(Received for publication, May 22, 1995; and in revised form, October 9, 1995)
From the
Transient rise in nuclear calcium concentration is implicated in
the regulation of events controlling gene expression. Mechanism by
which calcium is transported to the nucleus is vehemently debated.
Inositol 1,4,5-trisphosphate (InsP) and
inositol-1,3,4,5-tetrakisphosphate (InsP
) receptors have
been located to the nucleus and their role in nuclear calcium signaling
has been proposed. Outer nuclear membrane was separated from the inner
membrane. The two membrane preparations were, as best as possible,
devoid of cross contamination as attested by marker enzyme activity,
Western blotting with antilamin antibody, and electron microscopy.
InsP
receptor and Ca
-ATPase were located
to the outer nuclear membrane. InsP
receptor was located to
the inner nuclear membrane. ATP or InsP
induced nuclear
calcium uptake. External free calcium concentration, in the medium
bathing the nuclei, determined the choice for ATP or
InsP
-mediated calcium transport. We present a mechanistic
model for nuclear calcium transport. According to this model, calcium
can reach the nucleus envelope either by the action of ATP or
InsP
. However, the calcium release from the nucleus
envelope to the nucleoplasm is mediated by InsP
through the
activation of InsP
receptor, which is located to the inner
nuclear membrane. The action of InsP
in this process was
instantaneous and transient and was sensitive to heparin.
The mechanism of regulation of nuclear calcium signaling is vehemently debated currently(1) . Nuclear calcium signals control a variety of nuclear functions(2, 3) , including gene transcription(4, 5) , DNA synthesis, DNA repair, and nuclear envelope breakdown(6) . It has been shown that nuclear vesicle fusion requires nuclear calcium mobilization(7) . There are indications that nuclear and cytosolic calcium signals are differentially regulated and are independent of each other. For examples, nuclear calcium concentrations are higher than cytosolic ones in a number of cell systems (8, 9, 10, 11, 12) or lower in smooth muscle (13) and neuronal cells(14) . The source of nuclear calcium signal has been proposed to be cytosolic calcium stores(15) , whereas clear evidence has come forward favoring a nucleocytoplasmic barrier to calcium (16) or the nucleus being insulated from large cytosolic calcium changes(14) .
The nucleus contains an endoplasmic
reticulum-type calcium pump ATPase (17) , which attests to the
ATP-mediated nuclear calcium uptake(18) . The nucleus is
endowed with functional inositol trisphosphate receptors (19, 20) and the necessary machinery for
InsP(
)production(21) . The nucleus also
contains inositol 1,3,4,5-tetrakisphosphate receptors(22) ,
which mediates nuclear calcium entry in the presence of
InsP
(23) . All this information has been gathered
on the basis of work on isolated rat liver nuclei.
Nuclear envelope (24) consists of an inner membrane and an outer membrane
interrupted by nuclear pores(25) . The outer nuclear membrane
constitutes a continuum with the endoplasmic reticulum(26) ,
whereas the inner nuclear membrane forms a membrane envelope for the
nucleoplasm and is lined by nuclear lamina, which are the attachment
sites for nuclear lamins(27) . In the study presented in this
paper, we isolated the two membranes of the nuclear envelope, as best
as possible, free from cross contaminations from one and the other. The
criteria for the purity of the inner and the outer membrane were the
marker enzyme activity, electron microscopy, and Western blotting with
antilamin antibodies. We document data demonstrating that the inner
nuclear membrane preparation is right-side-in vesicular structure,
which is the site for InsPR location. In contrast, the
outer nuclear membrane, as isolated here, consists of membranous
sheaths with enriched mannose-6-phosphatase activity, devoid of
immunoreactivity with antilamin antibodies, and is the site for
location of high affinity InsP
R and
Ca
-pump ATPase. We also provide evidence that
depending upon the external free calcium concentrations, ATP or
InsP
mediates calcium transport to the nuclear envelope.
The inner nuclear membrane was incubated with antilamin
antibodies (1:100 dilution) for 3 h at room temperature. This was
centrifuged for 15 min at 12,000 g. The resulting
pellet was washed three times with a phosphate buffer saline (PBS). The
final pellet was suspended in PBS, incubated with secondary antibody
(anti-IgG labeled with colloidal gold, particle size 12 nm) at a 1:50
dilution, and incubated for 3 h at room temperature, washed three times
as before, fixed, embedded, and thinly sectioned. In order to render
antilamin antibody accessible to the site of antigen i.e. interior to the inner nuclear membranes, it was necessary to
suspend the inner nuclear membrane in a low salt medium containing 10
mM HEPES, pH 7.5, 10 mM KCl, and 10 mM MgCl
. This was centrifuged and the pellet was treated
as above. Electron microscopic examination was carried out with an EM
420 Phillips instrument.
Mannose-6-phosphatase activity was determined as described(30) . NAD pyrophosphorylase activity was determined according to Kornberg(31) , and NADPH cytochrome c reductase activity was measured by the reduction of cytochrome c as described(33) . Protein was determined according to Bradford (35) with bovine serum albumin as a standard.
The use of a high molarity sucrose in liver homogenization medium and maintaining still higher sucrose concentration in subsequent centrifugation step resulted in a nuclear preparation that was devoid of any plasma membrane or microsomal contaminants. The question of microsomal-nuclear association during nuclei purification process was adequately checked by comparing nuclear preparation and final postnuclear materials from two sets of starting liver homogenate, i.e. normal liver homogenate from x number of rats and the homogenate supplemented with microsomes prepared from the same number of rats (for details, see (19) ). Fig. 2illustrates well defined nuclear structure with intact nuclear pores (Fig. 2B).
Figure 2:
Electron microscopic examination of
nuclear membranes. A, the intact nuclei with nuclear envelope,
nucleolus and nuclear material. B, nuclear pores indicated by arrow. C, outer nuclear membranous sheaths obtained
after sodium citrate treatment followed by sucrose gradient
centrifugation. D, the inner nuclear membrane obtained after
sucrose gradient centrifugation of DNase-digested nuclear material. The
vesicular nature of inner nuclear membrane is preserved (E) as
in D, at higher magnification. F and G,
inner nuclear membrane suspended in a low salt medium causing rupture
of the membrane and accessibility of antilamin antibody to its target.
The lamin on nuclear lamina was labeled with colloidal gold (dark
points). Bar denotes length (in µm): A, 5.0; B, 0.5; C, 0.5; D, 2.0; E, 0.5; F, 0.1; G, 0.1. Magnifications were: A,
2500; B and C,
22,000; D,
6000; E,
20,000; F,
74,200; G,
124,000.
Western blotting with antilamin antibody revealed immunoreactive protein band exclusive to the inner nuclear membrane (Fig. 1). The outer nuclear membrane showed no immunoreactivity with the antilamin antiserum (Fig. 2, compare lane 4 with lane 5). This further attested to the lack of cross-contamination from one membrane to the other.
Figure 1: Western blotting with antilamin antiserum. Protein from the purified inner nuclear membrane (lanes 1-4) and outer membrane (lane 5) was electrophoresed on SDS-PAGE with 12% polyacrylamide and transferred to nitrocellulose membrane as described under ``Experimental Procedures.'' Antilamin antiserum used was a kind gift from Dr. A. M. Fry (Lausanne, Switzerland). No lamin immunoreactive protein was detected in outer membrane (lane 5, 40 µg of protein was loaded). Inner membrane protein on each lane was: lane 1, 1 µg; lane 2, 10 µg; lane 3, 20 µg; lane 4, 40 µg. Two immunoreactive proteins represent lamin A (66 kDa) and lamin B (68 kDa). Certain minor protein bands seen are due to nonspecific immunoreactivity.
Electron microscopic examination of inner nuclear membrane showed its vesicular nature (Fig. 1, D and E), whereas the outer membrane was represented by membranous sheath (Fig. 2C). The sidedness of the inner nuclear membrane was verified by immunoelectron microscopy using colloidal gold and antilamin antibody (Fig. 2, F and G). Lamin was spotted only on the inner side of the inner membrane (Fig. 2F) and only on one side of the membrane preparation (Fig. 2G). This tempted us to propose that the inner nuclear membranes, as purified, were right-side-in orientation. When the inner nuclear membrane was not treated with a low salt medium, antilamin antibody could not be accessible to the lamin lying on the nuclear lamina. No lamin was seen by colloidal gold immunochemistry, and the intact inner nuclear membrane vesicles as represented in Fig. 2E remained intact (data not shown) without low salt treatment. This provided additional support for the hypothesis that the inner membranes were vesicular in nature and were right-side-in.
Figure 3:
Western blotting with the antiserum
against purified rat brain InsPR. Inner membrane (IM) and outer nuclear membrane (OM) were
electrophoresed on 8% acrylamide and transferred to nitrocellulose
membrane. Procedure for Western blotting is described under
``Experimental Procedures.'' 10 µg of membrane protein
was loaded on each lane. Note that the 220-kDa protein band identified
with the inner nuclear membrane is the nuclear InsP
R, which
was phosphorylated by protein kinase C(20) .
Anti-InsP
R antibody was a kind gift from Dr. S. H.
Snyder.
The
specificity of InsP receptor located to the inner nuclear
membrane was analyzed by competitive displacement of
[
H]InsP
by various inositol
phosphates (Fig. 4). InsP
at 6.5 nM inhibited 50% of the [
H]InsP
binding and was the most potent inhibitor of binding (Fig. 4).
Binding was also inhibited, although to a lesser extent, by the 2,4,5
InsP
isomer, InsP
(1, 3, 4, 5) ,
InsP
(3, 4, 5, 6) , and
InsP
. The specific binding increased linearly with the
amount of inner membrane protein added in the binding assay and heparin
at 10 µg/ml completely abolished
[
H]InsP
binding (data not shown).
Scatchard plots (Fig. 5) of saturation isotherms showed a single
class of binding sites with K
= 5.5 nM and B
= 1205 fmol/mg protein (Fig. 5A). K
(7.0 nM) and B
(385 fmol/mg protein) were found with the
intact nuclear preparation (Fig. 5B). Almost no change
in the K
values of InsP
R located to
the inner nuclear membrane preparation as compared with the intact
nucleus attested to the functional efficiency of the InsP
R
in the inner nuclear membrane preparation. Moreover, increased B
values in the case of inner membrane indicated
a rise in receptor density upon membrane purification. No meaningful
[
H]IP
binding activity was found with
the outer membrane (data not shown).
Figure 4:
[H]InsP
binding to purified inner nuclear membrane.
[
H]InsP
binding was carried out in an
Eppendorf tube at 0 °C (on ice) for 10 min in a medium containing
50 mM Tris-HCl, pH 8.0, and 2 mM EDTA. The inner
membrane protein was 0.1 mg, and the total volume of assay medium was
400 µl. The binding was terminated by centrifugation (Beckman
Microfuge) at 12,000 rpm for 5 min, and the supernatant was removed by
aspiration. The concentration of [
H]InsP
was 1 nM plus various concentrations of competing
ligands. Each assay was carried out in quadruplicate. Numbers in parentheses on the panel denote the respective isomer
of various inositol phosphate derivatives.
Figure 5:
Scatchard plots of
[H]InsP
binding to the inner nuclear
membrane (A) and to the intact nuclei (B). The K
and B
were
determined by Scatchard analysis under classical binding conditions, i.e. by the use of progressively increasing concentrations of
[
H]InsP
. These experiments were
performed on two independent nuclear preparations in quadruplicate,
with replicates varying by <10%.
Figure 6:
Selective displacement of
[H]InsP
binding to the outer (A) and inner (B) nuclear membrane. The purified
membranes were used, and the binding assay was carried out in an
Eppendorf tube for 10 min at 0 °C (on ice) in a final volume of 400
µl. Details of binding conditions are described under
``Experimental Procedures.'' Nonspecific binding was
determined in the presence of 10 µM nonradioactive
inositol phosphates. Bound and free radioligands were separated by
centrifugation. [
H]InsP
concentration
was 1 nM.
Figure 7:
Scatchard plots of
[H]InsP
binding to the outer (A) and inner (B) nuclear membrane. These experiments
were carried at progressively increasing concentrations of
[
H]InsP
. Two independent membrane
preparations were used for this study. Each experiment was done in
quadruplicate, with replicates varying by
<10%.
Figure 8:
Western blot of
Ca-ATPase. Isolated nuclear membranes, inner (IM) and outer (OM), were subjected to SDS-PAGE with
8% polyacrylamide and transferred to nitrocellulose membrane. Antiserum
raised against endoplasmic reticulum Ca
-pump ATPase
(a kind gift from Dr. F. Wuytack, Leuven, Belgium) was used. The
immunocomplex was revealed by alkaline phosphatase Misty Purple
reagent. 10 µg of protein was loaded on each
lane.
Figure 9:
Inositol phosphate selectivity for nuclear Ca
uptake. Isolated nuclei were
incubated at 37 °C for 5 min in the presence of various inositol
phosphates at their indicated concentrations. Calcium chloride was
supplemented into the medium bathing nuclei so as to give 1 µM free calcium concentration according to Fabiato(34) .
Traces of
Ca
(2.0 µCi/ml; 1 Ci
= 37 GBq) were present. Calcium uptake was terminated by
filtering under vacuum over GF/B Whatman glass fiber filters, followed
by rapid rinsing with 3 ml of ice cold medium. Filters were transferred
to 5-ml Biofluor liquid scintillation vials, and the
Ca
trapped on the filters was determined
by spectrometry. Each experiment was carried out in quadruplicate.
Inositol phosphates were: 2,
InsP
(1, 3, 4, 5) , 100
nM; 3, InsP
(3,4,5,6), 100 nM; 4, InsP
, 100 nM; 5,
InsP
, 100 nM; 6, InsP
(2, 4
5), 5 µM; 7, InsP
(2,4,5), 100
nM; 8, InsP
(1,4,5), 5 µM; 1 is control.
Figure 10:
Ca
transport to the intact nuclei. Nuclei were incubated with either
1 mM ATP or 100 nM InsP
, and the amount
of
Ca
transported was determined as
described to the legend to Fig. 9. External free calcium
concentration, as indicated was maintained.
Figure 11:
Ca
movement in the inner space of inner nuclear membrane. The free
calcium concentration was maintained at 1 µM. InsP
(A) action on calcium flux was instantaneous and
transient. Heparin (10 µg/ml) inhibited InsP
-mediated
calcium entry. Ionomycin (B) could transport robust and
transient
Ca
. The concentration of
InsP
was 5 µM and that of ionomycin 2
µM. The final volume of each assay was 500 µl, and the
temperature was 37 °C. Details of calcium measurement are described
under ``Experimental Procedures.''
Ca
movement after a given time was
defined as the radioactivity at given time subtracting the
radioactivity at time 0. These experiments were carried out on four
independent inner nuclear membrane preparations in quadruplicate, with
replicates varying less than <10%.
It has been known for some time that calcium plays a crucial
role in nuclear function; however, precise nature of nuclear calcium
signaling has remained enigmatic. The existence of a separate and
discrete control mechanism for calcium transport across the nuclear
membrane has mainly relied on studies on isolated
nuclei(18, 19, 23, 36, 37) .
Isolation of nuclei free from mitochondrial, plasma membrane, and
microsomal contaminations has been carried out in many laboratories (23, 25, 36, 37, 38) .
InsPR(19) , InsP
R(23) , and
Ca
-ATPase (17) have been located to the
nucleus, but the distinct site of their location to the nuclear
membranes has remained elusive. Nuclear envelope (27) consists
of an outer membrane and an inner membrane, and the two nuclear
membranes, although continuous, remain nonidentical in their
surroundings, density, and protein compositions(30) . We have
succeeded in separating the two membranes of nuclear envelope, as best
as possible, without cross-contamination. Three lines of investigation
substantially favor this claim. First, the increase in NAD
pyrophosphorylase specific activity (Table 1) was associated with
the inner nuclear membrane as compared with the intact nuclei, whereas
specific activity of mannose-6-phosphatase and NADPH cytochrome c reductase was enhanced in the case of purified outer nuclear
membrane. Considering the percentage of total activity of NAD
pyrophosphorylase, about 26% of liver homogenate activity was seen with
isolated nuclei and about 6% was found with the inner membrane (Table 1). NADPH cytochrome c reductase activity (a
marker for microsomes) was less than 2% with the intact nuclei and was
almost negligible with the inner nuclear membrane. A differential
distribution of the three marker enzymes associated with the purified
inner and outer nuclear membranes attest to their separate entity. This
was further confirmed by Western blotting analysis with antilamin
antiserum (Fig. 1). The outer nuclear membrane showed no
immunoreactive protein bands with antilamin antiserum. Immunoreactivity
with antilamin antibody was only manifested with the inner nuclear
membrane. A third line of evidence for the observed difference between
inner and outer membrane is attested by electron microscopic
investigation (Fig. 2). The outer nuclear membrane constituted
membranous sheath like structures (Fig. 2C), whereas
the inner nuclear membrane was well defined vesicular structures. Based
on marker enzyme activity, Western blot analysis, and electron
microscopic studies, we have reasons to propose that the inner and the
outer membranes of the nuclear envelope were separated and purified, as
best as experimentally feasible, free from contaminations from one
membrane to the other. We are also tempted to postulate that NAD
pyrophosphorylase activity, initially reported as a nuclear marker
enzyme activity(22, 32) , may serve as a marker for
the inner nuclear membrane.
Concerning the sidedness of the inner nuclear membrane vesicles, the immunoelectron microscopic investigations were carried out employing antilamin antiserum with colloidal gold. Lamins were observed toward the inner side of the inner membrane, indicating that the inner nuclear membrane, as isolated here, were right-side-in. Another line of support for the intactness as well as right-side-in inner membrane vesicles is derived from the observation that colloidal gold immunoreactivity was not seen with the intact membrane preparation. This may be attributed to inaccessibility of antibody to the lamins lying on nuclear lamina. The lamins could only be identified when inner membranes were treated with low salt medium, which rendered the antilamin antibody accessible to nuclear lamina, a site for lamin attachment.
Specific
[H]InsP
binding was associated with
the inner nuclear membrane, which was sensitive to heparin, an
accredited inhibitor of InsP
R(39) . Of the various
inositol phosphates examined, InsP
-(1,4,5) isomer was the
most effective inhibitor of [
H]InsP
binding to the inner nuclear membrane (Fig. 4). Scatchard
analysis of saturation isotherms indicated a density of 1205 fmol/mg
protein and a high affinity (K
5.5 nM)
for these sites (Fig. 4A). The receptor density in the
inner nuclear membrane was 4 times higher as compared with the intact
nucleus (B
, 385 fmol/mg protein) without altered K
(7.0 nM). This further attests to the
efficiency of the procedure adopted for inner nuclear membrane
separation from the outer membrane. The
[
H]InsP
binding data were further
supported by Western blot analysis employing antibodies raised against
rat brain type I InsP
R (Fig. 3). A distinct 220-kDa
immunoreactive protein band was identified with the inner nuclear
membrane, which was absent from the outer membrane preparation. The
220-kDa protein band was earlier identified as nuclear
InsP
R and was a target for nuclear protein kinase C
phosphorylation(20) . It may be recalled here that the same
antibody recognized 260-kDa protein band in rat cerebellar extract or
rat microsomal fraction(20) . The location of InsP
R
to the nuclear membranes has been disputed. Patch-clamp studies
indicate its location to the outer nuclear membrane(40) . Data
documented in this paper are in agreement with the inner membrane
location of InsP
R in a confocal microscopic
study(37) . The inner membrane location of nuclear
InsP
R also found support from the study demonstrating
inhibition of nuclear vesicle fusion by blocking InsP
R
activation(41) . Lack of [
H]InsP
binding to the outer nuclear membrane (data not shown) coupled
with the absence of InsP
R immunoreactivity (Fig. 3)
provides strong argument favoring that the InsP
R is located
to the inner nuclear membrane.
Out of various inositol phosphates
tested, InsP(1, 3, 4, 5) was the most effective
and selective inhibitor of [
H]InsP
binding to the outer (Fig. 6A) or inner nuclear
membrane (Fig. 6B) preparation. The rank order of
displacement of radioligand with various inositol phosphates was:
InsP
(1,3,4,5), InsP
, InsP
,
InsP
(3,4,5,6), and InsP
. The Scatchard analysis
of saturation isotherms of the InsP
binding sites located
to the outer membrane (Fig. 7A) gave K
of 7.06 nM and B
of 3.6 pmol/mg
protein, corresponding to the high affinity binding site observed with
the intact nuclei(23) . The K
value was a
bit higher than the K
(1.55 nM) observed
with the intact nucleus. This may be attributed to the sodium citrate
treatment and consequent formation of membranous sheathlike
preparation. Likewise, the Scatchard analysis of the InsP
binding sites to the inner nuclear membrane gave a K
of 60.6 nM and B
value of 13.3 pmol/mg protein. The K
value
obtained with the inner nuclear membrane corresponded with the low
InsP
binding site observed with the intact
nuclei(23) . These results show that
[
H]InsP
binding sites are located to
the outer as well as to the inner nuclear membranes, but they are not
identical either qualitatively or quantitatively. The high affinity
putative InsP
R seems to be located to the outer nuclear
membrane. We have purified (
)nuclear high affinity
InsP
R protein, which has a molecular mass of 74 kDa on
silver-stained SDS-PAGE. The purified nuclear InsP
R gave a K
of 2.3 nM and actual B
of 11.2 nmol/mg protein. Proteins of multiple molecular mass have
been purified as InsP
R from pig cerebellum(42) ,
pig platelets(43) , and rat cerebellum(44) .
Functionally, the outer nuclear membrane InsP
binding
protein intervenes the action of InsP
in nuclear calcium
transport (discussed below), whereas InsP
binding site
located to the inner nuclear membrane does not seem to be involved in
the nuclear calcium movement.
Based on the immunoreactivity
employing endoplasmic reticulum anti-Ca-ATPase
antiserum, it may be proposed that Ca
-pump ATPase is
located to the outer nuclear membrane and not to the inner nuclear
membrane (Fig. 8). The exclusive location of
Ca
-pump ATPase to the outer nuclear membrane
indicated that this enzyme is common to microsomes and nuclei. Lanini et al.(17) suggested that nuclear
Ca
-ATPase is identical to the endoplasmic reticulum
Ca
-ATPase. Recent studies (45, 46, 47) utilizing thapsigargin and
2,5-di-(tert-butyl)-1,4-benzohydroquinone have pointed out
functional differences between nucleus and endoplasmic reticulum
Ca
-ATPase.
Fig. 9depicts selective nature
of InsP(1,3,4,5) isomer in mediating
Ca
transport to the isolated intact
nuclei. Other inositol phosphates, including InsP
,
InsP
, or InsP
, did not elicit nuclear calcium
transport. InsP
- or ATP-mediated calcium transport was only
observed with the intact nucleus (23) and not with the inner
nuclear membrane (data not shown). This has further provided support to
the location and function of Ca
-ATPase and
InsP
R to the outer nuclear membrane.
Having established
the location of Ca-ATPase and InsP
R to
the outer nuclear membrane and accepting the role of ATP and InsP
in calcium transport to the isolated nuclei, the pertinent
question addressed in this study was to distinguish between
ATP-mediated and InsP
-mediated calcium transport to the
nucleus (Fig. 10). According to the data depicted in Fig. 10, the external free calcium concentration in the medium
bathing the nuclei is one of the factors that determines the mode of
transport of calcium to the nucleus. For instance, free calcium
concentration below 0.1 µM may serve as a switch-on
mechanism for the Ca
-ATPase pathway by which ATP
intervenes calcium entry into the nucleus. This pathway appears to be
short circuited when the calcium levels exterior to the nucleus rises
above 1 µM. The InsP
-mediated nuclear calcium
transport is favored above 0.8 µM free calcium levels, and
calcium levels above 1 µM or up to 5-10 µM seemed effective for InsP
-mediated calcium transport
to the nucleus. Thus, it may be suggested that the mode of calcium
transport to the nucleus depends on the calcium concentration in the
external medium, which in the context of cell would account to
cytosolic calcium variations(14, 48, 49) . We
do not know if InsP
R acts as an InsP
-gated
calcium channel(50, 51) . We are tempted to speculate
that InsP
may orient cytosolic calcium waves to the
nucleus.
The inner nuclear membrane can serve as a model system to
address the role of InsP-mediated nuclear calcium
transients since InsP
R is located to this membrane. In fact
data are provided here demonstrating that InsP
-induced
calcium release was primarily directed toward the inner nuclear space.
The action of InsP
was instantaneous, transient, and
sensitive to heparin (Fig. 11A). In the absence of
InsP
, no meaningful calcium movement was observed,
vindicating the idea the inner membrane may serve as a barrier to free
calcium diffusion. Calcium was also transported to the inner nuclear
space by the action of ionomycin. The action of ionomycin was also
instantaneous and 5 times greater than that of InsP
. To
quantify the concentration of calcium transported by InsP
or ionomycin, we determined the diameter (2.0 ± 0.23
µm), volume (4.18 ± 1.6 µm
), and the number
of inner membrane vesicles (100
10
)/mg of protein.
Based on these values, in our experimental conditions, 1000 nmol of
calcium uptake/mg of protein was equivalent to 2.4 µM
(considering the added calcium concentration in the medium 2.08
mM). Accordingly, InsP
was able to deliver calcium
into the inner nuclear space to a tune of 2 µM. Likewise,
ionomycin was capable of transporting 10 µM calcium to the
inner nucleus space. Here the action of InsP
was sensitive
to heparin(39) , which is additional evidence for the
functional localization of InsP
R to the inner nuclear
membrane. The transient nature of calcium movement into the inner
nuclear space tempts us to speculate that the calcium diffuses out of
nucleus through the nuclear pores.
In conclusion, we present a
mechanistic model for calcium movement in and out of the nucleus.
Ca-ATPase and InsP
R are located to the
outer nuclear membrane and thus intervene in the action of ATP and
InsP
, respectively, in eliciting calcium transport to the
nuclear envelope. Calcium can be further released to the inner nuclear
space by the activation of InsP
R (located to the inner
membrane) by InsP
. Calcium can diffuse out from the
nucleoplasm by the nuclear pores. This model presupposes the generation
of InsP
within the nucleus, which awaits further studies,
although the machinery to generate InsP
is present within
the nucleus(21) . Studies have begun to discern that InsP
is capable of controlling calcium within the
nucleus(52) .