(Received for publication, August 22, 1996, and in revised form, December 30, 1996)
From the Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
The key signal transduction enzyme protein kinase
C (PKC) contains a hydrophobic binding site for alcohols and
anesthetics (Slater, S. J., Cox, K. J. A., Lombardi, J. V., Ho, C.,
Kelly, M. B., Rubin, E., and Stubbs, C. D. (1993) Nature
364, 82-84). In this study, we show that interaction of
n-alkanols and general anesthetics with PKC results in
dramatically different effects on membrane-associated compared with
lipid-independent enzyme activity. Furthermore, the effects on
membrane-associated PKC
differ markedly depending on whether
activity is induced by diacylglycerol or phorbol ester and also on
n-alkanol chain length. PKC
contains two distinct
phorbol ester binding regions of low and high affinity for the
activator, respectively (Slater, S. J., Ho, C., Kelly, M. B., Larkin,
J. D., Taddeo, F. J., Yeager, M. D., and Stubbs, C. D. (1996)
J. Biol. Chem. 271, 4627-4631). Short chain
n-alkanols competed for low affinity phorbol ester binding
to the enzyme, resulting in reduced enzyme activity, whereas high
affinity phorbol ester binding was unaffected. Long chain
n-alkanols not only competed for low affinity phorbol ester
binding but also enhanced high affinity phorbol ester binding.
Furthermore, long chain n-alkanols enhanced phorbol ester
induced PKC
activity. This effect of long chain
n-alkanols was similar to that of diacylglycerol, although the n-alkanols alone were weak activators of the enzyme.
The cellular effects of n-alkanols and general anesthetics
on PKC-mediated processes will therefore depend in a complex manner on
the locality of the enzyme (e.g. cytoskeletal or
membrane-associated) and activator type, apart from any
isoform-specific differences. Furthermore, effects mediated by
interaction with the region on the enzyme possessing low affinity for
phorbol esters represent a novel mechanism for the regulation of PKC
activity.
Driven by the need to improve anesthetic efficacy and to remove deleterious side effects, much effort has been expended in determining the site and mechanism of anesthetic action, yet both have remained elusive (1, 2). Current theories of anesthesia are based on the observations made by Meyer (3) and Overton (4) at the beginning of this century that anesthetic potency is a linear function of the solubility of the agent in olive oil or its hydrophobicity (1). Thus a site of anesthetic action must follow what is often referred to as the "Meyer-Overton rule," which requires the potency of an effect to be a linear function of anesthetic hydrophobicity for it to be critically involved in anesthesia. For many years the site of anesthetic action was thought to reside within the hydrophobic cell membrane interior (1). However, these "lipid theories" have been criticized on the grounds that the magnitude of effects on the physical properties of membrane lipids at clinically relevant concentrations are too small to be responsible for inducing anesthesia (2). The alternative "protein theory" proposes that a hydrophobic binding site for anesthetics resides within a protein (5). Strong support for this came from a series of important studies that showed that the soluble enzyme firefly luciferase contains a hydrophobic binding site for alcohols and anesthetics, which mediated effects on enzyme activity that obeyed the Meyer-Overton rule (5, 6).
The challenge now is to find a site for anesthetics on a central nervous system protein that could be relevant to the mechanism of anesthesia. Efforts in this direction have shown that a number of membrane proteins localized in the central nervous system are sensitive to clinically relevant concentrations of alcohols and anesthetics. Examples include voltage- and ligand-gated ion channels, synaptic receptors, and G-protein-mediated second messenger systems (e.g. Refs. 2 and 7-14). Most of the effects have been observed on intact cell systems; therefore, ascertaining which protein in a cascade of signaling events may actually be the target is difficult. In addition, membrane lipids are required for protein function in all of these systems, so it is difficult to discount a perturbation of the membrane being sensed by the protein leading to the observed effect. Furthermore, there is also the possibility that the membrane protein-lipid interface maybe involved in anesthetic action (1, 15, 16).
Strong support for the protein theory of anesthesia was provided in a
recent report from this laboratory (17), which showed that the
regulatory region of protein kinase C
(PKC),1 a key enzyme in intracellular
signal transduction (18, 19), contains a hydrophobic binding site for
alcohols and anesthetics. Furthermore, it has been shown that
inhibition of PKC by staurosporine decreases the EC50 for
anesthesia in tadpoles, supporting a role for the enzyme in the
anesthetic process (20, 21). The discovery of the n-alkanol
and anesthetic binding site was based on the finding that the in
vitro activity of a rat brain preparation consisting of the
"conventional," Ca2+-dependent PKC,
I/II, and
isoforms was inhibited, in a lipid-free assay, by a homologous series of n-alkanols along with
halothane and enflurane, with a potency that followed the Meyer-Overton rule (17). An inhibitory effect of anesthetics on rat brain Ca2+-dependent PKC has also been demonstrated
in other in vitro studies (22). The n-alkanols
and anesthetics were also observed to inhibit membrane-associated PKC
activity. However, the question remains whether this effect on
membrane-associated PKC is mediated by a hydrophobic
n-alkanol and anesthetic binding site similar to that
present on the lipid-free enzyme form and whether, in each case, a
common mechanism of action is involved.
The activity of PKC is regulated by interaction with co-factors,
including Ca2+ and anionic phospholipids, along with its
main natural activator, diacylglycerol (19). Binding of this activator
and the tumor-promoting phorbol esters has been shown to be confined to
two zinc finger motifs (cys1 and cys2), which constitute the conserved
C1 domain contained within the regulatory region of the enzyme
(23-28). Using peptide fragments, it has been shown that both of these
subdomains bind phorbol esters, the former with a relatively low
affinity (26). Furthermore, recent studies have provided evidence that this asymmetry in phorbol ester binding to the cys1 and cys2 subdomains is carried over into the intact enzyme (29, 30). Based on a highly
sensitive phorbol ester binding assay recently developed in this
laboratory (31), the low and high phorbol ester binding affinities for
these sites on intact PKC were directly determined. Using this
technique it was found that DAG interacts preferentially with the low
affinity phorbol ester binding site (31). Furthermore, this interaction
of diacylglycerol with the low affinity phorbol ester binding region
resulted in a cooperative enhancement of high affinity phorbol ester
binding. In keeping with this, the level of PKC activity induced by
diacylglycerol and phorbol ester together was found to be greater than
that achievable for either activator singly, each activator being
present at a concentration that when alone was sufficient to induce
maximal activity (31, 32).
In the present study, it was hypothesized that alcohol and anesthetics
might interact with either the low or high affinity activator binding
sites, or both, based on the finding that the alcohol and anesthetic
binding site on PKC is also located within the regulatory domain (17,
22). First, it was confirmed that alcohols and anesthetics inhibited
membrane lipid-independent PKC with a potency that was a linear
function of hydrophobicity. By contrast, effects of these agents on
membrane-associated PKC
differed markedly dependent on whether
activity was induced by phorbol ester or diacylglycerol and also on
n-alkanol chain length (i.e. hydrophobicity).
Thus, short chain alcohols inhibited membrane-associated activity
induced by either phorbol ester or diacylglycerol. However, long chain
alcohols and anesthetics potentiated phorbol esterinduced activity
but had biphasic effects on diacylglycerol-induced activity. Evidence
for direct competition between n-alkanols and phorbol esters
for low affinity phorbol ester binding is presented.
A peptide substrate corresponding to the PKC
phosphorylation site of myelin basic protein (QKRPSQRSKYL) was custom
synthesized by the Jefferson Cancer Institute Protein Chemistry
Facility. Lipids were obtained from Avanti Polar Lipids, Inc.
(Alabaster, AL), except
N-(5-dimethylaminonapthalene-1-sulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (dansyl-PE), which was from Molecular Probes (Eugene, OR). SAPD was
from Calbiochem. Protamine sulfate and
4-12-O-tetradecanoylphorbol-13-acetate (TPA) were from
Sigma. ATP was from Boehringer Mannheim, and
[
-32P]ATP was from DuPont NEN. The
n-alkanols and propofol were obtained from Aldrich. Enfurane
was from Anaquest (Madison, WI). All other chemicals were of analytical
grade and obtained from Fisher.
Recombinant
PKC (bovine brain) was prepared using the baculovirus Sf9 insect
cell expression system (33) and purified as described previously (34).
Assays of membrane-associated, lipid-dependent activity
followed a previously described method (32, 35). Large unilamellar
vesicles of defined size (100 nm) and composition (36), prepared as
described elsewhere (37), consisted of
1-palmitoyl-2-oleoylphosphatidylcholine (POPC):bovine brain
phosphatidylserine (4:1 molar, 150 µM), incorporating DAG
at a level of 4 mol % of total lipid concentration (POPC composition was modified accordingly) or 0.5 µM TPA as indicated. For
determinations of lipid-free activity, PKC
(~10 ng) was assayed in
the presence of 0.4 mg ml
1 protamine sulfate. Additions
of n-alkanols and anesthetics were made from concentrated
stock buffer solutions. Loss of volatile anesthetics due to evaporation
from reaction tubes has been previously determined under assay
conditions similar to those used here and found to be negligible
(17).
Binding was
quantified using fluorescence resonance energy transfer from PKC
tryptophans to the 2-(N-methylamino)benzoyl fluorophore of
SAPD, as described previously (31). Briefly, the fluorescence
intensities at the emission maxima of PKC tryptophans and SAPD (333 and
425 nm, respectively), obtained on excitation of the tryptophan
fluorophore at 290 nm, were determined using a PTI Alphascan
fluorimeter (Photon Technology International, Princeton, NJ). The assay
conditions were identical to those used for PKC activity
determinations, except that the assay volume was 2 ml, and the PKC
concentration was 5 ng/µl. Sapintoxin-D was titrated into the assay
system, and the fluorescence intensities were recorded as a function of
the phorbol ester concentration. The direct excitation of the SAPD
fluorophore was corrected for by measuring the fluorescence intensity
corresponding to each SAPD concentration in the presence of all assay
components (including alcohols or anesthetics where added) except
PKC
. This signal was then subtracted from the corresponding
fluorescence intensities observed in the presence of the enzyme,
therefore isolating the contribution of resonance energy transfer to
the observed signal. The resultant fluorescence intensity
(Fi) values were normalized to that observed in
the presence of all assay components except for the phorbol ester
(F0) according to: (Fi
F0)/F0. The Hill coefficients corresponding to low and high affinity SAPD binding were
obtained by fitting binding data using nonlinear least squares analysis
to a modified Hill equation (31).
Partitioning
of SAPD was determined from Stern-Volmer plots, as described previously
(38). Briefly, the emission fluorescence intensity at 530 nm of the
fluorophore-labeled phospholipid
1-palmitoyl-2-(7-nitrobenzo-2-oxa-1,3-diazole-4-yl)aminohexanoyl-PC (C6-NBD-PC; 1 mol % of the total lipid concentration) on
excitation at 470 nm was measured as a function of SAPD concentration.
Conditions were identical to those used in the activity and binding
assays and included all assay components, except PKC. The probe was also used to assess the effect of SAPD on head group organization by
measuring the fluorescence anisotropy (38).
In previous a study from this laboratory, evidence for a
hydrophobic binding site within the regulatory domain of PKC for alcohols and anesthetics was provided, based on an inhibitory effect of
these agents on a rat brain PKC,
, and
isoform mixture in a
lipid-free assay system (17). In the present study, the effects of a
homologous series of n-alkanols on membrane-associated phorbol ester compared with DAG-induced PKC activity were determined. The aim here was to determine the site of interaction of
n-alkanols and to gain some insight into the mechanism of
the effects of these agents on PKC activity, which has not been
resolved in previous studies. Based on the asymmetric binding of
phorbol esters and diacylglycerols to the two activator binding sites
on PKC
, it was predicted that interaction of alcohols and
anesthetics with these sites would result in disparate effects of these
agents on PKC activity, dependent on activator type. In this study, a single Ca2+-dependent PKC
isoform, prepared
using the baculovirus Sf9 insect cell expression system (33), was used
to circumvent the effects of variations in mixed isoform preparations
that tend to complicate the interpretation of results.
Lipid-independent PKC activity, induced by protamine sulfate, was
found to be inhibited in a concentration-dependent manner by a homologous series of n-alkanols. The relationship
between the concentration of each n-alkanol required to
inhibit activity by 50% (EC
50) and chain length was
linear (Fig. 1), confirming the existence of a
hydrophobic inhibitory site for n-alkanols on the single
PKC
isoform, as previously shown for the rat brain mixed isoform
preparation (17). In addition, as shown in Fig. 1 (inset),
it was also found that the general anesthetics enflurane and propofol
inhibited lipid-independent activity with potencies (EC
50) that fitted closely with those of
n-alkanols of comparable hydrophobicity (i.e.
octanol:water partition coefficient).
The chain length-dependent effects of n-alkanols
on membrane-associated PKC activity induced by DAG and phorbol ester
(TPA) are shown in Fig. 2. It was found that
n-alkanols with chain lengths less than that of
n-pentanol inhibited DAG-induced activity, whereas TPA
stimulation was unaffected. For n-alkanols with chain
lengths greater than that of n-hexanol, TPA-induced activity
was potentiated in a concentration-dependent manner,
whereas effects on DAG stimulation were biphasic, consisting of a
potentiation at low alcohol concentrations followed by a reduction in
this potentiation effect at higher alcohol concentrations. A plot of
the potency of the amplifying effect of long chain
n-alkanols on TPA-induced activity (n-alkanol concentration required to increase TPA-induced activity by 50%; EC+50) against n-alkanol chain length was linear
(Fig. 3). Furthermore, as shown in Fig. 3
(inset), the anesthetics enflurane and propofol behaved in a
manner equivalent to that of the long chain alcohols in that
TPA-stimulated activity was enhanced by these agents, with potencies
proportional to the corresponding value of the membrane:buffer
partition coefficient.
In a previous study, it was found that the dose-response curves for the
interaction of the phorbol ester SAPD to PKC and for the induced
activity were "dual sigmoidal," indicating the existence of low and
high affinity binding domains for this phorbol ester on PKC
(31).
This is again shown to be the case in the present study for TPA-induced
activity (Fig. 4A). Addition of the short
chain n-alkanol n-butyl alcohol resulted in an
inhibition of TPA-induced activity within a concentration range
corresponding to the low affinity phorbol ester interaction (~5
µM). This inhibition of membrane
lipid-dependent, TPA-induced PKC activity by short chain
n-alkanols was also observed in a previous study from this laboratory (17), although the inhibition was observed at a phorbol ester concentration of 500 nM, which under the present
assay conditions was unaffected by n-butyl alcohol. However,
the previous study was performed on a PKC
,
, and
isoform
preparation for which it has recently been shown that phorbol ester
affinity varies considerably (39, 40), which may explain the differing
sensitivity of TPA-induced activity to inhibition by short chain
n-alkanols. Furthermore, the present activity assay has also
been modified in a number of respects, principally in the use of myelin
basic protein peptide substrate instead of histone as a phosphate
acceptor and in the use of large unilamellar vesicles instead of small unilamellar vesicles.
By contrast to the inhibitory effect of the short chain
n-alkanol n-butyl alcohol, the representative
long chain n-alkanol n-octanol enhanced
TPA-induced activity (Fig. 4A). This was observed as both a
shift in the TPA dose-response curve to lower activator concentrations
and also as an increase in the maximal activity attained. Furthermore,
the TPA-induced activity dose-response curve observed in the presence
of n-octanol was single sigmoidal, contrasting with the dual
sigmoidal curve obtained in its absence. This suggests that long chain
n-alkanols compete with low affinity phorbol ester binding
(see below). The increase in TPA-induced activity by
n-octanol is similar to that previously observed to occur on
co-addition of DAG together with TPA (Fig. 4A; see Ref. 31).
The data presented in Fig. 4A also show that "basal"
membrane-associated PKC activity induced by Ca2+ in the
absence of activators was unaffected by short chain
n-alkanols, whereas long chain n-alkanols had a
small activating effect. This was, however, negligible compared with
the level of activity induced by phorbol esters or DAG.
Competition between n-alkanols and phorbol esters for
binding to PKC was determined using a binding assay based on
measurements of resonance energy transfer from the PKC
tryptophans
to the fluorescent phorbol ester SAPD, as described previously (31). SAPD has a structure similar to TPA and differs only in the presence of
an N-methylaminobenzoyl-fluorophore at the C-12 position,
and the two phorbol esters have similar activation dose-response curves (data not shown). This method for measuring phorbol ester binding has
several advantages over the commonly used [3H]phorbol
dibutyrate binding assay. First, the latter has much lower binding
affinity for PKC than SAPD (41), so that low affinity PKC-phorbol ester
interactions are not detected. Second, corrections of the data for
nonspecific binding to the membrane do not need to be made, since the
fluorescence resonance energy transfer signal is derived only from the
PKC-bound SAPD. Third, the binding assay is highly sensitive. The SAPD
binding isotherm obtained in the absence of n-alkanols was
double sigmoidal (Fig. 4B), indicating high and low affinity SAPD
binding to PKC
, as previously shown (31). It was necessary to use a
logarithmic abscissa in these plots to reveal the effects of
n-alkanols on high affinity binding. However, this results
in a distortion of the binding isotherm, making it appear that the low
affinity binding event has not plateaued. The binding data are
therefore replotted on a linear abscissa in Fig. 4B, inset,
to clarify that both high and low affinity interactions of SAPD with
the enzyme are saturable.
The effects of n-butyl alcohol and n-octanol on SAPD binding were found to closely correspond to the chain length-dependent effects on TPA-induced activity. Thus, n-butyl alcohol inhibited low affinity SAPD binding, consistent with the inhibition of TPA-induced activity over a concentration range corresponding to this low affinity SAPD interaction (Fig. 4B). Similarly, low affinity SAPD binding was also inhibited by the long chain n-alkanol n-octanol (Fig. 4B). However, the long and short chain n-alkanols had markedly different effects on high affinity SAPD binding, which was found to be unaffected by n-butyl alcohol but enhanced by n-octanol (Fig. 4B). This enhancement of high affinity SAPD binding by n-octanol is consistent with the enhancement of TPA-induced activity (Fig. 4A) and again is similar to the effect of DAG on phorbol ester binding (Fig. 4B; see Ref. 31).
The finding that n-butyl alcohol and n-octanol
inhibited low affinity SAPD binding suggested that these
n-alkanols may share a common site(s) of action. Consistent
with this, the dose-dependent amplification of TPA
activation induced by n-octanol was inhibited by a fixed
concentration of n-butyl alcohol (Fig.
5A). A similar inhibition of the
n-octanol-induced amplification of phorbol ester activity
was obtained when the concentration of n-octanol was fixed
and the level of n-butyl alcohol varied (Fig.
5B).
The possibility has to be considered that the low affinity phorbol
ester binding region might be a nonspecific "site" resulting from a
perturbation of the membrane by the high SAPD (and/or TPA) concentrations used. Based on this, the observed inhibition of low
affinity SAPD binding by the n-alkanols may have resulted from a "stabilization" of this perturbation. However, we are able to discount this hypothesis for several reasons. 1) The low affinity SAPD binding region is saturable (Fig. 4B,
inset). 2) The resonance energy transfer signal observed
between 1 and 10 µM SAPD (the low affinity binding range)
was found in a previous study to be inhibited by TPA (31). This is
inconsistent with a perturbance of the membrane, since both TPA and
SAPD would have been expected to induce a similar nonspecific
perturbing effect, which would have resulted in an enhanced resonance
energy transfer signal, contrasting with the decrease observed. 3) It
was previously shown that, in contrast to TPA, its "nonactivating"
epimer 4-TPA inhibited PKC
activity induced by levels of SAPD
corresponding to its low affinity binding interaction (31). This isomer
of TPA differs only in the orientation of the 4-hydroxy moiety.
Therefore, again, had the low affinity phorbol ester binding site
corresponded to a nonspecific event, then both TPA and 4
-TPA would
have been expected to have similar effects on SAPD-induced activity. 4) The effects of SAPD or TPA at concentrations between 1 and 10 µM on the head group motional properties of the
fluorescent phospholipid C6-NBD-PC were negligible, based
on fluorescence anisotropy data (results not shown). This indicates
that the extent of nonspecific perturbation of this membrane region by
the phorbol esters is negligible. 5) SAPD partitioning into the
membrane was determined using C6-NBD-PC as a probe (Fig.
6). Since the level of quenching of the NBD fluorescence
is proportional to the concentration of SAPD in the vicinity of the NBD
fluorophore within the membrane, measurement of C6-NBD-PC
fluorescence intensity as a function of SAPD concentration
(Stern-Volmer plot) allows SAPD partitioning into the membrane to be
determined. The results show a linear dependence of SAPD partitioning
into bovine brain phosphatidylserine/POPC bilayers on added SAPD
concentration (Fig. 6). If the membrane were perturbed by SAPD (or TPA)
at the higher phorbol ester concentrations in such a manner as to
increase SAPD binding to a nonspecific membrane site, then a nonlinear
Stern-Volmer plot should have resulted. We conclude, therefore, that
the low affinity SAPD binding site does not result from a perturbation
of membrane structure by the phorbol ester itself. 6) The possibility
that the inhibitory effect of n-alkanols (and DAG) on low
affinity SAPD binding results from a stabilization of a nonspecific
perturbation of the membrane by the phorbol ester is ruled out by the
method of data analysis used; the resonance energy transfer signal,
corresponding to SAPD binding, was isolated from the raw observed
fluorescence signal by subtraction of the fluorescence intensities
obtained in the absence of PKC but in the presence of all other assay
components, including SAPD and n-alkanols. This therefore
cancels out any nonspecific effects on SAPD fluorescence arising from
the (minimal) perturbation of the membrane by either the phorbol ester
itself or by the n-alkanols.
It is also possible that the low affinity interaction of SAPD with
PKC may have resulted from an increase in membrane association of
the enzyme due to a perturbation of the membrane structure at high SAPD
concentrations. This possibility was investigated by determining the
level of membrane association based on measurements of resonance energy
transfer from PKC
tryptophans to dansyl-PE, which was incorporated
into membranes used in this study. It was found that the enzyme was
fully associated with the membranes even at SAPD concentrations below
that corresponding to low affinity binding (results not shown).
Therefore, the low affinity binding event could not have resulted from
increased membrane association due to a perturbation of the membrane
structure by the phorbol ester. Similarly, the enhanced level of
phorbol ester-induced activity in the presence of long chain
n-alkanols (or anesthetic) may also have resulted from an
enhanced level of membrane association. This possibility was ruled out
by the finding that the level of membrane association induced by
maximally activating concentrations of SAPD or TPA was unaffected by
long chain n-alkanols and DAG (results not shown).
In the present study, it is shown that n-alkanols
interfere directly with phorbol ester binding to membrane-associated
PKC, providing evidence that a hydrophobic binding region for these agents exists on both the membrane-associated and lipid-independent (protamine sulfate-activated) forms of the enzyme. However, the nature
of the effects of n-alkanols and anesthetics on
membrane-associated PKC
activity mediated by this interaction is
shown to differ according to activator type and n-alkanol
chain length. Although short chain n-alkanols have an
inhibitory effect, long chain n-alkanol and general
anesthetic interaction results in an enhanced level of high affinity
phorbol ester binding and enzyme activity. These differing effects on
phorbol ester binding to membrane-associated PKC
and on the induced
level of activity are summarized in Table I.
|
The observation that n-alkanols compete for low affinity
SAPD binding would be consistent with the binding site for these agents
being situated within the cys1 subdomain, if the low affinity phorbol
ester binding site is contained within this subdomain, as previously
proposed (31). Persuasive evidence that this may be the case has
recently been presented by Blumberg and co-workers (29) based on the
finding of differing dose-response curves for the phorbol ester-induced
translocation in NIH 3T3 cells of PKC mutants modified in either the
cys1 or cys2 subdomain. Evidence of nonequivalent binding to the two
cys subdomains was also presented in another study, which showed that
deleting either the first or second zinc finger of PKC
expressed in
yeast resulted in differing dose-dependent effects of
phorbol esters on yeast growth (30). It would therefore appear to be
unlikely that low affinity phorbol ester binding is a nonspecific
event. Furthermore, the possibility that the observation of low
affinity phorbol ester binding resulted from a perturbation of the
membrane by the activators or the n-alkanols and anesthetics
was ruled out by the finding that low affinity SAPD binding was
saturable. Also, the observation of our previous study that TPA and the
isomer 4
-TPA inhibits SAPD binding to this site is contrary to the
enhancement expected had the low affinity binding event been
nonspecific in nature. Finally, the finding that the quenching of
C6-NBD-PC fluorescence intensity by SAPD is a linear
function of phorbol ester concentration over a range corresponding to
low affinity binding argues against perturbation of the membrane in
such a manner as to increase SAPD binding to a nonspecific membrane
site. It is worth pointing out that although the phorbol ester
concentration required for interaction with the low affinity binding
site is relatively high compared with that used in most experimental
paradigms, the same cannot be said of the DAG and n-alkanol
levels, which are at physiologically and pharmacologically relevant
concentrations, respectively, and would be expected to occupy the low
affinity site and modulate activity accordingly.
The activator- and chain length-dependent effects of the
n-alkanols on membrane-associated PKC activity contrast
starkly with the inhibitory effect of the entire range of
n-alkanols on lipid-independent PKC
activity. The latter
inhibitory effect confirms the original observation made for a mixed
rat brain PKC isoform mixture (17). In the case of the
membrane-associated enzyme, the n-alkanols appear to have
two competing effects: an inhibitory effect and a distinct
"amplifying" effect on activity that is only revealed for longer
chain length n-alkanols. The observation that
n-butyl alcohol and n-octanol inhibited SAPD binding and the level of TPA-induced activity over a concentration range corresponding to low affinity SAPD binding provides direct evidence that these agents compete for the low affinity phorbol ester
interaction. That both long and short chain n-alkanols
interact with this low affinity phorbol ester binding region is further indicated by the observation that the amplifying effect of
n-octanol on TPA-induced activity could be reduced by the
addition of an equipotent level of n-butyl alcohol (Fig.
5).
In addition to competing for low affinity phorbol ester binding, the
long chain n-alkanol n-octanol, but not the short
chain n-alkanol n-butyl alcohol, increased the
magnitude of high affinity phorbol ester binding, which is consistent
with the observed enhancement of phorbol ester-induced PKC activity.
This closely resembles the effect of DAG, which also competes for low
affinity and enhances high affinity phorbol ester binding (Fig.
4B; see Ref. 31). Therefore, the amplification of phorbol
ester-induced activity by DAG, n-alkanols, and anesthetics
appears to proceed by a similar mechanism, involving competition for
low affinity phorbol ester binding along with both an increase in high
affinity phorbol ester binding and enzyme activity. The observation
that n-alkanols do not compete effectively with phorbol
esters for the high affinity phorbol ester binding site does not
preclude the possibility that these agents might be capable of
competing with DAG for this region. Competition for DAG binding to both
activator sites might provide an explanation for the divergent effects
of short and long chain alcohols on DAG-activated PKC activity,
producing the inhibitory and biphasic effects observed. Thus, long
chain alcohols could compete for DAG binding to the low affinity
phorbol ester binding site and enhance DAG binding to the high affinity
site. This would lead to enhanced activity, as found for TPA. However,
at higher concentrations, the long chain n-alkanols may
compete for DAG binding to the high affinity phorbol ester site, which
would lead to a reduction in the (potentiated) level of activity that
results from n-alkanol interaction with the low affinity
phorbol ester binding site. Both these effects competing together could
produce the observed biphasic effect on DAG-induced activity. By
contrast, displacement of DAG from either site by short chain alcohols
would result in inhibition, as observed. To test for competition
between alcohols and DAG for binding to the high and low affinity
phorbol ester binding sites, fluorophore-labeled DAG could be used,
which, although currently not available, might resolve this point.
The finding of a linear relationship between the potency of the amplification of phorbol ester-induced activity by long chain n-alkanols (n-heptanol to n-decanol) and chain length (Fig. 3) suggests that the low affinity phorbol ester interaction has a hydrophobic component. However, the observation that high affinity phorbol ester binding and the resultant level of activation was unaffected by the short chain n-alkanol n-butyl alcohol, even though this agent inhibited low affinity phorbol ester binding, suggests that for increased high affinity binding there is a minimum hydrophobic requirement that is only met by the longer chain n-alkanols.
Although the existence of a discrete binding site for
n-alkanols and anesthetics within PKC provides strong
support for the protein theory of anesthesia, the significance of the
magnitude of the effects of the n-alkanols on PKC activity
mediated by this site, obtained using a model system, cannot easily be
extrapolated to an impact of these agents on a PKC-regulated cellular
function. However, the 20-40% change in PKC activity observed in the
present study may lead to a much amplified effect on a function
(e.g. ion channel opening) that is downstream in a signaling
cascade controlled by the enzyme. Furthermore, these effects of long
chain n-alkanols and anesthetics occur well within the
pharmacologically relevant range (42, 43), supporting a role of PKC in
anesthesia. This is further supported by recent studies that showed
that inhibitors of the enzyme modulate the righting reflex of tadpoles,
a commonly used model for anesthesia (20, 21).
It is clear that the inhibitory effect of n-alkanols and
anesthetics on lipid-independent PKC activity obeys a Meyer-Overton type linear relationship between anesthetic hydrophobicity and potency,
whereas this was not found for membrane-associated DAG- or TPA-induced
enzyme activity. However, this does not preclude a role for
membrane-associated PKC in the anesthetic process if one accepts that
there may be more than one cellular anesthetic target. The results
presented here also suggest that the effects of n-alkanols
and anesthetics on PKC activity will differ according to the
subcellular distribution of the enzyme. For example, PKC associated
with cytoskeletal or other nonmembrane protein elements, as modeled by
protamine sulfate-activated PKC, will be inhibited, whereas
membrane-associated activity induced by DAG or phorbol ester may be
potentiated. The relevance of this should be seen in the context of the
importance of cytofilament-associated PKC in neuronal function, which
has only recently been appreciated. For example, recent studies have
shown that PKC
II and PKC
both associate with, and are
activated by, cytoskeletal F-actin (44, 45). In the case of PKC
this
interaction occurs in the nerve endings and mediates effects on
glutamate exocytosis (44).
The results of this study provide evidence of a discrete hydrophobic site on PKC that may have broader consequences for its regulation. Thus there is the intriguing prospect of more highly specific ligands, either naturally occurring or therapeutically intended synthetic compounds, that may provide control over PKC-mediated signal transduction pathways, an aspect we are currently pursuing. At present, the exact location of the low affinity phorbol ester binding domain on PKC is unclear, but alcohols and general anesthetics would appear to interact competitively with DAG and phorbol ester at this region.
We are extremely grateful to Dr. R. M. Bell
for providing the PKC baculovirus preparation.