(Received for publication, February 7, 1995; and in revised form, June 6, 1995)
From the
The kinetics of inositol 1,4,5-trisphosphate
(IP)-induced Ca
release of the
immunoaffinity-purified IP
receptor (IP
R),
reconstituted into lipid vesicles, was investigated using the
fluorescent Ca
indicator fluo-3. IP
R was
purified from mouse cerebellar microsomal fraction by using an
immunoaffinity column conjugated with an anti-IP
R type 1
(IP
R1) antibody. The immunoblotting analysis using
monoclonal antibodies against each IP
R type showed that the
purified IP
R is almost homogeneous, composed of
IP
R1. Ca
efflux from the proteoliposomes
was monitored as fluorescence changes of 10 µM fluo-3,
whose concentration was high enough to buffer released Ca
and to keep deviations of extravesicular free Ca
concentration within 30 nM, excluding the possibility of
Ca
-mediated regulation of IP
-induced
Ca
release. We also examined IP
-induced
Ca
release using 1 µM fluo-3, where the
deviations of free Ca
concentration were within 300
nM. At both fluo-3 concentrations, IP
-induced
Ca
release showed similar kinetic properties, i.e. little Ca
regulation of Ca
release was observed in this system. IP
-induced
Ca
release of the purified IP
R exhibited
positive cooperativity; the Hill coefficient was 1.8 ± 0.1. The
half-maximal initial rate for Ca
release occurred at
100 nM IP
. At the submaximal concentrations of
IP
, the purified IP
R showed quantal
Ca
release, indicating that a single type of
IP
R is capable of producing the phenomenon of quantal
Ca
release. The profiles of the
IP
-induced Ca
release of the purified
IP
R were found to be biexponential with the fast and slow
rate constants (k
= 0.3
0.7
s
, k
= 0.03
0.07 s
), indicating that IP
R has two
states to release Ca
. The amount of released
Ca
by the slow phase was constant over the range of
10-5000 nM IP
concentrations, whereas that
by the fast phase increased in proportion to added IP
. This
provides evidence to support the view that the fast phase of
Ca
release is mediated by the low affinity state and
the slow phase by the high affinity state of the IP
R. This
also suggests that the fast component of Ca
release
is responsible for the process of quantal Ca
release.
Inositol 1,4,5-trisphosphate (IP) (
)is
the second messenger derived from the hydrolysis of
phosphatidylinositol bisphosphate via activation of phospholipase C,
phospholipase C activity is enhanced by the activation of G
protein-linked and tyrosine kinase-linked cell surface membrane
receptors by various extracellular stimuli, such as hormones, growth
factors, neurotransmitters, odorants, lights, etc. (1) . The
IP
signal is converted into a Ca
signal
by binding to its specific receptor, i.e. the IP
receptor (IP
R), which is an IP
-induced
Ca
-releasing channel located on intracellular
Ca
stores such as the endoplasmic reticulum. This
IP
-mediated Ca
signaling plays a critical
role in a variety of cell functions, including fertilization, cell
proliferation, metabolism, secretion, contraction of smooth muscle, and
neural signals(1) . For these multiple cell signaling, the
mechanisms of transducing the IP
signal into a
Ca
signal, i.e. IP
-induced
Ca
release (IICR), may be diverse in each cell type.
There have been many studies on the kinetics of IICR; they describe
the channel-opening mechanism of the
IPR(2, 3, 4, 5, 6) ,
the regulation of IICR by modulators such as protein kinase
A(7, 8, 9) , ATP (10, 11) ,
GTP(12, 13) , and
Ca
(4, 14, 15, 16, 17, 18, 19, 20) ,
and the phenomenon of ``quantal Ca
release'' originally described by Muallem et
al.(21) , where submaximal concentrations of IP
cause the partial release of Ca
from
intracellular stores. There are often discrepancies in the properties
of channel opening and the effects of modulators on IICR among the
reports, because, in these studies, the arguments on some critical
points of IICR have often been complicated due to the different
experimental systems used, i.e. different micro-environments
surrounding the IP
R and Ca
pools assayed (e.g. different constitution of phospholipase C,
IP
-metabolizing enzymes, Ca
-binding
proteins, Ca
pumps, and modulators in each cell
type). For example, the degree of the cooperativity of Ca
release, which is an important issue for understanding the
channel opening mechanism, differed among the reports. Some reports
show no cooperativity of IICR(4, 15) , others show
positive cooperativity (n
= 2)(2, 5) (n
= 4)(3) . In
addition, recent molecular cloning studies have revealed that there are
at least three types of the IP
R from distinct
genes(22, 23, 24) . One of the major
arguments on IICR derives from the fact that multiple IP
R
types can coexist within a single cell type(25, 26) .
To further characterize the channel opening mechanism, the kinetic
study of IICR should be examined using a purified single type of the
IP
R.
The cerebellum is known to be the richest source of
IPR type 1 (IP
R1) among rodent tissues tested.
A recent immunohistochemical study indicated that rat cerebellum
contains three IP
R types whose expressing cell types are
quite distinct; IP
R1 is well known to be enriched in
Purkinje cells, IP
R type 3 (IP
R3) is present in
Bergmann glia and astrocytes, and IP
R type 2
(IP
R2) is also present, but not in neurons and
astrocytes(27) . The differential localization of each
IP
R type in cerebellar cell types indicates that most
IP
R
channel complexes in the cerebellum are
homotetramers within single cells. In the present study, we have
purified the cerebellar IP
R using an immunoaffinity column
coupled with an anti-IP
R1 antibody as described
previously(9) . The population of the purified IP
R
has been found to be almost homogeneous, containing little
IP
R2 and IP
R3. Therefore the results derived
from the analysis of this purified IP
R reflect the
properties of IP
R1. In this study, we have investigated the
kinetics of IICR mediated by the purified and reconstituted
IP
R using the fluorescent Ca
indicator
fluo-3 and have defined the cooperativity, quantal Ca
release, and biphasic nature of IICR.
Figure 1:
Immunoblots of the
immunoaffinity-purified IPR. The purified IP
R
was analyzed by Western blotting to investigate its homogeneity. The
same amounts of [
H]IP
binding
activity of cerebellar microsomal fraction and the purified
IP
R (1.5 pmol of IP
R/lane) were applied to the
gel, followed by immunoblotting with monoclonal antibodies 18A10,
KM1083, and KM1082 against IP
R1, IP
R2, and
IP
R3, respectively. Lanes 1, 3, and 5,
the solubilized cerebellar membrane fraction with 1% of CHAPS. Lanes 2, 4, and 6, the immunopurified
IP
R. The arrow indicates the position of
IP
R.
Figure 2:
Typical profile of IP-induced
Ca
release from proteoliposomes reconstituted with
the purified IP
R. Changes of fluorescence of the
Ca
indicator fluo-3 ([fluo-3] = 10
µM) were recorded after injection of IP
(500
nM). The total Ca
concentration was
estimated from the fluorescent intensity as described in the text. A, IP
-induced Ca
release from
the liposomes was followed by a constant leakage of Ca
(the solid line). B, the net IICR was obtained
by extrapolating and subtracting the constant Ca
leakage from the profile. C, the net IICR was found to
be well fitted by a biexponential (the solid line) with the
fast and slow rate constants.
where T represents a total amount of released
Ca, A is the amplitude of the fast and slow
components (percent) (A
+ A
= 100%), k is the rate
constant (s
), and t is time (s).
Figure 3:
Time course of IP-induced
Ca
release following the injection of different
IP
concentrations. IP
-induced Ca
release at different concentrations of IP
was
performed on a single batch of the proteoliposomes ([fluo-3]
= 10 µM). 5 µM (a), 200 nM (b), 70 nM (c), 40 nM (d) and 20 nM (e)
of IP
.
Figure 6:
Biexponential analysis of
IP-induced Ca
release: IP
dependence of the rate constants (A and B) and
the amplitudes (C and D). All profiles of IICR was
found to be biexponential, with the fast and slow rate constants as
described in the legend to Fig.1and in the text (). The fast (squares) and slow (circles)
rate constants (A and B) and the amplitudes of the
fast (squares) and slow (circles) (C and D) were plotted as a function of the concentration of
IP
. A and C were measured at 10
µM fluo-3 (values are mean ± S.D., n = 3-4; initial free Ca
concentration = 100 nM; deviations of free
Ca
concentration by the released Ca
= 10-30 nM) and B and D at 1 µM fluo-3 (values are mean ± S.D., n = 2-5; initial free Ca
concentration = 200 nM; deviations of free
Ca
concentration by the released Ca
= 150-300 nM).
Relative amounts of released Ca at
various concentration of IP
are shown in Fig.4, A (n = 3-4) and B (n = 2-5). The amount of released Ca
increased as a function of IP
concentration,
indicating that the single type of IP
R is capable of
producing the quantal response of Ca
release.
Figure 4:
The amounts of released Ca plotted as a function of IP
concentration. The
amounts of released Ca
were plotted as a function of
IP
concentration. The data were normalized to the amplitude
for 5.0 µM IP
. A, 10 µM fluo-3 (values are mean ± S.D., n =
3-4, initial free Ca
concentration = 100
nM, deviations of free Ca
concentration by
the released Ca
= 10-30 nM). B, 1 µM fluo-3 (values are mean ± S.D., n = 2-5, initial free Ca
concentration = 200 nM, deviations of free
Ca
concentration by the released Ca
= 150-300 nM).
The
initial rates of Ca release varied with IP
concentrations and saturated above 1 µM IP
at both fluo-3 concentrations of 10 µM (Fig.5A, n = 3-4;
deviations of [Ca
]
=
10-30 nM) and 1 µM (Fig.5B, n = 2-5;
deviations of [Ca
]
=
150-300 nM). Both half-maximal initial rates of IICR in
the presence of 10 and 1 µM fluo-3 occurred at 100
nM. We determined the degree of cooperativity of IICR by Hill
plotting (Fig.5C, n = 3-4 and
5D, n = 2-5). The slopes in the Hill plot over
the range of submaximal concentrations of IP
(20-200
nM) were calculated to be 1.8 ± 0.1 (Fig.5, C and D), indicating that the IICR of the purified
IP
R exhibited positive cooperativity. As the EC
value and the Hill coefficient of IICR at both concentrations of
fluo-3 were calculated to be the same, the changes of free
Ca
concentration by the released Ca
had no significant effect on the sensitivity for IP
and the cooperativity of IICR.
Figure 5:
Analysis of IP-induced
Ca
release. Initial rates were measured from the
initial and fast slope of IICR. A and B, normalized
initial rates of Ca
release were plotted as a
function of the concentration of IP
. C and D, analysis of initial rates by a Hill plot shows the
positively cooperativity of IICR. A and C were
measured at 10 µM fluo-3 (values are mean ± S.D., n = 3-4; initial free Ca
concentration = 100 nM; deviations of free
Ca
concentration by the released Ca
= 10-30 nM), B and D at 1
µM fluo-3 (values are mean ± S.D., n = 2-5, initial free Ca
concentration = 200 nM; deviations of free
Ca
concentration by the released Ca
= 150-300 nM).
Figure 7:
The amounts of released Ca by the fast and slow components of IP
-induced
Ca
release. The amounts of total released
Ca
in Fig.3and the amplitude of the two
components of IICR allowed us to calculate the amounts of released
Ca
by the fast (squares) and slow (circles) components. A, 10 µM fluo-3
(values are mean ± S.D., n = 3-4; initial
free Ca
concentration = 100 nM;
deviations of free Ca
concentration by the released
Ca
= 10-30 nM). B, 1
µM fluo-3 (values are mean ± S.D., n = 2-5; initial free Ca
concentration = 200 nM; deviations of free
Ca
concentration by the released Ca
= 150-300 nM).
In this study, we have investigated the
kinetics of IICR of the immunoaffinity-purified and reconstituted
IPR, excluding the possibility of modulation of IICR by
factors other than Ca
and IP
itself.
Furthermore, as the immunoaffinity-purified IP
R showed very
strong immunoreactivity with the monoclonal antibody against
IP
R type 1 and little with the monoclonal antibodies
against IP
R types 2 and 3, the population of the
immunoaffinity-purified IP
R was almost homogeneous of
IP
R1 but contained very small amounts of IP
R2
and IP
R3. Therefore the results derived from the analysis
of this purified IP
R reflect mainly the properties of
IP
R1. Due to the absence of IP
metabolizing
enzymes, in our system, applied IP
doses should be constant
throughout each experiment. However, we must consider the regulation of
the IP
R by changes in free Ca
concentration. Feedback regulations of IICR by the released
Ca
have been observed in permeabilized cells (14, 16) and microsomal
systems(4, 15) . On the other hand, the high
concentrations of Ca
chelators and Ca
indicators caused artificial effects on IICR in those
experiments(18, 34) . In this study, to avoid problems
concerning the regulation of IICR by the released Ca
,
we used high enough concentration of fluo-3 to keep extravesicular free
Ca
concentration within 100-130 nM. We
also used 1 µM fluo-3, 200-500 nM free
Ca
concentration, to compare the effect of changes of
extravesicular free Ca
by IICR on the kinetics of
Ca
release. At both fluo-3 concentrations, where the
extravesicular free Ca
concentration changed from 100
to 130 nM (10 µM of fluo-3) and from 200 to 500
nM (1 µM of fluo-3), the kinetics of the
Ca
release was essentially the same, indicating
little feedback regulation by the released Ca
in our
system. We also observed similar kinetics of Ca
release at the initial extravesicular free Ca
concentration of 300 nM (data not shown), where
Ca
release using cerebellar microsomes was shown to
be inhibited(35) . Feedback regulation by the released
Ca
or the regulation by Ca
outside
of pools may be mediated by the action(s) of other molecules which can
sense changes of Ca
concentration.
The
dose-response curves of IICR mediated by the purified IPR
showed that the amount of released Ca
increased as a
function of IP
concentration (Fig.4) and provided
evidence to suggest that the purified IP
R, which was
chiefly composed of IP
R1, can exhibit the quantal response
of Ca
release. Ferris et al.(36) have also reported that conventionally purified and
reconstituted cerebellar IP
Rs showed the quantal response
and suggested that heterogeneity of IP
R types was a
possible mechanism underlying quantal Ca
release.
However, it is likely that the quantal Ca
release is
an intrinsic property of IP
R1.
Recently,
heterogeneity of IPR densities in pools, which had equal
sensitivity to IP
, was reported to be responsible for
biphasic Ca
release(39) . If this is the
reason for biphasic nature of IICR, the amplitudes of the fast and slow
components in the curve fitting should be independent to the IP
concentrations, and the ratio of the amounts of released
Ca
by the fast and slow components must be constant.
Because in such an assumption, the amplitudes and the ratio of the
amounts of released Ca
should reflect the
distribution of such heterogeneity, i.e. proportion of
IP
-sensitive Ca
pools with high and low
density of IP
R reflect the amplitudes and the ratio of
amounts of the released Ca
by the fast and slow
phases, respectively. However, in our experiments, the amplitudes of
the fast and slow components and the ratio of the total released
Ca
were dependent on IP
concentrations,
indicating that the biphasic nature of Ca
release was
not due to such heterogeneity of receptor density. A possibility of
heterogeneity in the size of individual Ca
pools was
also excluded by the same reasons and by the direct observation using
electron microscopy as described under ``Results.'' The
present study has demonstrated that the purified IP
R has
two states with different affinity for IP
, i.e. a
low affinity and a high affinity state. This could arise from
alternative splicing leading to the production of variants of
IP
R1(40) . Alternatively, there may be two
different states of a single IP
R due to an
IP
-dependent inactivation or a
Ca
-dependent interconversion.
In the absence and
presence of changes in extravesicular free Ca concentrations, we observed the biphasic nature of IICR,
indicating that the changes in the free Ca
concentration may not be responsible for the biphasic kinetics.
However, we cannot rule out the possibility that the released
Ca
causes an instantaneous and local rise in
Ca
concentrations near the channel pore, which cannot
be promptly chelated by fluo-3, and would mediate the interconversion
of the two states. If the released Ca
instantaneously
rises near the channel pore, in cooperation with IP
, the
inactivation of the IP
R reported by Hajnoczky et al.(41) may be caused by the interconversion of
IP
R. This hypothesis could be supported by the observations
in Fig.3of (41) , where the degree of inactivation
(interconversion) varied with the cytosolic free Ca
concentrations during the preincubation with IP
and
IP
R, whereas no significant change in IICR at various
cytosolic free Ca
concentrations were observed
without preincubation.
We demonstrated here the positive
cooperativity of IICR and the quantal Ca release
phenomenon of IICR by the purified IP
R, which was mainly
composed of a single type of IP
R (IP
R1), and
the biphasic nature of IICR, which had kinetically two states to
release Ca
. The purification and reconstitution of
other types of IP
Rs may reveal new insights into IICR and
may allow us to relate any differences in the kinetic properties of
IICR to the differences in the structure of the different types of
IP
R. Also, this will allow us to observe the effects of
modulators, such as protein kinase A, ATP, Ca
, etc.
on type-specific IICR.