(Received for publication, July 13, 1995; and in revised form, September 5, 1995)
From the
The effects of the alkali metal ions Na,
K
, Rb
, and Cs
on
ATP-dependent Ca
uptake,
[
H]Inositol 1,4,5-trisphosphate
(InsP
) binding, and quantal InsP
-induced
Ca
release were investigated using rat cerebellar
microsomes. Both the ion species and concentration affected the ability
of the microsomes to support Ca
uptake with
K
being most effective (3.8 nmol of
Ca
/min/mg at 100 mM K
). The
order of efficacy of the other ions was as follows: K
> Na
> Rb
=
Cs
Li
. The binding of
[
H]InsP
to cerebellar microsomes was,
however, affected little by the presence of these ions. All these
alkali metal ions (except Li
) supported
InsP
-induced Ca
release at concentrations
above 25 mM; however, the extent of Ca
release (expressed as a percent Ca
release
compared with that released by the ionophore A23187) was dependent upon
the ion species present. Again K
was more potent than
the other ions at facilitating InsP
-induced Ca
release (order of efficacy: K
> Rb
> Na
> Cs
), although the
concentration of InsP
required to induce half-maximal
Ca
release (IC
) was not significantly
altered. Over the ion concentration range tested (25-100
mM), the extent of InsP
-induced Ca
release with both K
and Rb
increased in a linear fashion, while Na
showed
only a slight increase and Cs
showed no increase over
this range. The effect of K
concentration on quantal
Ca
release was to alter the extent of release rather
than the IC
InsP
concentration. Using
stopped-flow techniques, the effects of InsP
and
K
concentrations on the kinetics of
InsP
-induced Ca
release were shown to
exhibit a monoexponential process in this microsomal preparation. The
rate constants for Ca
release increased with
InsP
concentration (0.11 s
at 0.02
µM InsP
to 0.5 s
at 40
µM InsP
); however, the relationship between
the fractional extent of release and rate constants for release did not
change in a similar way with InsP
concentration. Although
the fractional extent of Ca
release increased with
K
concentration, the rate constants for release over
this K
concentration range were unaffected. This
observation leads us to question the role of K
as a
counter ion required for Ca
release, and we therefore
postulate a role for K
(and the other alkali metal
ions) as a ``co-factor'' required for channel opening.
Inositol 1,4,5-trisphosphate (InsP) (
)is
a second messenger which activates a Ca
channel
usually located within specific intracellular Ca
stores of many cell types(1) . Several studies have so
far demonstrated that in order for InsP
-induced
Ca
release to occur univalent metal ions such as
K
are
required(2, 3, 4, 5) . An early
proposal suggested that InsP
-induced Ca
efflux from Ca
stores is electrogenic and can
only occur if accompanied by a K
influx to
counterbalance Ca
movement, thus alleviating a
buildup in membrane potential(2, 3) . The inhibition
of InsP
-induced Ca
release by
K
channel blockers such as tetraalkylammonium cations
and nonyltriethylammonium has indicated that InsP
-sensitive
Ca
stores must also contain K
channels(2) . However, in a more detailed study where an
extensive range of K
channel blockers was used, it was
concluded that the inhibition of InsP
-induced
Ca
release by these blockers was likely to be due to
a direct interaction with the InsP
-sensitive
Ca
channel itself rather than inhibition of any
putative K
channels, since the addition of
K
ionophores valinomycin or gramicidin D, which would
supply an alternative route for K
influx, failed to
reverse the inhibition of InsP
-induced Ca
release by these K
channel
blockers(6, 7) .
From studies using permeabilized
hepatocytes, Joseph and Williamson (3) showed that the chloride
salts of the alkali metals K, Na
,
Rb
, and Cs
were essentially similar
in supporting InsP
-induced Ca
release.
Their study also showed that salt concentration affected Ca
release with optimal concentration being
40 mM;
however, the release process was not affected by changes in osmotic
strength (3) .
The fact that the purified cerebellar
InsP-sensitive Ca
channel alone could
also support Ca
movements when reconstituted into
sealed liposomes and exposed to InsP
(8, 9, 10) must indicate that separate
K
channels are not required for Ca
release, although the possibility exists that the
InsP
-sensitive Ca
channel may also have
intrinsic K
channel
activity(11, 12) . One alternative possibility could
be that alkali metal ions may stimulate the InsP
-sensitive
Ca
channel by acting as ``co-factors''
stimulating particular steps in the mechanism of channel opening
instead of/or as well as acting as a counter-ion(13) . This
hypothesis therefore needs further investigation.
It is clear that
alkali metal ions such as K are essential for
InsP
-induced Ca
efflux, but it is not
clear as yet how these ions cause this activation. In this study we
undertake a detailed investigation to characterize the effects of
alkali metal ions on the cerebellar InsP
-sensitive
Ca
channel, by investigating their effects on
[
H]InsP
binding and the kinetics of
quantal InsP
-induced Ca
release.
Fluo-3 was obtained from Sigma, InsP from
Calbiochem, and [
H]InsP
from DuPont
NEN. All alkali metal chlorides were obtained from Aldrich.
Rat
cerebellar microsomes were prepared essentially as described in (14) and (15) with minor modifications. Briefly,
15-20 cerebella were minced and homogenized in 10 volumes of cold
buffer (0.32 M sucrose, 5 mM Hepes, 0.1 mM phenylmethylsulfonyl fluoride, 0.03 mM benzamidine, 5
µM leupeptin A, and 10 µM pepstatin, pH 7.2).
The homogenate was centrifuged at 500 g for 10 min.
The resulting pellet was homogenized in 5 volumes of buffer and again
centrifuged at 500
g for 10 min. The supernatants were
pooled and the mitochondria removed by centrifugation at 10,000
g for 20 min. The microsomal pellet was obtained by
centrifuging the remaining supernatant for 1 h at 100,000
g. The pellet was resuspended in the Hepes-sucrose buffer to a
concentration of
15 mg/ml, snap-frozen in liquid nitrogen, and
stored at -70 °C.
Ca uptake and
InsP
-induced release from cerebellar microsomes were
measured using fluo-3 as described elsewhere(16) , with some
modifications. Rat cerebellar microsomes (0.3 mg/ml) were suspended in
a buffer containing Tris phosphate (40 mM), creatine kinase
(10 µg/ml), phosphocreatine (10 mM, Tris salt), fluo-3
(250 nM), and the appropriate concentration of alkai metal ion
as the chloride salt, pH 7.2, at 37 °C. Ca
uptake
was initiated by the addition of 1.5 mM Mg-ATP and the
fluorescence change monitored on a Perkin-Elmer LS-50B
spectrofluorimeter, with excitation at 506 nm and detecting the
emission at 526 nm. After ATP-dependent Ca
loading,
further Ca
uptake was inhibited by the addition of
between 0.2 and 0.5 mM sodium orthovanadate (which inhibited
>90% of the Ca
pumps(7) ) and InsP
at the appropriate concentration was added. Total Ca
accumulated within the microsomes was measured by
permeabilization with Ca
ionophore A23187 (12.5
µg/ml).
Fluorescence intensity was related to
[Ca] by the following equation given in (16) ,
On-line formulae not verified for accuracy
where K is the dissociation
constant for Ca
binding to fluo-3 at pH 7.2 and 37
°C. F is the fluorescence intensity of the sample and F
and F
are the
fluorescence intensities of the sample in 1 mM EGTA and 2.5
mM CaCl
, respectively.
Under standard
conditions (100 mM KCl, pH 7.2 and 37 °C) we have shown
the dissociation constant for Ca binding to fluo-3 to
be 900 nM(17) ; however, both the alkali metal ion
present and its concentration also affect this dissociation constant.
The dissociation constants for fluo-3 binding to Ca
were therefore measured in the appropriate alkali metal salt at a
variety of concentrations by monitoring the change in fluorescence in a
10 mM Hepes/Tris buffer, pH 7.2, 37 °C using 250 nM fluo-3 and varying the free Ca
concentration by
known concentrations EGTA and CaCl
as calculated by the
``ION'' computer program developed by Fabiato(18) .
It must be noted that the pH of the EGTA and CaCl
solutions
were adjusted with Aristar Tris (from BDH) to avoid alkali metal ion
contamination.
The rapid measurements of InsP-induced
Ca
release were monitored using an Applied
Photophysics stopped-flow spectrofluorimeter (model SX 17 MV), exciting
the sample at 505 nm and measuring the emission above 515 nm using a
cut-off filter. The microsomes were in the same buffer as described
previously and Ca
accumulation was followed on a
conventional spectrofluorimeter. Once sufficient Ca
loading had occurred, further accumulation was inhibited with
orthovanadate and microsomal/fluo-3 suspension added to syringe A of
the stopped-flow apparatus. Syringe B was filled with InsP
at 10 times the experimental concentration required as the mixing
ratio of syringe A to B was 10:1, to avoid introducing substantial
Ca
contamination when mixing the microsomes/fluo-3
suspension with InsP
. The fluorescence data were initially
adjusted by comparing the changes on the stopped-flow apparatus with
identical experiments undertaken on a conventional fluorimeter, such
that these traces could be related to fractional Ca
release. These traces were then analyzed using nonlinear
regression analysis programs supplied by Applied Photophysics. The time
courses for Ca
release for the microsomal preparation
used in this study could be fitted well to a monoexponential process
using the following equation,
On-line formulae not verified for accuracy
where A is the fractional amount or extent of release
and k is the rate constant which defines this release process.
Maximal amount of Ca release (1.0) was defined as
that released by 40 µM InsP
. Over the
Ca
concentration range in which
InsP
-induced release was monitored, the fluorescence
changes, when related to Ca
concentrations, were
around the K
value for Ca
binding to fluo-3. Over this range of fluorescence is linearly
related to Ca
concentration (linear regression
coefficient r > 0.99).
The binding of
[H]InsP
to cerebellar microsomes was
carried out as described in (14, 15, 16) .
0.5 mg of rat cerebellar microsomes was suspended in 0.5 ml of buffer
containing 50 mM Tris/HCl, pH 8.3, 1 mM EDTA, with
the appropriate concentration of alkali metal chloride salt and doped
with 0.02 µCi of [
H]InsP
.
Specific binding was measured at 40 nM InsP
(the K
value for InsP
binding
cerebellar microsomes under our experimental conditions) and
nonspecific binding measured in the presence of 10 µM excess cold InsP
. After the addition of the microsomes
to the buffer, the mixture was allowed to incubate at 4 °C for 15
min. Bound InsP
was separated from free by centrifugation
at 15,000
g for 20 min and the pellets then
solubilized in 0.5 ml of Solvable (DuPont), added to Ultima flow
scintillant and the radioactivity determined by liquid scintillation
spectrometry.
Before using fluo-3 to measure InsP-induced
Ca
release in the presence of different alkali metal
ions at different concentrations, the effects of these ions on the
dissociation constant of Ca
binding to fluo-3 were
determined. Fig. 1shows that the K
value for Ca
binding to fluo-3 is
dependent upon the ionic concentration and type of metal ion present.
The K
value varies from 230 nM in the absence of added ions to 900 nM in the presence of
100 mM KCl, the latter being identical to the previously
determined value(17) . Both K
,
Na
, and Li
ions affected the K
for Ca
binding to
fluo-3 in a similar fashion. The change in K
for Ca
binding to fluo-3 in the presence
of Cs
and Rb
at concentrations up to
100 mM salt was substantially lower than the values obtained
with K
(varing from 230 nM to 550
nM). In all subsequent experiments the changes in
Ca
concentrations were calculated using the
appropriate K
for the metal ion and
concentration used in each experiment. However, in buffers containing
cerebellar microsomes, no additional affect on the K
for Ca
binding to fluo-3 was observed.
Figure 1:
The effects of alkali metal ion
concentration and species on the dissociation constant (K) of Ca
binding to
fluo-3. Each K
value was measured at pH
7.2, 37 °C in the presence of the appropriate metal ion and
concentration. The K
was calculated by
altering the [Ca
] free, by varying the EGTA
and CaCl
concentration and measuring the changes in fluo-3
fluorescence as described in (17) . Each Ca
titration was carried out three times and the mean value plotted.
The K
values for the metal ions are
represented as follows:
, K
;
,
Na
;
, Rb
;
,
Cs
; and
,
Li
.
Prior to monitoring the effects of alkali metal ions on
InsP-induced Ca
release, the microsomes
were first loaded with Ca
by activating the
microsomal Ca
-pump (Ca
-ATPase) with
ATP. If no alkali metal ions were present in the assay buffer, little
or no Ca
uptake could be measured (<0.1 nmol of
Ca
/min/mg), also extremely poor Ca
uptake was also observed in the presence 100 mM Li
(
0.2 nmol of
Ca
/min/mg). In our system K
was the
most effective alkali metal ion (3.8 ± 0.2 nmol of
Ca
/min/mg at 100 mM K
);
however, all other alkali metal ions used (except Li
)
could support Ca
-pump activity to a level which could
sufficiently load the microsomes with Ca
prior to
performing release experiments (Na
, 3.1 ± 0.2;
Rb
, 1.9 ± 0.3; Cs
, 2.0
± 0.2 nmol of Ca
/min/mg at 100 mM alkali metal ion, respectively). We also ensured that in all
experiments Ca
accumulation into the microsomal
vesicles was allowed to reach similar levels, before further uptake was
inhibited by the addition of up to 0.5 mM orthovanadate.
Fig. 2shows the effects of the alkali metal ions
Na, K
, Rb
, and
Cs
(all at 100 mM concentration) on quantal
InsP
-induced Ca
release. It is clear that
the amount of InsP
-induced Ca
release
(measured as a percent of that releasable with A23187) is dependent
upon the type of metal ion present, with K
able to
support the greatest amount of release (15.7% Ca
release at maximal InsP
concentration).
Rb
was the next most potent ion (causing 11.8%
InsP
-induced Ca
release), while
Na
and Cs
supported lower levels of
Ca
release (9.3 and 6.7% release, respectively).
Although the concentration of InsP
required to cause
half-maximal InsP
-induced Ca
release
(IC
) varied between 1.0 and 2.0 µM for the
metal ions tested at 100 mM (Na
, 1.0 ±
0.2 µM; K
, 1.3 ± 0.3
µM; Rb
, 2.0 ± 0.8 µM;
Cs
, 1.1 ± 0.4 µM), the standard
errors for the IC
values were such that no significance
could be placed on these small variations. Fig. 2also shows
that in this preparation the concentration of InsP
required
to reach maximal release differed with the type of metal ion present.
Both K+ and Rb
required
10 µM InsP
to reach maximal levels of Ca
release, while Na
and Cs
appear
to require lower InsP
concentrations (
3
µM) to attain their maximal levels.
Figure 2:
The effect of alkali metal ions on quantal
InsP-induced Ca
release. Each curve
represents the effect of 100 mM:
, K
;
, Na
;
, Rb
; and
,
Cs
on InsP
-induced Ca
release measured as a percent of Ca
released by
InsP
(0.01-20 µM) compared with that
released by A23187 (12.5 µg/ml). The data are presented as the mean
± S.E. of three or more
determinations.
Table 1shows
that different alkali metal ions do not significantly alter the
affinity of the receptor for InsP, since little or no
effect was observed on the amount of
[
H]InsP
bound to the cerebellar
membranes in the presence of these ions when measured using 40 nM InsP
(the K
value for
InsP
binding to cerebellar microsomes under our
experimental conditions). There was also little effect of metal ion
concentration upon [
H]InsP
binding,
as measured using K
. A small decrease in the amount of
InsP
bound was observed, however, in the absence of any
added metal ion (12.3 pmol/mg) compared with 100 mM K
(14.7 pmol/mg).
Fig. 3shows the
effects of alkali metal ion concentrations on InsP-induced
Ca
release measured at 20 µM InsP
. InsP
-induced Ca
release increases in an essentially linear relationship with
increasing K
and Rb
concentration
over the range 25-100 mM. However, Cs
has effectively reached the maximum level of
InsP
-induced Ca
release by 25 mM as this release remains constant over the whole concentration
range tested. A small rise was observed with Na
on
InsP
-induced Ca
release, but this appears
to saturate at between 75 and 100 mM concentration. Fig. 3shows that although the alkali metal ions K
and Rb
support higher levels of
InsP
-induced Ca
release than the other
ions when measured at 100 mM ion concentration, this
difference is in fact slightly reversed at the lower ion
concentrations.
Figure 3:
Metal ion concentration on
InsP-induced Ca
release. Each curve
represents the effect of increasing the metal ion concentration of:
, K
;
, Na
;
,
Rb
; and
, Cs
on Ca
release induced by 20 µM InsP
.
Ca
release is expressed as percent of that released
by InsP
compared with that induced by A23187. Each data
point represents the mean ± S.E. of three or more
determinations.
Fig. 4shows the effects of varying
K concentration on quantal InsP
-induced
calcium release. Increasing the K
concentration
increases the percent Ca
released by
InsP
. There also appears to be a small decrease in the
IC
values of InsP
concentrations required for
Ca
release with increasing K
concentration (2.0 ± 0.5 µM at 25 mM
K
, 1.9 ± 0.3 µM at 50 mM K
, 1.4 ± 0.4 µM at 75
mM K
, and 1.3 ± 0.3 µM at
100 mM K
concentrations, respectively), but
again when the standard errors for these values are taken into account,
this appears to have little significance.
Figure 4:
K concentration on
quantal InsP
-induced Ca
release. Each
curve represents the effect of K
at 100 mM
(
), 75 mM (
), 50 mM (
), and 25
mM (
) concentration on InsP
-induced
Ca
release measured as percent release compared with
A23187. The InsP
concentration was varied between 0.01 and
20 µM. The data represent the mean ± S.E. of three
or more determinations.
Several studies looking at
the time course for Ca release induced by InsP
using permeabilized cells have shown it to be biphasic in nature,
comprising a fast and slow component(19, 20) . Here an
investigation of rapid InsP
-induced Ca
release from rat cerebellar microsomes was undertaken. Fig. 5A shows the effects of increasing InsP
concentration from 0.02 to 40 µM on Ca
release measured using a stopped-flow spectrofluorimeter at 37
°C and 100 mM KCl. The time courses for Ca
release were plotted as fractional InsP
-induced
Ca
release, where maximal release was set to the
percent Ca
release observed at 40 µM
InsP
. As shown in Fig. 5A the
Ca
release data can be simply fitted to a
monoexponential process (solid line). However, the data
presented here could also equally well be fitted to a biexponential
processes comprising two rate constants and two amplitudes where the
values are similar in both cases. As a monoexponential equation could
be used to fit all experimental conditions described here (i.e. varying InsP
and K
concentrations),
our analysis was confined to using the simplest mathematical function
describing this process. At low InsP
concentrations maximal
release is reached between 10 and 15 s after addition, while at high
concentrations maximal release is reached after about 5 s. Fig. 5B shows that the rates and amplitudes of
InsP
-induced Ca
release are dependent
upon the InsP
concentration added. The rate constants
determined here appear to be 5-10-fold lower than previously
reported for InsP
-induced Ca
release in
permeabilized hepatocytes and basophilic leukemia
cells(19, 20) , but are considerably faster than those
obtained for the purified cerebellar InsP
receptor
reconstituted into liposomes(8, 9, 10) . Fig. 5B also shows a variation in the relationship
between the amplitudes and the rate constants with InsP
concentration. In the microsomal preparation used for this part
of the study, the maximum amount of Ca
release
(amplitude) required
1 µM InsP
; however,
the rate constant for this process still had not reached its maximum
level by 40 µM InsP
.
Figure 5:
Kinetics of Ca release
as a function of InsP
concentration. A,
stopped-flow traces of InsP
-induced Ca
release measured with (from top to bottom) 40,
1.0, 0.5, and 0.02 µM InsP
, respectively. The solid lines represent monoexponential fits to the experimental
data, with the rate constants (
) and amplitudes (
) given in B. The time courses for Ca
release represent
the average of between 6 and 10
experiments.
Fig. 6A
shows the InsP-induced Ca
release
time course (using 1 µM InsP
) at different
K
concentrations (25-100 mM). In some
of the time courses in this figure a split time base was used to
enhance the amount of data points collected within the first 2 s. A
1-ms time filter was also used to to reduce the signal-to-noise ratio.
Again all the data could be fitted to a monoexponential equation. Fig. 6B shows that although the amplitude (fractional
amount of Ca
release) increases in a linear
relationship with K
concentration (see also Fig. 2), the rate constants for Ca
release are
essentially unaffected.
Figure 6:
Kinetics of InsP-induced
Ca
release as a function of K
concentration. A, stopped-flow traces of
InsP
-induced Ca
release measured in the
presence of (from top to bottom) 100, 75, 50, and 25
mM K
, respectively. The solid lines represent the best monoexponential fits to the experimental data,
with the rate constants (
) and amplitudes (
) given in B. The time courses for Ca
release represent
the average of between 8 and 12
experiments.
Fluo-3 is a commonly used fluorophore for measuring
Ca fluxes in intact and permeabilized cells as well
as subcellular
fractions(14, 15, 16, 22) . The
affinity of this dye for Ca
is greatly dependent upon
both ion species and ion concentration present in the medium. Most
workers in the field using fluo-3 tend to use either 400 nM(21) or 900 nM(16, 17) as the
dissociation constant for Ca
binding in order to
calculate changes in Ca
concentration. However, as
illustrated, here the K
values can vary
substantially from 225 to 900 nM dependent upon the ion
present and its concentration and as such will affect the calculated
free Ca
concentrations. As pH and Mg
concentration also affect the affinity of fluo-3 for
Ca
, (
)we must stress the importance of
using the appropriate K
value for fluo-3
depending on the experimental conditions used. These observations
should also serve as a warning to experimentalists attempting to draw
conclusions from small differences in the calibrated Ca
concentrations inside cells.
The rate of ATP-dependent
Ca uptake into cerebellar microsomes is dependent
upon the ion species and concentration. K
was the best
ion for Ca
uptake being twice as effective as
Rb
at the same concentration. Li
was
extremely poor at eliciting uptake (approximately 5% of the rate as
that for K
), and therefore no InsP
-induced
Ca
release experiments were undertaken with this ion.
We noted that at least 20 mM alkali metal ion concentration
was required to attain a sufficient level of Ca
uptake into the microsomes in order for Ca
release experiments to be undertaken. These results are
comparable with those observed by Muallem et al.(2) using rat liver microsomes and probably relate to the
fact that alkali metal ions, in particular K
,
stimulate the microsomal Ca
-ATPase. K
ions have been shown to stimulate the sarcoplasmic reticulum
SERCA1 isoform of the Ca
-ATPase by increasing the
rate of the dephosphorylation step (E2P - E2)(23) . Since the kinetic properties of the
endoplasmic reticulum Ca
-ATPase are similar to the SR
type(24) , it is likely that K
stimulation of
ATP-dependent Ca
uptake by the microsomal
Ca
-ATPase is by a similar mechanism.
K is also most effective at stimulating InsP
-induced
Ca
release when measured at 100 mM concentration. However, the potency of K
compared
with the other ions tested was diminished at concentrations below 50
mM. The stimulation of InsP
-induced Ca
by K
increased linearly with concentration up to
100 mM, which was the maximum concentration tested in this
study. This observation directly contrasts with the study of Joseph and
Williamson(3) , which showed that in rat permeabilized
hepatocytes K
stimulated InsP
-induced
Ca
release optimally at 40 mM, while at
higher concentration Ca
, release was inhibited. The
only significant effects of the metal ion species and ion concentration
on quantal InsP
-induced Ca
release was on
the percent or extent of release, as the IC
values for
Ca
release with InsP
concentration and
[
H]InsP
binding levels were affected
little. The fact that here we observe little effect of K
on [
H]InsP
binding, while in
the paper by Hannaert-Merah et al.(28) shows a
2-3-fold decrease in affinity with K
, most
probably reflects differences in experimental conditions used in both
studies (i.e. binding studies were undertaken at 4 °C
rather than 20 °C).
In this study the rate of
InsP-induced Ca
release from rat
cerebellar microsomes was resolved using stopped-flow techniques and
shown to be slower than earlier reports using permeabilized rat
basophilic leukemia cells and rat
hepatocytes(19, 20) , but considerably faster than the
rates observed for the cerebellar InsP
receptor
reconstituted into liposomes(8, 9, 10) . The
differences in the rates of Ca
release using
different cell types may reflect differences in InsP
receptor isoforms present in these cells. From immunological
studies using isoform specific antibodies, the cerebellum appears to
express mainly the type I isoform, while hepatocytes express mainly the
type II isoform(25) . It is as yet unknown what isoforms are
present in basophilic leukemia cells. The differences observed between
the Ca
release rates from cerebellar microsomes
compared with cerebellar InsP
receptors reconstituted into
liposomes may well reflect a difference in the receptor density in
membrane vesicles between the two systems, which in turn may affect the
rates of release (26) . The difference in the rates observed
here compared with those in other studies is unlikely to be due to any
effects of Ca
pumps, since >90% of the
Ca
pumps were inhibited prior to InsP
addition. We have calculated the rate constant for Ca
uptake into the microsomes following orthovanadate inhibition to
be <0.01 s
. Since the rate of
InsP
-induced Ca
release through the
InsP
sensitive channel is much faster than the rate of
Ca
uptake in this system, especially after
orthovanadate inhibition, the rate of InsP
-induced
Ca
release is affected little whether the
Ca
pumps were fully inhibited or 90% inhibited, since
even at lowest InsP
concentration used the rate constant
for Ca
release was calculated to be >0.1
s
.
In this preparation the release process with
low InsP concentration reached completion after 10 s,
whereas with high InsP
concentration (40 µM)
the maximal amount of release was reached after 5 s. The fact that the
rates of InsP
-induced Ca
increase with
InsP
concentration can be used to argue against the model
for quantal Ca
release which assumes all-or-none
release from Ca
stores which have heterogeneous
sensitivities to InsP
(5) . (For a detailed
description of the current models for quantal Ca
see (12) . As has already been made apparent by Hirose and
Iino(26) , this rather simplistic model would therefore imply
that only the extent of Ca
release would vary with
InsP
concentration as this would reflect the number of
stores recruited to release Ca
, while the rate
constants for Ca
release should remain unaffected
unless other factors such as variability in the channel density between
distinct Ca
stores also occurs. However, the fact
that the rate of Ca
release is more sensitive to
InsP
concentration than the extent or amplitude of
Ca
release (illustrated by the fact that in Fig. 5B the amplitude or extent of Ca
release has reached a maximum by 1 µM InsP
, while the rate constant still appears to
increase beyond 40 µM InsP
) would also argue
against a more elaborate version of the all-or-none model for quantal
Ca
release which assumes that the stores are not only
heterogeneous with respect to their sensitivities to InsP
but also have heterogeneous receptor densities. In this case the
concentration of InsP
required to reach both the maximum
amount of Ca
release and the maximum rate of
Ca
release should be the same.
In this preparation
of rat cerebellar microsomes we found that InsP-induced
Ca
release could be fitted assuming a simple
monoexponential process at all InsP
concentrations and
K
concentrations used; however, a biexponential
process with similar rate components and amplitudes for the two
components could also be fitted equally well. In investigations of the
rate constants of InsP
-induced Ca
release
using different preparations of cerebellar microsomes, we have
concluded that the rate constants are consistently lower than those
previously reported in other studies, using permeabilized
cells(19, 20) . However, they are consistently similar
between cerebellar microsomal preparations (varying between 0.5 and 1.7
s
with 20-40 µM
InsP
). In some microsomal preparations
InsP
-induced Ca
release can only be
successfully fitted to a biexponential process consisting of two
independent monoexponential components(27) , while other
preparations, such as the one used in this study, can be fitted equally
well to a monoexponential process. As yet the reason for this variation
between preparations remains unknown, although since there is also a
variation in the levels of Ca
released by
InsP
, the IC
values and the cooperativity of
InsP
-induced Ca
release between
cerebellar microsome preparations, the variability may be due to subtle
differences in membrane preparations. A related phenomenon was recently
reported by Hannaert-Merah et al.(28) , who showed
that the kinetics of InsP
binding and dissociation to
cerebellar microsomes was either monophasic or biphasic depending on
the cerebellar microsome preparation used.
From the kinetic data
presented here, the only affect of varying K concentrations seems to be on the extent of Ca
release rather than on any effects on the rate constants for
release. Since it has been reported previously that there is
heterogeneity between InsP
-sensitive Ca
stores(5) , one plausible explanation for this
observation might be that these stores are also heterogeneous in their
sensitivities to K
, such that some stores will be able
to respond to InsP
and release Ca
at low
K
concentration while other stores require higher
K
concentration before Ca
release
occurs. An alternative explanation, which at present cannot be ruled
out, is the possibility that as InsP
-sensitive
Ca
channels slowly desensitize after being opened by
the addition of InsP
(29) , K
could
slow down this desensitization step, thus increasing the amount of
Ca
release without necessarily affecting the rate of
release. It is unlikely that the effects of K
we have
observed on quantal InsP
-induced Ca
release are due substantially to an increase in the rate of
InsP
dissociation from its receptor as suggested by
Hannaert-Merah et al.(28) .
As K concentration does not affect the rate of
InsP
-induced Ca
release, this may have
implications in assessing the possible role of K
as a
counter ion. Although some studies have tried to monitor changes in
Rb
uptake into cerebellar microsomes upon
addition of InsP
, such changes have not been
detected(6) . We have also tried to monitor changes in
K
uptake into microsomes upon exposure to
InsP
, using flame spectrophotometry with no success. Since
the cerebellar InsP
receptor can be purified and
reconstituted into sealed vesicles and still retain Ca
channel activity(8, 9, 10) , this must
imply that if K
ions are required as a counter ion
during Ca
release then the channel itself must be an
antiporter allowing Ca
to flow in one direction,
while K
moves in the opposite direction(11) .
Although several studies using electrophysiological approaches have
shown the InsP
receptor to be weakly permeable to both
K
and Na
(30, 31) ,
they have only been shown to move in the same direction as
Ca
and not in the opposite direction as would be
required here. If the InsP
receptor was an antiporter, then
by analogy with other co-transporters such as the the
Na
/Ca
exchanger(32, 33) , changing the concentration
of one ion should have a direct effect on the rate at which the other
ion is transported as long as neither are at saturating concentrations.
However, the fact that changing the concentration of K
has no effect on the rate of Ca
release
(measured at InsP
concentrations where the rate of
Ca
release is not maximal) must imply that
K
is unlikely to be acting as a counter ion for
Ca
release. This therefore leaves us to postulate a
more direct role for K
and the other alkali metal ions
in affecting the mechanism of channel opening, possibly by acting as a
co-factor. This possibility of the InsP
receptor containing
a putative K
binding site which affects channel
function obviously requires further investigation.