(Received for publication, July 27, 1994; and in revised form, November 29, 1994)
From the
A number of thiol-reactive agents induce repetitive
Ca spiking in cells by a mechanism thought to involve
sensitization of the inositol 1,4,5-trisphosphate receptor
(IP
R). To further define the basis of this interaction, we
have studied the effect of several thiol-reactive agents on
[
H]IP
binding, IP
-gated
channel activity, and conformation of the IP
R in membranes
from hepatocytes, cultured WB rat liver epithelial cells, and
cerebellum microsomes. At 4 °C, the organomercurial thiol-reactive
agent mersalyl markedly stimulates (3-4-fold)
[
H]IP
binding to permeabilized
hepatocytes. The closely related molecule, thimerosal, has only a small
stimulatory effect under these conditions, and GSSG or N-ethylmaleimide are without effect. The stimulatory effect of
mersalyl was associated with a decrease in K
of the IP
R with no change in B
. Mersalyl was without effect on
detergent-solubilized hepatocyte binding sites or on the
[
H]IP
binding activity of cerebellum
microsomes. In contrast to thimerosal, which potentiates
IP
-mediated Ca
release, mersalyl blocked
IP
-gated Ca
channels. Mersalyl
pretreatment of WB membranes altered the pattern of immunoreactive
receptor fragments generated upon subsequent cleavage of the receptor
with proteinase K. This effect was not reproduced by thimerosal and was
also not observed in experiments on cerebellum microsomes. We conclude
that the WB cell and brain IP
receptors are differently
regulated by modification of thiol groups. Reaction of the WB cell
IP
receptor with mersalyl alters its conformation and
modifies the accessibility of sites on the protein that are cleaved by
proteinase K. In the presence of mersalyl, the receptor has high
affinity for IP
but is inactive as a Ca
channel. This contrasts with the high affinity receptor/active
Ca
channel induced by thimerosal, suggesting that
even closely related thiol agents may interact at different thiol
groups.
Intracellular Ca mobilization occurring in
response to agonist stimulation of cells is mediated by the interaction
of inositol 1,4,5-trisphosphate (IP
) (
)with a
specific receptor/Ca
channel(1) . At least
three different receptor isoforms have been identified by molecular
cloning(2, 3, 4, 5) . A domain model
of the receptor has been proposed in which binding of IP
to
the N-terminal region of the receptor initiates a conformational change
in the protein that gates a Ca
channel comprising
six-transmembrane domains located in the C-terminal
region(6, 7) . It has been directly demonstrated that
the purified IP
R is a functional Ca
channel(8, 9) .
Ca transients in single cells stimulated with suboptimal
concentrations of agonists occur as repetitive
spikes(1, 10) . A number of models have been proposed
to explain the complex behavior of Ca
signals
recorded from individual cells. Experimental evidence suggests that the
feed-back regulation of the IP
R plays a central role in the
initiation and propagation of Ca
spikes. Small
elevations of Ca
above resting levels have been shown
to enhance IP
-mediated Ca
release(11, 12, 13) . and
Ca
sensitization of the IP
R to endogenous
levels of IP
has been proposed as one mechanism of
Ca
wave propagation (14, 15) . The
thiol-reactive agents t-butylhydroperoxide and thimerosal have
also been shown to promote repetitive Ca
spiking in
several cell types(15, 16, 17, 18) .
In both instances, it is believed that the effects of these agents are
related to a sensitization of the IP
R to endogenous levels
of IP
. In the case of t-butylhydroperoxide,
enhanced levels of oxidized glutathione (GSSG) are thought to underlie
the sensitization. GSSG has been shown to decrease the half-maximal
concentration of IP
required for Ca
release from permeabilized hepatocytes(19) . Low
concentrations of thimerosal have also been shown to potentiate
IP
-mediated Ca
release in several
experimental
systems(20, 21, 22, 23, 24) .
The exact mode of action of thiol reagents on the IP
R has
not been delineated, and it is not known if all of these agents have a
common site of action. We have attempted to address this question by
comparing the effects of several different thiol-reactive agents on
[
H]IP
binding and
IP
-gated channel activity. In the present study we report
that mersalyl and thimerosal, two structurally related organomercurial
thiol-reactive agents, have different effects on the function of the
isoforms of the IP
R in brain and liver. The data suggest
the presence of several distinct reactive thiols that are important in
regulating IP
R function.
Microsomal membranes were prepared from rat cerebellum homogenates by differential centrifugation as described previously (28) and stored at -80 °C in the same buffer as WB cell membranes.
The effect of several thiol-reactive agents on
[H]IP
binding to
saponin-permeabilized hepatocytes is shown in Fig. 1. The
incubation period with thiol reagent and
[
H]IP
was 5 min and was performed at
4 °C in a Mg
-free medium to minimize hydrolysis
of [
H]IP
. Under these conditions, a
marked stimulation (4-5-fold) of
[
H]IP
binding was observed in the
presence of mersalyl, an organomercurial thiol-reactive agent. Two
structurally related molecules, thimerosal and p-chloromercurophenylsulfonate, also stimulated
[
H]IP
binding, but the degree of
stimulation was much lower than observed with mersalyl. Increasing the
concentration or incubation time did not enhance the stimulatory effect
of thimerosal (data not shown). Oxidized glutathione or the
thiol-alkylating agent, N-ethylmaleimide, were without effect
on [
H]IP
binding at 4 °C.
Figure 1:
The effect of different sulfydryl
agents on IP binding to permeabilized hepatocytes at 4
°C. Isolated hepatocytes were permeabilized with saponin and
incubated with [
H]IP
(5 nM)
in the presence or absence of the given concentration of thiol-reactive
reagent for 5 min at 4 °C (see ``Experimental
Procedures'' for additional details). The specific IP
bound to the hepatocytes was measured with a membrane filtration
assay. The data shown are from three to six separate experiments with
each assay done in triplicate.
Ca has been shown to stimulate IP
binding to the hepatic IP
R(32) . It has been
proposed that Ca
mediates a conversion of receptors
from a low affinity (active) form to a high affinity (inactive)
form(33, 34) . Hilly et al.(20) have
shown previously that the stimulatory effect of Ca
and thimerosal on IP
binding to permeabilized
hepatocytes are not additive. Fig. 2A demonstrates that
the stimulation of [
H]IP
binding by
mersalyl is dose-dependent with maximal effects being observed at
100-200 µM mersalyl. Ca
(buffered
at 10 µM concentration) stimulated IP
binding
to a lesser extent than a maximal concentration of mersalyl and the
effect of both agents were not additive. Scatchard analysis of the
binding data (Fig. 2B), indicated that mersalyl
stimulated [
H]IP
binding by
increasing the affinity of the IP
receptor without altering
the maximal number of binding sites. In three experiments the
respective apparent K
and B
values were 27 ± 3 nM and 125 ± 6 fmol/mg
protein under control conditions, and 4.7 ± 0.3 nM and
98 ± 3 fmol/mg protein in the presence of 100 µM mersalyl. The effect of mersalyl is qualitatively similar to the
effect of Ca
on the binding affinity of the hepatic
IP
R(33) .
Figure 2:
The effect of mersalyl on Ca sensitivity and binding affinity for IP
. A,
[
H]IP
binding was measured as
described in Fig. 1in the presence of increasing concentrations
of mersalyl in the presence or absence of added Ca
.
The amount of total Ca
added to the buffer containing
1 mM HEDTA was adjusted to yield a free concentration of 10
µM as measured in parallel determinations with a
Ca
-sensitive mini-electrode. B, the amount
of [
H]IP
bound to permeabilized
hepatocytes was measured in the absence of Ca
and in
the presence and absence of 100 µM mersalyl with
increasing concentrations of unlabeled IP
present in the
incubation medium. The displacement curves obtained in the presence and
absence of mersalyl are shown as a Scatchard
plot.
We have shown previously that the
stimulatory effect of Ca on IP
binding in
permeabilized hepatocytes is lost after detergent solubilization of
membranes(26) . The experiment in Fig. 3was carried out
to determine if this was also the case with mersalyl. Permeabilized
hepatocytes were treated with mersalyl and then washed. The washed
mersalyl-treated hepatocytes retained an enhanced IP
binding activity (Fig. 3A). These hepatocytes
were then solubilized with Triton X-100, and binding activity was
measured in extracts exposed to increasing concentrations of mersalyl (Fig. 3B). The addition of mersalyl to Triton X-100
extracts prepared from control hepatocytes had no significant effect on
[
H]IP
binding. Binding activity in
Triton X-100 extracts of mersalyl pretreated membranes was only
slightly higher than control extracts and the further addition of
mersalyl produced a dose-dependent inhibition of binding. These results
indicate that, as with Ca
, the stimulatory effect of
mersalyl cannot be observed after detergent solubilization. The reason
why mersalyl pretreatment of membranes causes mersalyl to inhibit
[
H]IP
binding in detergent extracts
is presently not clear. A possibility is that mersalyl binding to the
receptor in membranes alters the conformation of the protein (see
below) in a manner that exposes an additional mersalyl-reactive thiol
group after detergent solubilization that is inhibitory to ligand
binding.
Figure 3:
The
effect of mersalyl on [H]IP
binding
to detergent solubilized binding sites. Panel A, hepatocytes
(5 mg of protein/ml) were permeabilized with saponin and an aliquot was
incubated with [
H]IP
in the absence
(
) or presence (
) of 100 µM mersalyl for 5
min. The amount of [
H]IP
bound was
determined (control cells). The remaining permeabilized cells were
divided into two equal portions, and one portion was incubated with 100
µM mersalyl for 5 min. Both aliquots were centrifuged (2
min, 80
g), washed once in hepatocyte resuspension
buffer-HEDTA, and resuspended in this buffer to 5 mg protein/ml. The
[
H]IP
binding activity of these cells
were measured (washed cells). Panel B, the control (
) and
mersalyl pretreated cells (
) were solubilized by addition of 1%
(w/v) Triton X-100. Insoluble material was removed by centrifugation
(25,000
g; 10 min), and
[
H]IP
binding to the solubilized
extracts was measured in the presence of increasing concentrations of
mersalyl as described
previously(26) .
We have examined the functional effect of mersalyl and
thimerosal on IP-mediated Ca
channel
function in Fig. 4and Fig. 5. In order to avoid the
known inhibitory effect of thiol-reactive agents on
Ca
-ATPase (16, 22, 35) , we
have utilized an assay method based on the ability of Mn
to traverse the IP
-activated Ca
channel in a retrograde manner and quench the fluorescence of
Fura-2 compartmentalized in intracellular stores(36) . All the
fluorescence measurements were carried out at 4 °C in order to
permit comparison to the ligand binding data. The addition of
Mn
to permeabilized Fura-2-loaded hepatocytes,
pretreated with the Ca
pump inhibitor thapsigargin,
produced a rapid quenching of cytosolic Fura-2 released from the
permeabilized cells. The subsequent addition of 1 µM IP
(a maximal dose) produced an additional quench
corresponding to the entry of Mn
into the
IP
-sensitive compartment (Fig. 4). Further entry of
Mn
into the IP
-insensitive compartment
could be observed after addition of ionomycin. When the permeabilized
hepatocytes were pretreated with mersalyl a complete inhibition of the
IP
-mediated quench was observed (Fig. 4). The total
pool size of the intracellular stores was not altered by mersalyl
pretreatment. Greater than 95% inhibition of IP
-mediated
Mn
quench was observed with IP
concentrations in the range 0.1-10 µM (data
not shown). The inhibitory effects of mersalyl were also noted at 37
°C, although the effects were less marked than at 4 °C. (
)Previous studies have shown that the potentiating effect
of thimerosal on IP
-induced Ca
release is
seen only at suboptimal IP
concentrations(22, 23) . In agreement with these
studies, thimerosal at 4 °C markedly potentiated the Mn
quench mediated by 10 nM IP
(Fig. 5, lower panel) with a much smaller effect on the responsiveness
to 500 nM IP
(Fig. 5, upper
panel).
Figure 4:
The effect of mersalyl on
IP-mediated Mn
quenching of
compartmentalized Fura-2 in intracellular stores. Hepatocytes were
loaded with Fura-2/AM, washed, and permeabilized at 4 °C as
described under ``Experimental Procedures.'' The
permeabilized cells were pretreated with mersalyl (100 µM)
for 5 min before addition of Mn
. All recordings were
made at 4 °C.
Figure 5:
The effect of thimerosal on
IP-mediated Mn
quenching of
compartmentalized Fura-2 in intracellular stores. Hepatocytes were
loaded with Fura-2/AM, washed, and permeabilized at 4 °C as
described under ``Experimental Procedures.'' The
permeabilized cells were pretreated with thimerosal (100
µM) for 5 min before addition of Mn
. All
recordings were made at 4 °C.
A possible mechanism of action of mersalyl is that
binding of this agent to a free thiol group on the IPR
alters the conformation of the protein, resulting in a form of the
receptor with an inactive Ca
channel and a high
affinity for ligand. To try to detect a conformational change in the
protein in its native membrane environment, we have looked for changes
in the pattern of immunoreactive fragments generated after addition of
proteases. Such experiments are facilitated by using membranes that
contain higher levels of immunoreactive IP
R than found in
hepatocytes. Fig. 6shows the effect of mersalyl on
[
H]IP
binding to membranes prepared
from WB rat liver epithelial cells and rat cerebellum. Both membranes
are known to contain relatively high levels of immunoreactive type-I
IP
R(31, 37) . Only the receptor in WB
membranes showed a stimulation of [
H]IP
binding by mersalyl, and these were used in subsequent studies.
Figure 6:
The effect of mersalyl on IP binding to WB-cell and rat cerebellum membranes A comparison of
the effect of mersalyl on [
H]IP
binding to WB and rat cerebellum microsomes is shown. Assay
conditions were as described for Fig. 1except that the final
assay volume was reduced to 250 µl and triplicate 75-µl samples
were removed for filtration. The results are the mean ± S.E. of
three observations.
Fig. 7A shows the pattern of fragments observed
after proteinase K digestion of WB cell membranes, as visualized with
an antibody raised to amino acids 401-414 in the N-terminal
region of the type-I IPR. In addition to several
intermediate digestion products, a prominent proteolytic product of
37.1 ± 2.2 kDa (n = 4) was formed with
progressive proteinase K digestion under control conditions. This band
was not observed after proteinase K treatment of WB cell membranes that
had first been treated with mersalyl and reisolated by centrifugation.
The protease fragmentation pattern of cerebellum IP
R was
substantially different from that observed with WB cell membranes,
although a 35.4 ± 0.4 kDa (n = 3) polypeptide
was also formed in cerebellar membranes (Fig. 7B). In
agreement with the results of binding data, pretreatment of cerebellum
membranes with mersalyl had no effect on the protease cleavage pattern.
Figure 7:
The effect of mersalyl pretreatment on
protease susceptibility of IP receptors in WB and
cerebellum membranes. A, WB cell membranes were prepared,
preincubated with mersalyl (200 µM), and subjected to
cleavage with proteinase K as described under ``Experimental
Procedures.'' The membranes treated with protease for the
indicated periods of time were reisolated by centrifugation and
denatured with SDS-PAGE sample buffer, and approximately 80 µg of
protein was electrophoresed on a 10% gel. Immunoblotting was carried
out with an affinity-purified IP
R antibody raised to amino
acids 401-414 of the rat type-I IP
R. The arrows indicate the position of the intact IP
R in WB
membranes and a major 37-kDa fragment that forms after proteinase K
digestion. B, the same experiment shown in panel A was repeated using rat cerebellum
microsomes.
Fig. 8A shows that thimerosal, at an equivalent
concentration, does not mimic the action of mersalyl. The effect of
mersalyl on the protease cleavage pattern of the WB IPR was
completely prevented by inclusion of an excess of dithiothreitol. The
37-kDa immunoreactive fragment in both WB cell and cerebellum was
associated with the membrane fraction. This reflected a peripheral
association with the membrane, since we were able to remove this
fragment by washing the membranes with 0.1 M Na
CO
, pH 11.0 (data not shown). The
supernatant fractions obtained after proteinase K digestion of control
and mersalyl-treated WB membranes were analyzed for the presence of the
37-kDa KEEK-reactive fragment (Fig. 8B). The absence of
the 37-kDa cleavage product in WB membranes after mersalyl pretreatment
was not the result of a selective loss of this fragment into the
soluble fraction. The combination of mersalyl and thimerosal together
produces a pattern of protease digestion which is the same as obtained
with mersalyl alone (Fig. 8C). This observation
supports the conclusion that the two sulfydryl reagents interact with
different thiol groups.
Figure 8:
The effect of thimerosal and
dithiothreitol on proteinase K generation of 37-kDa fragment. A, WB cell membranes (1 mg of protein/ml) were incubated in
Buffer A under the following conditions: lane 1, control; lane 2, + mersalyl (200 µM); lane
3, + thimerosal (200 µM); lane 4,
+ mersalyl + dithiothreitol (20 mM). After 10 min of
incubation at 4 °C, the membranes were pelleted by centrifugation
(100,000 g; 20 min), resuspended to 1 mg of
protein/ml, and treated with proteinase K (10 µg/mg protein) for 15
min at room temperature. The protease-treated membranes were again
pelleted by centrifugation, quenched in SDS-PAGE sample buffer, and
immunoblotted as described for Fig. 5A. B, WB
membranes were treated with and without mersalyl and then digested with
proteinase K as described for panel A. After centrifugation
the supernatants were precipitated with 10% trichloroacetic acid. The
protease-digested membrane fraction and the trichloroacetic
acid-precipitated supernatant fractions were analyzed on 10% SDS-PAGE
and Western-blotted with KEEK antibody. C, WB cell membranes
were treated as in A under the following conditions: lane
1, control; lane 2, + mersalyl (200
µM); lane 3, + thimerosal (200
µM); lane 4, + mersalyl + thimerosal.
Only the lower part of the immunoblots are shown. The arrow indicates the location of the 37-kDa immunoreactive
fragment.
Several recent studies have examined the effect of thimerosal
on IP binding and IP
-dependent Ca
release. Low concentrations of thimerosal were found to
potentiate IP
-mediated Ca
release from
adrenal cortex microsomes(21) , permeabilized
hepatocytes(20) , cerebellum microsomes (20, 22) , and permeabilized A7r5 smooth-muscle
cells(23) . In adrenal cortex and hepatocytes, the potentiation
by thimerosal was accompanied by a large stimulation of IP
binding reflecting an increased affinity of the IP
R.
On the basis of these data, it has been concluded that the high
affinity form of the receptor induced by thimerosal is functionally
active and is distinct from the high affinity form of the receptor
induced by Ca
, which is functionally less active as a
Ca
channel(33) . Our data show that the
related thiol-reagent, mersalyl, has a marked stimulatory effect on
IP
binding and behaves more like Ca
, in
that it generates a high affinity form of the receptor that is
functionally inactive. These studies reinforce the idea that the
IP
R can exist in several non-equivalent high affinity
states that may have high or low Ca
conductance (20, 38) . It should be noted that the stimulatory
effect of thimerosal on IP
binding has not been observed in
all studies (e.g.(22) and (23) ), and only a
modest stimulation of binding was observed in permeabilized hepatocytes
under our assay conditions at 4 °C. We attribute this difference to
reaction of the sulfydryl reagents with separate thiol groups on the
receptor rather than to differences of reactivity with the same thiol
group for several reasons. First, incubation of permeabilized
hepatocytes at 4 °C for longer periods with higher concentrations
of thimerosal did not enhance the effect on
[
H]IP
binding. Second, the effects of
mersalyl and thimerosal on channel function and protease digestion of
the receptor are clearly different. Third, the combination of mersalyl
and thimerosal together produces a pattern of protease digestion which
is the same as obtained with mersalyl alone (Fig. 8C).
Other factors, such as temperature, may modify the reactivity of
individual thiol groups. For example, we have found that the effect of
thimerosal on ligand binding could be enhanced, and the effect of
mersalyl diminished, by preincubating the permeabilized hepatocytes at
37 °C with the thiol agents prior to measurement of binding at 4
°C. ()The elevation of temperature does not, however,
qualitatively modify the effect of the sulfydryl agents on channel
function (Footnote 2 and data not shown). A temperature-dependent
alteration in the accessibility of N-ethylmaleimide-reactive
thiol groups on the hepatic IP
R has also been documented
previously(39) . Whereas low concentrations of thimerosal
stimulate IP
-mediated Ca
release, higher
concentrations (>10 µM) have been found to inhibit the
release process in some systems(22, 23) . In part,
this is due to an inhibition of the Ca
pump and to an
increase in the passive Ca
permeability of the
endoplasmic reticulum membrane(22, 23) . Using the
Mn
quench assay, a biphasic dependence on thimerosal
concentrations up to 100 µM was not observed in the
present study.
Mersalyl stimulates IP binding to liver
and WB cell membranes but is without effect on cerebellum microsomes.
The full spectrum of IP
R isoforms in each of these tissues
has not been established unequivocally. However, it is known that the
cerebellum contains predominantly type-I IP
R (3, 40) and that non-neuronal cells contain an
alternative transcript of the type-I isoform that has a 40-amino acid
deletion(41, 42) . These ``long'' and
``short'' forms of the type-I IP
R have been shown
to differ in their regulation by Ca
and
cAMP-dependent phosphorylation(41) . It has recently been
reported that the liver expresses low amounts of type-III
IP
R mRNA and much higher amounts of type-I and type-II
IP
R mRNA(43) . By contrast, the WB cell contains
predominantly types I and III IP
R and very little of any
other isoform, as judged by the 90% immunodepletion of
[
H]IP
binding sites from WB cell
extracts by a combination of type-I and type-III specific antibodies. (
)Thus the difference in reactivity toward mersalyl of the
hepatocyte/WB cell and cerebellum IP
R could be accounted
for by the selective insensitivity of the type-I (long form) to the
sulfydryl agent or alternatively, to tissue-specific differences in
ancillary sulfydryl reagent-sensitive regulatory proteins.
It is
somewhat surprising that two such closely related organomercurial
agents should have different effects on the hepatic IPR.
Both molecules are anionic and, in the case of mersalyl, known not to
penetrate mitochondrial membranes(44) . The inhibitory effects
of mersalyl are believed to be the result of the formation of a
mercaptide bond with free thiol groups in a protein(45) . With
thimerosal, the mercury is already linked to sulfur, and it is
therefore possible that reaction with protein thiols may generate mixed
disulfides. In studies on the rate of reaction of the free thiols of
ovalbumin with several mercaptide forming agents, it was observed that
mersalyl was more reactive than other agents such as p-chloromercurobenzoate (46) . These variations in
reactivity were attributed to differences in the degree of steric
hindrance around the reactive mercury in these molecules(46) .
Therefore, differences in the chemistry of reaction and/or
accessibility to reactive thiol groups may underlie the distinctive
effects of the two agents.
The current model of IP gating of the Ca
channel in the IP
R
proposes that ligand binding is associated with a large conformational
change in the protein(6) . In the present study, we have shown
that mersalyl pretreatment of WB cell membranes effectively eliminates
the appearance of a 37-kDa immunoreactive receptor fragment generated
by proteinase K cleavage. We interpret these data to indicate that
reaction of a thiol group on the receptor induced a conformational
change in the protein that exposed the region of the receptor
containing the antibody epitope (amino acids 401-414) to cleavage
by proteinase K. Two findings suggest that this conformational change
is linked to the effect of mersalyl on
[
H]IP
binding. First, mersalyl does
not affect [
H]IP
binding in
cerebellum microsomes and also does not alter the formation of any of
the proteolytic fragments in this system. Second, thimerosal does not
mimic the effect of mersalyl on [
H]IP
binding or the appearance of 37-kDa proteinase K fragment. The
antibody epitope falls within the region thought to be involved in
ligand binding(2) . However, IP
itself had no
effect on the pattern of proteinase K digestion fragments and did not
prevent the effect of mersalyl (data not shown).
[H]IP
binding to Triton X-100
solubilized extracts (Fig. 3B) or heparin-agarose
column eluates (data not shown) are not affected by mersalyl (data not
shown). Mersalyl may continue to react with thiol groups on the
receptor under these conditions and still have no effect on binding,
since removal of the protein from its membrane environment may grossly
alter regulation of the binding site(26) . However, the present
data do not exclude the possibility that mersalyl or other sulfydryl
agents interact with ancillary regulatory proteins rather than reacting
directly with the receptor. Despite this, it is clear that reaction of
specific thiol groups on the receptor, or a regulatory protein, has
profound consequences on the function of the IP
receptor/ion channel. These thiols have varied sensitivity to
sulfydryl reagents, and reactivity is also different between different
IP
R isoforms. Identification of these functionally
important cysteine residues and characterization of their role in
ligand binding/ion channel function of the receptor remains a
challenging objective.