(Received for publication, April 20, 1995; and in revised form, December 4, 1995)
From the
Incubation of cerebellar microsomes with D-myo-inositol 1,4,5-trisphosphate (InsP)
(0.01-1 µM), at 4 or 20 °C in a cytosolic-like
medium devoid of Ca
and Mg
,
followed by InsP
removal, induced an increase in InsP
binding determined with 1 nM [
H]InsP
. At 20 °C, and pH
7.1, maximal stimulation (1.5-2.5-fold) was obtained with 1
µM InsP
, and the EC
was 60
± 5 nM. Several lines of evidence suggested that the
activating site is identical with the InsP
binding site:
(i) activation and binding exhibited the same inositol phosphate
specificity; (ii) addition of decavanadate, a competitive inhibitor of
[
H]InsP
binding, to the preincubation
mixture, prevented the activating effect of InsP
; (iii) the
concentration of InsP
giving half-maximal activation was
close to that giving half-maximal InsP
binding. The time
course of activation was found to be much slower than that of binding.
While a t
less than 0.4 s has been measured
recently at neutral pH and 20 °C for binding of 0.5 nM [
H]InsP
(Hannaert-Merah, Z.,
Coquil, J.-F., Combettes, L., Claret, M., Mauger, J.-P., and Champeil,
P.(1994) J. Biol. Chem. 269, 29642-29649), a 20-s
preincubation with 1 µM InsP
was required to
half-maximally stimulate binding. Under the present conditions, the
InsP
-induced binding increase was only partially
reversible. However, this effect was not blocked by antiproteases
suggesting that it did not involve proteolysis. Taking advantage of the
marked difference in the kinetics of InsP
binding and
InsP
-dependent activation, we performed binding experiments
on a short period (3 s) to determine the effect of InsP
pretreatment on the binding parameters. The data showed that this
treatment increased the affinity of the receptor without changing the
number of binding sites (control: K
= 107 nM, B
=
28 pmol/mg of protein; after preincubation with 1 µM InsP
: K
= 53
nM, B
= 32 pmol/mg of protein).
The two states of the receptor bound InsP
with a Hill
coefficient close to 1 on a 3-s scale. In agreement with the effect of
InsP
pretreatment, equilibrium binding experiments
performed on 10-min incubations revealed an apparent positive
cooperative behavior (apparent Hill coefficient = 1.6; apparent K
= 66 nM). These results
report a new regulatory process of the InsP
receptor in
cerebellum occurring independently of Ca
and on a
relatively long time scale.
The second messenger D-myo-inositol
1,4,5-trisphosphate (InsP) (
)mobilizes
intracellular Ca
by activating a receptor/channel
located in the endoplasmic reticulum (Taylor and Richardson, 1991;
Ferris and Snyder, 1992; Berridge, 1993). This process may be crucial
in the genesis of repetitive calcium spikes which characterize the
cellular calcium response to many stimuli (Berridge, 1993). Several
genes encoding a family of InsP
receptors have been
identified, and the molecular diversity of the receptor is amplified
further by alternative splicing. In many tissues, diverse isoforms of
the receptor are produced in different amounts
(Südhof et al., 1991; Nakagawa et
al., 1991; Ross et al., 1992; Blondel et al.,
1993; De Smedt et al., 1994). In the cerebellum, most
InsP
receptor mRNAs encode type I receptor (Furuichi et
al., 1989; De Smedt et al., 1994). The high concentration
of type I receptor in Purkinje cells of cerebellum, has facilitated its
purification, and the receptor has been proposed to be an homotetramer
(Supattapone et al., 1988; Maeda et al., 1990, 1991).
Analysis of its cDNA sequence has suggested overall structural
organization in three basic domains: an amino-terminal InsP
binding domain, a carboxyl-terminal Ca
channel
domain, and a linking domain containing sites for regulatory processes.
This structure has been subsequently confirmed for other isoforms
(Furuichi et al., 1989; Mignery and
Südhof, 1990; Miyawaki et al., 1991;
Südhof et al., 1991; Maranto, 1994).
Studies of InsP binding and InsP
-induced
Ca
release (IICR) with permeabilized cells or diverse
receptor preparations including microsomes have allowed the
identification of a variety of regulatory mechanisms for the InsP
receptor. Ca
is centrally involved in the
control of IICR: submicromolar concentrations of Ca
activate, whereas higher concentrations desensitize the channel
(Iino, 1990; Finch et al., 1991; Bezprozvanny et al.,
1991; Combettes et al., 1994). In central nervous tissue,
Ca
inhibits InsP
binding through a
Ca
-binding protein, calmedin (Danoff et al.,
1988), whereas, in peripheral tissues, high Ca
concentrations transform the receptor into an inactive form which
displays a high affinity for InsP
(Pietri et al.,
1990; Rouxel et al., 1992; Watras et al., 1994). ATP
has been shown to stimulate channel activity through specific sites on
the receptor (Ferris et al., 1990; Maeda et al.,
1991; Bezprozvanny et al., 1991; Combettes et al.,
1994). Several protein kinases, including A, C, G, and
Ca
/calmodulin-dependent enzymes, phosphorylate the
receptor (Ferris and Snyder, 1992; Joseph and Ryan, 1993; Komalavilas
and Lincoln, 1994; Koga et al., 1994). Oxidizing sulfhydryl
reagents such as thimerosal have been shown to increase the sensitivity
of the Ca
store by increasing the affinity of the
InsP
receptor (Missiaen et al., 1991; Renard et al., 1992; Hilly et al., 1993). In addition,
recent observations suggest that InsP
exerts effects on its
receptor different from the mere rapid opening of the Ca
channel. It has been shown that IICR activation in permeabilized
hepatocytes is followed by a period of inactivity dependent on the
duration of exposure to InsP
(Hajnóczky and Thomas, 1994). Furthermore,
it was also observed that preincubation with InsP
potentiates the stimulation by thimerosal of the Ca
channel activity of the purified cerebellum receptor
reconstituted in phospholipid vesicles (Kaplin et al., 1994).
The present studies report that occupancy of InsP
binding
sites in sheep cerebellum microsomes induces a time-dependent increase
in the affinity of InsP
receptor for its ligand. In
contrast with results by Hajnóczky and
Thomas(1994) on permeabilized hepatocytes, this InsP
effect
was independent of Ca
, suggesting that it represents
a previously undescribed process.
Sheep cerebellar microsomes were prepared as described
previously (Hannaert-Merah et al., 1994). The microsomal
preparation was resuspended in homogenization buffer (5 mM Hepes, 250 mM sucrose, 10 mM KCl, 1 mM -mercaptoethanol, 10 µg/ml leupeptin, 10 µM pepstatin A, 0.2 mM phenylmethylsulfonyl fluoride, pH 7.4
at 4 °C) and then frozen in liquid N
and stored at
-80 °C. Membranes were thawed and diluted in an ice-cold
cytosolic-like medium (MI) containing 110 mM KCl, 20 mM NaCl, 1 mM NaH
PO
, 1 mM EDTA, 25 mM Hepes/KOH (pH 7.1), and 10 µg/ml
leupeptin. Where indicated, membranes were washed and resuspended in
the same medium. Preincubation of membranes (0.2-1.0 mg/ml
protein) with InsP
(1 nM to 1 µM) was
also conducted in MI, supplemented with other agents as indicated. In
the first series of experiments, InsP
was removed by
centrifuging the preincubation mixture at 36,000
g for
1 h and washing membranes twice with ice-cold MI. A more rapid washing
procedure was used in subsequent experiments. The membrane suspension
preincubated with or without InsP
was layered onto a
Whatman GF/C glass fiber filter and then washed with 10 ml of MI. The
vacuum pump was adjusted to give an outflow rate of 1 ml/s, except
where indicated. The binding activity of membranes was then measured
directly on the filter, as described below.
[H]InsP
binding was measured by
mixing microsomes with an appropriate volume of a binding medium
consisting of MI supplemented with 1 nM [
H]InsP
, 0.1 mg/ml bovine serum
albumin, and the indicated concentrations of unlabeled
InsP
. Nonspecific binding was determined in the presence of
10 µM InsP
. Binding was performed either in a
test tube or on a GF/C glass fiber filter. In the former case, the
binding mixture (final volume: 0.5 ml) was incubated for 10 min at 4 or
20 °C. Four hundred microliters of the mixture were transferred
onto a GF/C glass fiber filter which was then rinsed with 1 ml of an
ice-cold washing medium (25 mM Hepes, 250 mM sucrose,
1 mM NaH
PO
, 1 mM EDTA, pH
7.1). To measure [
H]InsP
binding to
membranes absorbed onto the GF/C filter, 0.5 ml of the binding medium
was added to the filter, the vacuum pump being either off or running
according to the desired contact time with
[
H]InsP
. Except where indicated, the
filter was shortly rinsed with 1 ml of the ice-cold washing medium. As
previously reported (Rouxel et al., 1992; Hannaert-Merah et al., 1994), InsP
dissociates very quickly from
its receptor in a cytosolic-like medium at neutral pH and 20 °C (t
< 0.4 s). Therefore, the rinsing step was
not performed with MI but with a Hepes buffer containing 250 mM sucrose and cooled to 4 °C. In addition, the perfusion rate of
outflow was adjusted so that the washing lasted less than 0.5 s. This
procedure removed almost all [
H]InsP
trapped in the GF/C glass fiber filter. An adsorption of
[
H]InsP
to this type of filter is
possible, but was prevented by the addition of 1 mM EDTA to
the binding medium. Excess fluid was removed from the filter under
vacuum before transfer into a counting vial. Radioactivity was measured
in a scintillation counter. Total binding and nonspecific binding were
determined at least in triplicate, and the results were expressed as
means ± S.E. The competitive binding curves and the
dose-response curve for InsP
-dependent binding increase
were fitted according to a one-site model, using Inplot Software
(GraphPAD). Kinetics of InsP
-dependent binding increase
were fitted to a simple exponential behavior.
[H]InsP
(17-21 Ci/mmol) was
purchased from DuPont NEN. Nonradioactive InsP
was obtained
from Calbiochem. Decavanadate was prepared as described previously
(Föhr et al., 1989). All other products
were from Sigma or Boehringer Mannheim.
A crucial point in this type of experiment was to
remove carefully the nonradioactive InsP present in the
preincubation mixture, as [
H]InsP
binding might be reduced by residual InsP
leading to
an underestimation of the InsP
-dependent activation. An
experiment was therefore performed in which the volume of the washing
medium was varied. When membranes were preincubated with 1 µM InsP
, the lowest subsequent binding of
[
H]InsP
was observed in the absence
of washing; that is, the condition for which the highest contamination
of the filter with unlabeled InsP
was expected. Washing the
filter with MI increased the binding of
[
H]InsP
to the membranes, the maximal
binding being attained at about 3-5 ml of washing medium.
Increasing the volume of the washing medium to 30 ml did not change
[
H]InsP
binding. When membranes were
preincubated without InsP
, washing the filter with up to 30
ml of MI had no effect on [
H]InsP
binding. These results indicate that unlabeled InsP
carried over from the preincubation mixture could be removed from
the filter easily and the InsP
remaining after 5 ml of
washing does not reduce the subsequent
[
H]InsP
binding when measured with 1
nM [
H]InsP
. An additional
experiment to determine the amount of residual InsP
after a
washing with 10 ml of MI (the standard protocol) was performed. In this
experiment, the preincubation mixture containing both cerebellar
membranes and 1 µM unlabeled InsP
was
supplemented with 10 nM [
H]InsP
. The results indicate
that a two-thousandth of the initial
[
H]InsP
remained on the filter,
corresponding to 1 pmol of InsP
(final concentration of 2
nM after addition of binding mixture on the filter). From the
[
H]InsP
displacement curve by
nonradioactive InsP
, we conclude that this residual
InsP
was about 10-fold lower than the InsP
required to significantly reduce
[
H]InsP
binding measured with 1
nM [
H]InsP
. Thus, 10 ml of
MI were adequate to wash membranes.
Secondly, as heparin
is a potent inhibitor of InsP binding to its receptor
(Taylor and Richardson, 1991), we examined whether this agent was able
to block the activating effect of InsP
. Unfortunately,
pre-exposure of the cerebellar membranes to heparin led to an
irreversible inhibition of InsP
binding activity. A similar
effect was reported previously for microsomes from bovine adrenal
cortex at 4 °C in InsP
binding assays (Guillemette et al., 1989). However, in contrast with these studies, we
were unable to recover more than 30% of
[
H]InsP
binding by diluting and
washing cerebellar microsomes at 37 °C. Therefore, we repeated the
same type of experiments with decavanadate, another agent described as
a competitive antagonist of InsP
receptor
(Föhr et al., 1989; Taylor and Richardson,
1991). As illustrated in Fig. 1, A and B, 3
µM decavanadate inhibited InsP
binding
measured with 0.1 µM [
H]InsP
by 91% (A) and greatly reduced the activation by 0.1
µM InsP
(B). Cerebellar membranes
preincubated with 3 µM decavanadate alone did not exhibit
a modified binding activity. Thus, decavanadate was entirely removed by
washing membranes with MI and was unable to mimic the activating effect
of InsP
.
Figure 1:
Blockade by decavanadate of the
increase in binding induced by InsP. Experiments were
performed at 4 °C. Microsomes were prepared as described under
``Experimental Procedures'' and washed with ice-cold MI as
indicated in Table 1. A,
[
H]InsP
binding was performed in a
test tube by incubating microsomes for 10 min in binding medium
containing 20 nCi/ml [
H]InsP
, 0.1
µM InsP
, and decavanadate (DV) as
specified. Bound [
H]InsP
was measured
by filtering 0.4 ml of the incubation mixture. Results are expressed as
percent of [
H]InsP
binding measured
in the absence of decavanadate. B, microsomes were
preincubated for 10 min in MI with or without 0.1 µM InsP
and 3 µM decavanadate as indicated.
Five hundred-microliter aliquots of the preincubation mixtures were
transferred onto GF/C glass fiber filters, membranes were washed, and
InsP
binding was measured on a filter with 1 nM [
H]InsP
. Data are means of one
experiment representative of two.
Figure 2:
Effect of increasing InsP
concentrations in the preincubation mixture on subsequent
[
H]InsP
binding. Experiments were
performed at 20 °C. Microsomes were preincubated for 10 min in MI
in the absence or presence of InsP
at the specified
concentration. Five hundred-microliter aliquots of the preincubation
mixture were used to measure the InsP
binding on GF/C glass
fiber filters with 1 nM [
H]InsP
, as described under
``Experimental Procedures'' and in Table 1. Results are
expressed as percent of [
H]InsP
binding of microsomes preincubated without InsP
. Data
points are means of three different
experiments.
Figure 3:
Time course of activation of
[H]InsP
binding during preincubation
of microsomes with InsP
. The experiment was carried out at
20 °C. For 2-s and 5-s pretreatments, 1-ml aliquots of the diluted
microsome preparation were layered on GF/C glass fiber filters, and
adsorbed microsomes were perfused manually at 0.5 ml/s with 1 ml or 2.5
ml of MI containing 1 µM InsP
. Pretreatments
of 10 s to 2 min were performed by applying 1 ml of the InsP
solution onto the filters with the vacuum pump being off.
Five-minute pretreatments were conducted in test tubes. After the 5-min
incubation, 1 ml of preincubation mixture was transferred onto GF/C
filters. In all cases, microsome pretreatment was stopped by passing 10
ml of MI through the filter. [
H]InsP
binding to microsomes was measured on the filters as described
under ``Experimental Procedures'' and in Table 1.
Controls were performed according to the same protocols except that
InsP
was omitted from the preincubation medium. Results are
expressed as percent increase of [
H]InsP
binding as compared to the corresponding control. Data points are
means of one experiment representative of
two.
Figure 4:
Effect
of pre-exposure of microsomes to InsP on
[
H]InsP
binding parameters. A, the experiment was carried out at 20 °C. Microsomes
were preincubated for 10 min in MI with (
) or without (
) 1
µM InsP
. One-ml aliquots of the preincubation
mixtures were layered onto GF/C glass fiber filters. Adsorbed
microsomes were washed with 10 ml of MI and then perfused for 3 s with
0.5 ml of binding mixture containing 1 nM [
H]InsP
(20 nCi/ml) and the
indicated unlabeled InsP
concentrations. The filters were
not rinsed with ice-cold Hepes/sucrose buffer to remove free ligand;
however, the data were corrected for nonspecific binding determined
with 10 µM InsP
. Results are expressed as
percent of [
H]InsP
binding determined
for control membranes without unlabeled InsP
. Simulated
curves were constructed for a one-site model and n
= 1. B, Scatchard plots obtained from the same
data as in A. Data points are means of one experiment
representative of two.
From the data reported above, we anticipated that measurement of
[H]InsP
binding in the presence of
increasing InsP
concentrations on a time long enough to
allow activation by InsP
should express positive
cooperativity. As illustrated by the squares in Fig. 5,
binding values (B) measured at InsP
concentrations, shown previously to trigger the activation
process during preincubation, were higher than that determined with 1
nM [
H]InsP
alone (B
). These results proved such positive
cooperativity directly. When transformed into a direct coordinate
system and fitted to the Hill equation, half-maximal binding was
calculated to be 66 nM InsP
and the apparent Hill
coefficient to be 1.6 (data not shown). In 5 different experiments
performed at 4 or 20 °C, we measured a maximal B/B
value of 1.31 ± 0.06.
Figure 5:
[H]InsP
displacement curve by unlabeled InsP
obtained from 10-min
incubations. Comparison with displacement curves obtained from 3-s
incubations of microsomes pretreated with or without 1 µM InsP
. Experiments were performed at 20 °C. For
10-min incubations (
), cerebellar microsomes were added to a
binding medium (final volume, 0.5 ml), consisting of MI supplemented
with 1 nM [
H]InsP
and
increasing concentrations of unlabeled InsP
. Four hundred
microliters of the incubation mixture was then transferred on GF/C
glass fiber filters to measure the amount of bound
[
H]InsP
. Results are expressed as
percent of [
H]InsP
binding determined
without the addition of unlabeled ligand. Displacement curves obtained
from 3-s perfusions of microsomes pretreated for 10 min with (solid
line) or without (dashed line) InsP
were
determined from data presented in Fig. 4. Data points are means
of one representative experiment.
The present studies show that in sheep cerebellar microsomes
the affinity for InsP of its receptor was markedly
increased during exposure to InsP
over a period of 2 s to 2
min. As the total number of InsP
binding sites was not
changed by this treatment, it appears that these sites were converted
to a state of higher affinity. Several lines of evidence indicate that
the activating effect of InsP
resulted from an interaction
of InsP
with the same receptor. Firstly, the selectivity of
the activating site was the same as that of the
[
H]InsP
binding site and that
previously reported for the InsP
receptor in cerebellum
from other species and peripheral tissues (Nahorski and Potter, 1989;
Mourey et al., 1990; Südhof et
al., 1991; Maeda et al., 1991; Rouxel et al.,
1992). Secondly, decavanadate, a competitive inhibitor of InsP
receptor (Föhr et al., 1989; Taylor
and Richardson, 1991), prevented the activation by InsP
.
Thirdly, the EC
value for activation at 20 °C (60
nM) was close to the apparent K
value
determined for [
H]InsP
binding in a
10-min incubation (66 nM). Whereas the sites for activation
and binding appear to be identical, the former effect developed much
more slowly than the latter. We conclude from these observations that
the activation process results from prolonged occupancy of the
InsP
binding site.
Most of the experiments in the
present work were performed with a 10-min preincubation of cerebellar
microsomal membranes with nonradioactive InsP, followed by
washing and determination of [
H]InsP
binding after a short incubation period. The resulting increase
of affinity observed in these two-step experiments was confirmed in
binding measurements performed after longer (10 min) incubation
periods, which revealed an apparent positive cooperative behavior (Fig. 5) and hence also indicate that InsP
stimulates its own binding. Consistent with the slow activation
by preincubation with InsP
(Fig. 3), this behavior
was not observed in experiments in which this incubation was only
2-3 s at 20 °C, as indicated by the Hill coefficient value
close to 1 determined with untreated membranes (Fig. 4, A and B). Considered together, these characteristics of
InsP
binding to its cerebellar receptor are reminiscent of
properties of a hysteretic protein for which the apparent cooperative
behavior results from slow conformational transition upon binding of
its ligand. Positive cooperative behavior has not been reported
previously for InsP
binding to cerebellum receptor.
However, it has been proposed that upon interaction with its binding
site, InsP
elicits a large conformational change in its
receptor (Mignery and Südhof, 1990). Recently, this
conformational change has been suggested to alter accessibility of
thimerosal to certain sulfhydryl groups (Kaplin et al., 1994).
In peripheral tissues, elevation of cytosolic Ca above its resting concentration (100-200 nM)
increases the affinity of the InsP
receptor for its ligand
(Hilly et al., 1993; Marshall and Taylor, 1993) by reducing
the dissociation rate constant (Hilly et al., 1993). When the
free Ca
concentration reaches 0.5-1
µM, these receptors are converted into a high affinity
inactive state characterized by low rates of association and
dissociation of InsP
(Pietri et al., 1990; Hilly et al., 1993; Watras et al., 1994). In contrast, the
InsP
-dependent conversion of sheep cerebellum InsP
receptor to a higher affinity state was observed in the presence
of 1 mM EDTA, that is, at nanomolar free Ca
concentrations. In this tissue, Ca
inhibits
InsP
binding (Worley et al., 1987; Hannaert-Merah et al., 1994), an effect which has been proposed to be
mediated by the Ca
-binding protein, calmedin (Danoff et al., 1988). However, the InsP
activating effect
cannot be due to removal of inhibitory influence by calmedin, since 1
mM EDTA has been reported to prevent and reverse the
inhibition by this protein (Worley et al., 1987; Joseph et
al., 1989). Furthermore, we found that sheep cerebellar microsomes
preincubated with free Ca
concentrations up to 100
µM and then washed with 10 ml of MI containing 1 mM EDTA did not exhibit a lower [
H]InsP
binding as compared with membranes exposed to nanomolar
Ca
concentrations (data not shown). Therefore, we
conclude that the activation by InsP
occurs independently
of Ca
. However, this does not preclude any regulatory
influence of Ca
on this process.
The ability of
InsP to increase its own binding in microsomes washed with
MI, indicates that molecules involved in this process are tightly
associated with membranes. Several membrane-associated proteins have
been proposed to interact with the cerebellum InsP
receptor, including ankyrin (Joseph and Samanta, 1993;
Bourguignon et al., 1993) and calmedin which, however, is not
involved as discussed above. Another possibility is that the transition
induced by InsP
involves changes in interaction between the
InsP
receptor subunits. Evidence for an association between
subunits of InsP
receptors of adjacent cisternae of smooth
endoplasmic reticulum has been obtained in immunocytological studies of
Purkinje cells (Satoh et al., 1990; Otsu et al.,
1990; Villa et al., 1991; Takei et al., 1992, 1994).
Alternatively, it may be possible that the activating effect of
InsP
might involve a covalent modification of the
InsP
receptor, e.g. a change in the
phosphorylation state. Cerebellum InsP
receptor has been
shown to be phosphorylated by several protein kinases (Ferris and
Snyder, 1992; Koga et al., 1994).
A major characteristic of
the affinity increase of the cerebellum InsP receptor
following InsP
pretreatment is the slowness of its kinetics (t
= 20 s; Fig. 3) as compared
with that of the InsP
binding (see above) and IICR (t
= 0.2 s with 0.15 µM InsP
; Combettes et al.(1994)). This
difference implies that the increase in InsP
receptor
affinity, resulting from InsP
binding, occurs after
Ca
efflux has been completed and thus affects
subsequent events only. Recently, it has been reported that
pre-exposure of permeabilized hepatocytes to InsP
is
followed by a time-dependent inactivation of IICR
(Hajnóczky and Thomas, 1994). The time course for
[
H]InsP
binding activation in the
present studies (t
= 20 s, Fig. 3)
is very similar to that of the InsP
-induced inactivation
described by Hajnóczky and Thomas (t
= 15 s). However, in contrast to our
results on cerebellar microsomes, inactivation by InsP
in
permeabilized hepatocytes was dependent on the presence of
Ca
and was accelerated by increasing the
Ca
concentration up to 1 µM. With
respect to the effect of InsP
pretreatment of cerebellar
microsomes on the function of the InsP
receptor (IICR),
further studies will be required. The slow kinetics of the increase in
InsP
affinity suggests that it will be dependent on a
prolonged increase in the level of InsP
in intact cells,
and that, therefore, it reflects a long-term regulation process. The
same effect might also be attained with repetitive increases in the
cellular level of InsP
if the InsP
binding
activation is slowly reversible in intact cells, as suggested by the
present in vitro conditions. Interestingly, such a situation
might be encountered in long-term potentiation and long-term
depression, two important models of synaptic plasticity, induced by
tetanic and/or repetitive or prolonged stimulation of synapses (Madison et al., 1991; Bliss and Collingridge, 1993; Artola and Singer,
1993). In cerebellum, long term depression is well known to occur at
the parallel fiber-Purkinje cell synapses (Ito, 1989; Daniel et
al., 1992; Conquet et al., 1994). Evidence has been
obtained for involvement of the InsP
/Ca
signaling system in these processes (Kato, 1993; Kasai and
Petersen, 1994). Many different cell types respond by repetitive
Ca
spikes to sustained application of agonists acting
through InsP
. Characteristically, the agonist-stimulated
accumulation of InsP
consists in a rapid peak followed by a
much lower but sustained phase (Willars and Nahorski, 1995). We
hypothesize that such a pattern of InsP
accumulation may
lead to an increase in the affinity of the InsP
receptor
for InsP
and therefore facilitate the generation of
Ca
oscillations while InsP
level is
increased only slightly. Repetitive Ca
spikes have
been observed at basal InsP
concentration following
intracellular injection of thimerosal, a thiol alkylating agent which
sensitizes the Ca
stores to InsP
(Bootman et al., 1992), by increasing the affinity of the InsP
receptor (Hilly et al., 1993). Consistent with our
hypothesis, studies in intact cells have shown that
InsP
-dependent Ca
mobilization may also
be sensitized by a prior InsP
injection or agonist
activation (Parker and Miledi, 1989).
In summary, the present
studies show that in sheep cerebellar microsomes a prolonged exposure
of InsP to its receptor, converts this protein to a state
exhibiting higher affinity. This phenomenon indicates that, upon
binding, InsP
not only opens the Ca
channel (a rapid process) but also initiates a slower regulation
of the protein.