in Ca
binding and this
slightly changed substate may not reveal the expected tryptophan
fluorescence increase. The small shift observed in Ca
affinity at 37 °C favors this interpretation as opposed to a
total loss of Ca
binding in the presence of an
anesthetic. Alternatively, insertion of the anesthetic and the
resulting rearrangement may affect not Ca
binding but
the coupling between the Ca
binding and induction of
a proper conformation that in the active enzyme is detected as the
Ca
-dependent change in tryptophan fluorescence.
Anesthetics effects on the conformational change (normally reflected in
Ca
-dependent changes in fluorescence of either
tryptophan or FITC) correlate well with their effects on the
Ca
-ATPase activity in support of the interpretation
that anesthetics prevent the enzyme from assuming a functionally proper
active conformation. This suggestion is consistent with the observation
that two activation pathways of the enzyme, by dimerization of enzyme
monomers and by calmodulin binding to enzyme monomers, are inhibited by
anesthetics to a comparable extent implying a ``basic''
defect in the activation mechanism. In contrast, the two pathways are
affected distinctly different by two other groups of general
anesthetics, barbiturates and alkanols (Kosk-Kosicka et al.,
1995a).
These compounds with the structural properties of
detergents are postulated to inhibit Ca
-ATPase
monomers by binding to an exposed nonpolar patch on the enzyme surface
to which calmodulin binds to activate monomers and which is not easily
accessible in enzyme dimers.
In contrast to
Ca-ATPase, two other plasma membrane ATPases, the
Na
,K
-ATPase and
Mg
-ATPase, require at least 3-5 times higher
concentrations of volatile anesthetics.
Several other
membrane proteins that have been reported as potential targets for
anesthetics, including voltage-gated sodium, potassium, and calcium
channels, are half-maximally inhibited only at halothane concentrations
ranging from 4 to 30 times higher than its clinical potency, as
summarized in a recent review of the last 10 years of search for the
target of anesthetic action (Franks and Lieb, 1994). From among many
proteins shown to be affected by the anesthetics only ligand-gated ion
channels, including GABA receptors and possibly nicotinic acetylcholine
receptor, are also targeted at clinical anesthetic concentrations.
Studies performed in many laboratories imply GABA
receptors
channel complex as a major target for the general anesthetics (for
review, see Franks and Lieb(1994)). The pharmacological target is
expected to be affected by anesthetics at their clinical concentrations
(
1 MAC) because the dose-response curves for the induction of
anesthesia with volatile anesthetics are very steep (concentrations 20%
above 1 MAC anesthetize almost all subjects) and because concentrations
2-4 times higher have damaging side effects on the organism. Our
findings indicate that the anesthetics action on the
Ca
-ATPase is selective at their pharmacological
concentrations. It remains to be tested whether anesthetic action on
the Ca
-ATPases and the receptors at the molecular
level is exerted by their binding in protein interior. Our hypothesis
cannot be verified at present without x-ray structure data.
The
finding that volatile anesthetics inhibit the
CaATPases in vitro at their clinical
concentrations while several other membrane proteins are affected by
the anesthetics only at concentrations significantly exceeding those
used in the clinical situation opens a possibility that the enzyme is
perturbed during anesthesia and this anesthetic action has important
physiological consequences. The consequences would be multiple as so
many cellular events and cascades depend on precisely controlled
intracellular Ca
concentrations, ranging from
regulation of cell shape in erythrocytes, to neuronal transmission to
depression of contractile force in heart.
In conclusion, we propose
that the observed correlation between the attenuation of the
functionally important conformational change of the enzyme and
inhibition of its Ca-ATPase activity could be
explained by permeation of the protein molecule by volatile anesthetic
and stabilization of its nonactive conformational substate. We view
this process as an example of a general phenomena of discriminating
binding of small ligands in protein interior.