Cyclic ADP-ribose activates caffeine-sensitive calcium
channels from sea urchin egg microsomes
Claudio F.
Pérez1,
Juan José
Marengo1,
Ricardo
Bull1, and
Cecilia
Hidalgo1,2
1 Instituto de Ciencias
Biomédicas, Facultad de Medicina, Universidad de Chile, and
2 Centro de Estudios
Científicos de Santiago, Santiago 9, Chile
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ABSTRACT |
Adenosine 5'-cyclic diphosphoribose [cyclic
ADP-ribose (cADPR)], a metabolite of
NAD+ that promotes
Ca2+ release from sea urchin egg
homogenates and microsomal fractions, has been proposed to act as an
endogenous agonist of Ca2+ release
in sea urchin eggs. We describe experiments showing that a microsomal
fraction isolated from Tetrapigus
nyger sea urchin eggs displayed
Ca2+-selective single channels
with conductances of 155.0 ± 8.0 pS in asymmetric
Cs+ solutions and 47.5 ± 1.1 pS in asymmetric Ca2+ solutions.
These channels were sensitive to stimulation by
Ca2+, ATP, and caffeine, but not
inositol 1,4,5-trisphosphate, and were inhibited by ruthenium red. The
channels were also activated by cADP-ribose in a
Ca2+-dependent fashion. Calmodulin
and Mg2+, but not heparin,
modulated channel activity in the presence of cADP-ribose. We propose
that these Ca2+ channels
constitute the intracellular
Ca2+-induced
Ca2+ release pathway that is
activated by cADP-ribose in sea urchin eggs.
calcium release channels; ryanodine receptors; intracellular
calcium; oocytes; endoplasmic reticulum; calmodulin; intracellular
channels
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INTRODUCTION |
RELEASE OF Ca2+
from intracellular stores has a central role in egg development after
fertilization (19, 30). Consequently, the characterization of the
physiological agonists that elicit Ca2+ release in oocytes is a
highly relevant topic that is the subject of active investigation. In
the case of sea urchin eggs, three independent
Ca2+ release pathways, sensitive
to inositol 1,4,5-trisphosphate
(IP3), ryanodine, or nicotinic
acid adenine dinucleotide phosphate, have been described (5, 7, 12,
21). In addition, adenosine 5'-cyclic diphosphoribose (cADPR)
promotes Ca2+ release in sea
urchin egg homogenates and microsomes (14, 23, 25). Agents that inhibit
Ca2+ release through the ryanodine
receptors-Ca2+ release channels
(RyR-channels) of sarcoplasmic reticulum (SR) block cADPR-induced
Ca2+ release in oocytes, and
agonists of Ca2+ release from SR,
such as Ca2+, caffeine, and
ryanodine, also release Ca2+ from
sea urchin egg homogenates (12, 14). High concentrations of caffeine or
ryanodine, by depleting the same stores, desensitize the releasing
activity of cADPR in intact eggs and in egg homogenates. Subthreshold
concentrations of caffeine or ryanodine, which enhance the releasing
effects of subsequent doses of these agonists, also enhance the
releasing effect of subthreshold doses of cADPR and vice versa (3, 12,
20).
These similarities between the properties of the cADPR- and
ryanodine-sensitive Ca2+ release
pathways indicate that sea urchin eggs have a common Ca2+-induced
Ca2+ release route sensitive to
caffeine, cADPR, and ryanodine. Accordingly, cADPR has been proposed to
act as an endogenous agonist in sea urchin eggs (9, 14, 25), and it may
act as an agonist of Ca2+ release
in a variety of other cells as well (26). However, stimulation of
Ca2+ release by cADPR in sea
urchin eggs may involve at least two other proteins, besides the
putative ryanodine receptors, that change their affinity toward cADPR
in the presence of caffeine (29), suggesting that cADPR probably acts
indirectly on the sea urchin egg putative RyR-channels. Furthermore,
there is no information, to our knowledge, of the properties at the
single channel level of the Ca2+
release channels through which cADPR-activated release takes place in
sea urchin eggs.
We have isolated a microsomal fraction from sea urchin eggs, and using
planar lipid bilayers, we have investigated the presence of
cADPR-sensitive Ca2+ channels in
the isolated microsomes. Single channel experiments revealed channels
in the microsomes that were activated by
Ca2+, ATP, caffeine, and cADPR.
The activation of the channels by cADPR was modulated by
Ca2+, calmodulin, and
Mg2+ and was blocked by ruthenium
red.
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EXPERIMENTAL PROCEDURES |
Isolation of sea urchin egg microsomes.
Eggs were collected from seawater after electrostimulation of sea
urchins (Tetrapigus nyger) into
their coelomic cavity at room temperature. All subsequent steps were
done at 4°C. After removal of jelly coats (10), eggs were washed
once in Ca2+-free artificial
seawater. This first wash was followed by two brief low-speed
sedimentations in sucrose buffer (0.3 M sucrose, 0.1 M KCl, 20 mM
3-(N-morpholino)propanesulfonic acid, pH 7.0, plus protease
inhibitors: 10 µg/ml leupeptin, 2 µg/ml pepstatin A, 5 mM
benzamidine, 100 µg/ml soybean trypsin inhibitor). Eggs were
homogenized in a Dounce homogenizer and sedimented at 2,000 g for 20 min, and the resulting
supernatant was sedimented at 12,000 g
for 20 min. The pellet was discarded, and the supernatant was
sedimented at 100,000 g for 60 min. To
remove the egg pigment echinochrome, the
100,000-g pellet was resuspended in
sucrose buffer to a protein concentration of ~10 mg/ml and
fractionated in an agarose column (Bio-Gel A-0.5 m, Bio-Rad)
equilibrated with sucrose buffer. The column exclusion fraction,
containing the microsomes, was sedimented at 100,000 g for 60 min, and the pellet was
resuspended in sucrose buffer, quickly frozen in liquid nitrogen, and
stored at
80°C.
Ca2+ release
studies.
Ca2+ release was measured in
microsomes actively loaded with
Ca2+. For this purpose the
isolated sea urchin egg microsomes were added at a protein
concentration of 0.5 mg/ml to a fluorometer cuvette containing 1 ml of
a solution of 1 mM Mg-ATP, 8 mM phosphocreatine, 4 U/ml of creatine
kinase, 0.1 M KCl, and 20 mM 3-(N-morpholino)propanesulfonic acid-tris(hydroxymethyl)aminomethane (Tris), pH 7.2. To measure the
concentration of free Ca2+
([Ca2+]), 0.5 µM
fluo 3 was added to this solution, and fluo 3 fluorescence was
determined using a Shimadzu RF-540 spectrofluorometer with excitation
and emission wavelengths of 506 and 526 nm, respectively. Because after
vesicle addition, external
[Ca2+] reached
5-10 µM, no extra Ca2+ was
added to the incubation solution. After 30 min of incubation with
Mg-ATP at 22°C, external
[Ca2+] decreased to
0.3-0.4 µM. At this point, different agonists of Ca2+ release channels were added,
and the changes in fluo 3 fluorescence were followed as a function of
time. Calibration curves of fluo 3 fluorescence vs.
[Ca2+] were done using
solutions of known
[Ca2+], which was
determined with a Ca2+ electrode.
Bilayer experiments.
Planar phospholipid bilayers were formed from 5:3:2 palmitoyloleoyl
phosphatidylethanolamine-phosphatidylserine-phosphatidylcholine. Fusion
of vesicles to negatively charged Muller-Rudin membranes was performed
as described previously (4), with slight modifications. Sea urchin egg
microsomes were added to the cis
compartment solution, containing 5 ml of 200 mM CsCl, 0.1 mM
CaCl2, and 25 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-Tris, pH 7.4. The other
(trans) compartment contained 25 mM
HEPES-Tris, pH 7.4. After channel fusion, evidenced by the emergence of
current fluctuations a few minutes after the addition of the
microsomes, the cis compartment was
perfused with five times the compartment volume (a total volume of 25 ml) of a solution containing 25 mM HEPES-Tris, pH 7.4. After perfusion,
200 mM Cs-methanesulfonate, 0.5 mM Ca-HEPES, and sufficient
N-hydroxyethylethylenediaminotriacetic acid or ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid to give the desired free
[Ca2+], which was
always checked with a Ca2+
electrode, were added to the cis
compartment. The trans compartment was
supplemented with 50 mM Cs-methanesulfonate after fusion. Voltage was
applied to the cis compartment, and
the trans compartment was held at
virtual ground through an operational amplifier in a current-to-voltage
configuration. Unless indicated otherwise, most single channel records
illustrated here were obtained in asymmetric Cs-methanesulfonate
solutions (200 mM cis/50 mM
trans) at room temperature
(22-24°C), with membranes held at 0 mV. For analysis, data
were filtered at 0.5 kHz using an eight-pole low-pass Bessel filter
(model 902 LPF, Frequency Devices, Haverhill, MA) and subsequently
digitized at 2 kHz with an acquisition system (Axotape, Axon
Instruments, Foster City, CA).
Because the channels studied displayed four conductance levels, plus
rapid and complex kinetics, the normalized mean current (P*o) was used as an index
of channel activity and was calculated as follows
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(1)
|
where
Imax is the
channel maximal current amplitude and
Imean is the mean
current amplitude. Mean channel current and fractional times spent in
each subconductance state
(Pi) were
always calculated from steady-state records lasting
150 s. pClamp 6.0 (Axon Instruments) was used for the former calculations, and Transit
software (Baylor College of Medicine, Houston, TX) was used for the
latter. Furthermore, to establish that the four equal current levels
observed corresponded to true subconductance states and not to four
separate single channels, the following analysis was performed. For a
binomial system made of four independent units, the frequency of
occurrence of each conductance level is given by the following set of
equations
|
(2)
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(3)
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(4)
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(5)
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(6)
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In Eqs. 2-6,
Po corresponds to
the open probability of each unit and
Pc to the
probability of the closed state. To calculate Po,
Eq. 7 was used
|
(7)
|
From the calculated
Po the binomial
frequencies for the open conductance states were estimated, and the
resulting theoretical frequency values were compared with the
experimental distribution of frequencies using a
2 test.
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RESULTS |
IP3- and cADPR-induced
Ca2+ release
from sea urchin egg microsomes.
The sea urchin egg microsomes isolated according to the procedures
described above actively accumulated
Ca2+ at 22°C after addition of
1 mM Mg-ATP (not shown). The uptake of
Ca2+ ceased after 20-30 min
of incubation, when the
[Ca2+] of the
extravesicular solution reached 0.3-0.4 µM. At this point, addition of 5 µM IP3 to the
actively loaded vesicles produced transient
Ca2+ release, and subsequent
addition of 1 µM cADPR induced a new transient
Ca2+ release of smaller magnitude
(Fig.
1A).
The inverse sequence of agonist addition, first cADPR and then
IP3, produced a transient Ca2+ release after addition of
cADPR and a second release response to 5 µM
IP3 smaller (Fig.
1B) than the release response
obtained when 5 µM IP3 was added
before cADPR (Fig. 1A). These
results indicate that the isolated microsomes have release pathways
responsive to IP3 and cADPR.
Addition of the Ca2+ ionophore
A-23187 at the end of the experiment produced in all cases a marked
increase in extravesicular
[Ca2+] (Fig. 1). This
response to the ionophore indicated that the vesicles had actively
accumulated significant amounts of
Ca2+ and that the increase in
external [Ca2+]
observed after addition of cADPR or
IP3 corresponded in effect to
Ca2+ release through pathways in
the microsomal membranes.

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Fig. 1.
Effect of successive additions of inositol 1,4,5-trisphosphate
(IP3) and adenosine
5'-cyclic diphosphoribose (cADPR) on
Ca2+ release from sea urchin egg
microsomes. Free Ca2+
concentration ([Ca2+])
in extravesicular solution was monitored by measuring fluo 3 fluorescence. Before induction of
Ca2+ release, vesicles were
actively loaded with Ca2+ by
incubation for 30 min at 22°C with 1 mM Mg-ATP.
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Sea urchin egg microsomes displayed caffeine-sensitive
Ca2+ channels.
Using planar lipid bilayers, we investigated the presence of
Ca2+ channels in the isolated
microsomes. For this purpose, we used experimental conditions similar
to those routinely employed to detect ryanodine-sensitive
Ca2+ channels in SR vesicles, such
as pH 7.4 and high concentrations (up to 50 mM) of cations,
Cs+ or
Ca2+, in the
trans compartment (see
EXPERIMENTAL PROCEDURES). In the standard recording conditions (200 mM
Cs+
cis/50 mM
Cs+
trans) and in the presence of 12 µM Ca2+ in the
cis solution, only single channels
that exhibited
Imax of
4-4.2 pA, with a low
P*o of 0.007, were
observed (Fig. 2A, top
trace). Channel activity increased to a
P*o of 0.041 after
addition of 2 mM caffeine to the cis
solution (Fig. 2A, bottom trace).
Consistent channel activation by caffeine was observed in all single
channels tested.

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Fig. 2.
Single channel records showing channel activation after addition of
caffeine (A) or cADPR
(B) to
cis solution. Presence of 4 equal
subconductance states (S1-S4)
is shown in detail in bottom traces in
B, plotted with a time scale amplified
5-fold. P*o, normalized
mean current.
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In contrast to the consistent activation of channel activity by
caffeine, not all channels were consistently activated by ryanodine.
After addition of 50-100 µM ryanodine over 12 µM
Ca2+ in the
cis solution, a distinct increase in
channel Po was
observed only in three of seven single channel experiments. Also, all
our attempts to measure specific
[3H]ryanodine binding
to sea urchin egg microsomes were unsuccessful. For these reasons, we
have not included the effects of ryanodine on single channel activity
in this report.
Effect of cADPR on single
Ca2+ channel
activity.
Addition of cADPR to the cis solution
containing 12 µM Ca2+ produced
more stimulation of channel activity than 2 mM caffeine. After
cis addition of 2 and 5 µM cADPR,
channel activity increased from a basal
P*o of 0.006 to a
P*o of 0.039 and 0.125, respectively (Fig. 2B). At this
level of activity it became evident that cADPR-activated channels
exhibited a complex behavior. Channel activity presented bursting
kinetics, and fluctuations among a single closed state and four
near-equal subconductance open states were clearly discernible with an
amplified time scale (Fig. 2B, bottom
traces). Subconductance states, observed as well in
native ryanodine-sensitive Ca2+
channels from SR (1), were consistently observed in all channels studied. Several channel experiments
(n = 4) revealed that, in the presence
of 12 µM Ca2+ in the
cis solution,
P*o increased as a
function of cADPR concentration, reaching 0.095 ± 0.011 at 5 µM
cADPR, the highest concentration tested. Significant channel activation
over the control condition became noticeable at
0.5 µM cADPR (Fig. 3A, filled
symbols).

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Fig. 3.
A: effect of cADPR concentration
([cADPR]) in cis solution
on channel activity. Effect of increasing [cADPR] on
channel normalized mean current
(P*o, ) was evaluated
in several single channel experiments
(n = 4) in which single channel
activity was recorded in asymmetric Cs-methanesulfonate solutions (200 mM cis/50 mM
trans). In 1 single channel
experiment ( ), effect of cADPR was recorded in a higher
Cs-methanesulfonate gradient (300 mM
cis/50 mM
trans).
Imean and
Imax, mean and
maximal current amplitudes. B: effect
of [cADPR] in cis solution
on fractional time spent in each subconductance state
(Pi). Single
channel activity was recorded in asymmetric Cs-methanesulfonate
solutions (300 mM cis/50 mM
trans). When this higher
Cs-methanesulfonate gradient was used, data were filtered at 2 kHz and
digitized at 10 kHz. Inset: relative
distributions of all
Pi values. Four
subconductance states (1-4) are
labeled in order of increasing current.
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To analyze in more detail the effects of cADPR on the distribution of
subconductance open states, an experiment was performed using a higher
Cs-methanesulfonate gradient (300 mM
cis/50 mM trans) to increase channel current
and thus increase the signal-to-noise ratio of the experimental
records. An increase in
P*o by cADPR as low as 0.2 µM was observed when 300 mM Cs+
was used in the cis solution (Fig.
3A, open circles). This observation suggests that increasing Cs+ in
the cis solution makes the channels
more sensitive to stimulation by cADPR and is consistent with previous
reports showing that the effect of agonists on mammalian
ryanodine-sensitive Ca2+ channels
is modulated by ionic strength (8, 31). In these conditions, a
comparison of the fractional time spent in each subconductance state
(Pi) for the
control and in the presence of two different concentrations of cADPR,
0.2 or 0.5 µM, revealed that cADPR increased channel activity by
increasing all individual Pi values (Fig.
3B). However, as reflected in the
percent distribution of the states (Fig. 3B,
inset), the highest conductance state seemed to be
somewhat more prevalent in the presence of cADPR. Further analysis of
these experimental records in terms of the frequencies for the open
conductance states (see EXPERIMENTAL PROCEDURES) showed that they did not follow the
pattern expected for a binomial distribution. In fact, as shown in
Table 1, a comparison of the experimentally
determined open substate frequencies (data of Fig.
3B) with those predicted by
Eqs. 2-6 revealed that in all
these cases the
2 test detected
a significant difference between both frequencies. These findings
indicate that the four current levels recorded in cADPR-activated
channels are not due to a complex formed by four independent single
channels.
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Table 1.
Comparison between experimental and predicted binomial frequencies for
different conductance states of a cADPR-activated channel
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Channels recorded in asymmetric
Ca2+ gradients (12 µM
Ca2+
cis/43 mM
Ca2+
trans) were likewise activated by
cis addition of 1 µM cADPR (Fig.
4A).
Because 43 mM Ca2+, the
concentration present in the trans
compartment in these experiments, effectively blocks SR
ryanodine-sensitive Ca2+ channels
at the cytoplasmic surface (8, 31), the observed channel activity most
likely corresponds to channels fused with their cytosolic side oriented
toward the cis compartment.

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Fig. 4.
A: current records showing 2 representative cADPR-activated channels conducting
Ca2+ or
Cs+. Single channel activity was
recorded in asymmetric Ca-HEPES solutions (12 µM
cis/43 mM
trans) or asymmetric
Cs-methanesulfonate solutions (200 mM
cis/50 mM
trans).
B: current-voltage
(Imax-V)
plots for channels recorded in asymmetric Cs-methanesulfonate solutions
(200 mM cis/50 mM
trans; ;
n = 4) or asymmetric Ca-HEPES
solutions (12 µM cis/43 mM
trans; ;
n = 2). Values are means ± SE.
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A plot of maximal current values vs. voltage, obtained in channels
activated by cis addition of
micromolar cADPR in 12 µM Ca2+,
revealed oocyte channel conductances of 47.5 ± 1.1 pS in asymmetric Ca2+ solutions and 155.0 ± 8.0 pS in asymmetric Cs+ solutions
(Fig. 4B). The displacement of the
Imax-voltage
curves toward the right after trans
addition of Ca2+ to channels
recorded in Cs+ (data not shown)
implies that the channels were more selective for
Ca2+ than for
Cs+. The higher selectivity for
Ca2+ than for monovalent cations
such as Cs+,
K+, or
Na+ is a well-known feature of
mammalian ryanodine-sensitive Ca2+
channels (8). For practical reasons, because higher currents were
obtained in Cs+, making readily
visible the different subconductance states and increasing the
differences between the lowest subconductance state and baseline noise
(as shown in Fig. 4A), all
subsequent channel experiments were done in asymmetric
Cs+ solutions (200 mM
cis/50 mM
trans).
Lack of effect of IP3 on channel
activity.
Addition of 2 µM IP3 to the
cis solution containing 12 µM
Ca2+ had no effect on channel
activity and did not interfere with subsequent channel activation by 5 µM cADPR (Fig. 5A,
middle traces). In contrast, decreasing
[Ca2+] in the
cis solution from 12 to 0.72 µM in
the continuous presence of 2 µM
IP3 and 5 µM cADPR produced a
significant reduction in channel activity, decreasing
P*o from 0.115 to 0.005 (Fig. 5A, bottom
trace). This decrease in channel activity, which took
place when [Ca2+] in
the cis solution was lowered to levels
that optimize the Ca2+ release
response to IP3 in sea urchin egg
homogenates (5), indicates that these cADPR-activated channels are not
responsive to IP3. In addition,
this reduction implies that stimulation by cADPR requires >0.72 µM
Ca2+ in the
cis solution, as reported in further
detail below.

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Fig. 5.
A: single channel records showing lack
of channel activation by cis addition
of 2 µM IP3 and subsequent
stimulation of channel activity by cis
addition of 5 µM cADPR. Stimulatory effect of cADPR disappeared after
cis
[Ca2+] was lowered to
0.7 µM. B: single channel records
showing stimulation of channel activity by
cis addition of 2 µM cADPR and lack
of further stimulation by subsequent
cis addition of 4 µg/ml calmodulin
(CaM); channel activity decreased markedly after
cis addition of 20 µM ruthenium
red.
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Effects of calmodulin, ruthenium red, and heparin on channel
activity.
In a separate experiment, addition of 4 µg/ml (0.24 µM) calmodulin
to a channel already activated by 2 µM cADPR in 12 µM
cis Ca2+ did not modify channel
activity (Fig. 5B, 3rd trace). Yet,
further addition of ruthenium red produced a marked inhibition,
lowering P*o from 0.063 to
0.005 (Fig. 5B, bottom trace).
Likewise, no effect of cis addition of
calmodulin on the activity of cADPR-activated channels was observed
when the [Ca2+] of the
cis compartment was lowered to 0.72 µM (records not shown). Because cADPR-activated
Ca2+ release in sea urchin egg
microsomes has an absolute requirement for calmodulin (22, 23, 27),
endogenous calmodulin may be associated with the channels fused in the
bilayers. That this is most likely the case is shown by the experiments
described below.
Addition of heparin to the cis
solution containing 12 µM Ca2+
plus 5 µM cADPR had a marginal stimulatory effect on channel activity and did not interfere with subsequent channel inhibition by ruthenium red. Thus channels (n = 2) that, after
addition of 5 µM cADPR to the cis
solution containing 12 µM Ca2+,
increased their activity from a
P*o of 0.010 ± 0.001 to 0.083 ± 0.026, on addition of 300 µg/ml heparin increased
their P*o only to 0.125 ± 0.019. This small increase in channel activity brought about by
300 µg/ml heparin was not statistically significant. Further
increasing heparin to 600 µg/ml had a negligible effect on channel
activity, as reflected by a
P*o of 0.127 ± 0.028. Subsequent addition of 30 µM ruthenium red decreased channel activity
to a P*o of 0.008 ± 0.004.
Ca2+
dependence of channel activity.
In the absence of cADPR, varying cis
[Ca2+] from 0.7 to 270 µM had a modest effect on single channel activity (Fig.
6, open circles);
P*o increased from
<0.003 in 0.7 µM Ca2+ to
~0.01 in 12 µM Ca2+. Channel
P*o remained constant at
12-30 µM cis
Ca2+. Further increasing
[Ca2+] to 270 µM
produced some decrease in
P*o, to an average of
0.006.

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Fig. 6.
Effect of cis
[Ca2+] on
P*o. Experiments were done
in absence ( ) or presence ( ) of 1 µM cADPR in
cis solution. Values are means ± SE, with number of determinations in parentheses. Lines have
no theoretical meaning.
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In contrast, in the presence of 1 µM cADPR, increasing
cis
[Ca2+] to 12 µM had
a marked stimulatory effect on channel activity (Fig. 6, filled
circles), as reflected by an increase in
P*o to ~0.035. As
observed in the absence of cADPR, channel activity remained constant at
12-30 µM cis
Ca2+. Further increasing
cis
[Ca2+] to 60 µM
reduced P*o to ~0.020.
In the experimental conditions used to record channel activity, 1 µM
cADPR did not activate the channels at
0.7 µM
Ca2+ (Fig. 6). Yet, as shown in
Fig. 1, 1 µM cADPR was effective in eliciting
Ca2+ release from sea urchin egg
microsomes actively loaded with
Ca2+ and bathed in solutions
containing ~0.4 µM Ca2+.
Furthermore, concentrations of cADPR as low as 10 nM have been reported
to activate Ca2+ release from sea
urchin egg homogenates actively loaded with Ca2+ (13). Because vesicular
Ca2+ release and bilayer
experiments were done in different conditions, such as the presence of
millimolar Mg2+ and ATP in the
external solution when vesicular release was measured, we investigated
whether cADPR stimulation of channel activity was modified by addition
of ATP or Mg2+ to the
cis solution.
Stimulation of channel activity by ATP.
Addition of 2 mM ATP to the cis
solution containing 12 µM Ca2+
produced a significant stimulation of
P*o, from 0.007 to 0.022 (Fig. 7). Subsequent addition of 0.5 µM
cADPR did not stimulate further channel activity but produced a small
reduction in channel P*o
to 0.015 (not shown). Even increasing cADPR to 2 µM in the presence
of ATP did not stimulate channel activity, because
P*o remained at 0.015 (Fig. 7, top, 3rd trace). After
extensive washing of the cis
compartment (see EXPERIMENTAL
PROCEDURES) and addition of 12 µM
Ca2+ to the
cis solution, the channel regained its
low activity, with a P*o
of 0.005. At this point, addition of 1 µM cADPR increased
P*o to 0.032 (Fig. 7,
bottom). These results indicate that
the stimulatory effect of ATP on channel activity did not add to that
of cADPR and was wholly reversible. In concordance with these results,
several experiments revealed that cis
addition of 1-2 mM ATP to cADPR-activated channels did not
stimulate further channel activity (data not shown).

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Fig. 7.
Effect of ATP on activity of a single
Ca2+ channel. Addition of 2 mM ATP
to cis solution containing 12 µM
Ca2+ produced a marked stimulation
of P*o from 0.007 to 0.022 (A,
top and middle
traces). Subsequent addition of 2 µM cADPR to
cis solution produced a small
reduction in channel P*o,
to 0.015 (A, bottom
trace). After extensive washing of
cis compartment, channel regained its
low activity in 12 µM Ca2+, with
P*o = 0.005, and was
substantially activated to a
P*o of 0.032 by further
addition of 1 µM cADPR (B).
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Stimulation of channel activity by cADPR plus
Mg2+.
To analyze the effects of Mg2+ on
cADPR-activated Ca2+ channels,
cis
[Ca2+] was held at 24 µM, a concentration that was within the range of
[Ca2+] that produced
optimal channel activation. To avoid changes in [Ca2+] after
Mg2+ addition, ethylene
glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid was used as a buffer. Addition of 1 µM cADPR to a single Ca2+ channel increased
P*o from 0.003 to 0.020 (Fig.
8A). Subsequent addition of 1 mM Mg2+
to the cis solution markedly enhanced
channel activity, increasing P*o 12.8-fold, from 0.020 to 0.256 (Fig. 8A). Addition of
Mg2+ in the absence of cADPR had
no effect on channel activity (not shown).

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Fig. 8.
Single channel records showing channel activation by
cis addition of 1 µM cADPR and
subsequent addition of 1 mM Mg2+.
Channels similarly activated by 24 µM
cis
Ca2+ plus 1 µM cADPR responded
differently after addition of 1 mM
Mg2+ to
cis solution. Channel in
A was more activated after addition of
1 mM Mg2+, with an increase in
P*o from 0.020 to 0.256, than channel in B, in which
P*o was increased from
0.012 to only 0.053.
|
|
Yet, not all channels studied displayed the same marked activation by
Mg2+. In the example of the single
channel illustrated in Fig. 8B, after
addition of 1 µM cADPR in 24 µM
cis
Ca2+, the channel increased its
activity from a P*o of
0.009 to 0.012 (Fig. 8B,
top and middle
traces). Subsequent addition of 1 mM
Mg2+ produced only a 4.4-fold
stimulation of channel activity, to a
P*o of 0.053 (Fig.
8B, bottom trace).
The difference between these two types of responses is further
illustrated in Fig. 9. All channels that
presented a more marked activation by
Mg2+ (Fig. 9, filled squares) were
clearly activated by a cADPR concentration as low as 0.1 µM, with an
apparent saturation at 1-2 µM cADPR. In contrast, the channels
less activated by Mg2+ required
>0.5 µM cADPR for discernible activation and did not seem to
saturate at a cADPR concentration as high as 2 µM (Fig. 9, open
circles). These results indicate that, in the combined presence of 24 µM cis
Ca2+ and 1 mM
Mg2+, some of the channels
markedly increased their sensitivity to activation by cADPR. This
finding suggests that one of the additional components present in
Ca2+ release experiments makes the
channels more responsive to cADPR. However, the differences in the
requirements for higher cis
[Ca2+] to observe
channel activation by cADPR in bilayers, as opposed to release
experiments, cannot be attributed to
Mg2+. When
cis
[Ca2+] was decreased
to the submicromolar range, channels were not consistently activated by
addition of cADPR, even in the presence of 1 mM
Mg2+ (data not shown).

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Fig. 9.
Effect of cis [cADPR] on
channel activity measured in presence of 1 mM
Mg2+ plus 24 µM
Ca2+ in
cis solution. Channel activity is
expressed as P*o, and
values are means ± SE, with number of determinations in
parentheses. Lines have no theoretical meaning. and , Channels
that were more or less activated, respectively, by addition of 1 mM
Mg2+ to
cis solution containing 1 µM cADPR
and 24 µM Ca2+, as described in
Fig. 8. Inset: effect of increasing
concentrations of Mg2+ on activity
of a single channel, expressed as
P*o. In addition to
variable [Mg2+],
cis solution contained 1 µM cADPR
and 24 µM Ca2+.
|
|
A study of the response of the channels less sensitive to activation by
Mg2+ showed that concentrations of
Mg2+ >1 mM were less effective
in enhancing channel activation by cADPR, and channel activity in 9 mM
Mg2+ was lower than in its absence
(Fig. 9, inset).
Imax was reduced as well by >7 mM Mg2+ (data not
shown).
Effects of calmodulin antagonists plus
Mg2+ on channel
activity.
Channels activated by cADPR were not affected by calmodulin addition
(Fig. 5), even at a concentration (4 µg/ml) that induces near full
activation of Ca2+ release in sea
urchin egg microsomes (22, 23, 27). Because calmodulin seems to be
essential for activation of Ca2+
release by cADPR in these microsomes (23), the lack of effect of
calmodulin on single channel activity may reflect the presence of
endogenous calmodulin tightly associated with the channel protein. To
test this hypothesis, we examined the effects of the calmodulin antagonist W-7 (17) on channel activity. A single channel that displayed a P*o of 0.274 in the presence of 24 µM Ca2+, 1 mM Mg2+, and 2 µM cADPR in the
cis solution (Fig.
10, top
trace) decreased its activity to a
P*o of 0.047 after
cis addition of 10 µM W-7 (Fig. 10,
middle trace). To test for the
specificity of this effect, 10 µM calmodulin was subsequently added
to the cis solution. After addition of
this large excess of calmodulin, the channel increased its
P*o to 0.223, which is
similar to that measured before W-7 addition (Fig. 10,
bottom trace). Inhibitory effects
comparable to those obtained with W-7 were observed with use of
compound R-24571 (calmidazolium), a different calmodulin antagonist
(data not shown). These results support the above hypothesis that
channels fused in the bilayer contain sufficient endogenous calmodulin
to support channel activity.

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Fig. 10.
Single channel records showing channel inhibition by calmodulin
antagonist W-7. Top trace: channel
activity measured in presence of 2 µM cADPR, 1 mM
Mg2+, and 24 µM
Ca2+ in
cis solution. Middle
trace: channel activity after addition of 10 µM W-7
to cis solution.
Bottom trace: channel activity after
addition of 10 µM calmodulin to cis
solution.
|
|
 |
DISCUSSION |
The results described here demonstrate that microsomes isolated from
T. nyger eggs contain
Ca2+ channels that were activated
by cis addition of caffeine,
micromolar Ca2+, or millimolar
ATP. Channels were also activated by
cis addition of micromolar cADPR, and
stimulation by cADPR was modulated by cis
[Ca2+] and by
Mg2+.
Comparison with mammalian RyR-channels.
Activation by caffeine, ATP, and cis
[Ca2+] in the
micromolar range (8, 11, 12) is characteristic of vertebrate
RyR-channels. Furthermore, as described for RyR-channels isolated from
mammalian skeletal muscle (11), the egg channels displayed a
bell-shaped Ca2+ dependence of
P*o in the presence of
cADPR. In addition, the egg channels were inhibited by ruthenium red
and displayed multiple subconductance states, behaviors displayed as
well by the mammalian RyR-channels (1, 8). On the basis of these similarities, it is tempting to assign the cADPR-activated channels from sea urchin eggs to the ryanodine receptor family. Although the
conductance of the cADPR-activated egg channels (50 pS) was lower than
the conductances described for RyR-channels from mammalian skeletal and
cardiac muscle (100-120 pS) (8), this difference may not be very
significant, because variations in the conductance of the mammalian
channels have been reported (2). Yet, in contrast to the well-reported
effects of ryanodine on RyR-channels (8, 31), we did not observe
consistent modulation of the sea urchin channels by ryanodine. We have
no explanation for this lack of consistent effects of ryanodine on
channel activity.
Channels activated by cADPR were not activated by
IP3.
The egg channels activated by cADPR were not
IP3-gated channels, since addition
of IP3 (2 µM) to the
cis chamber had no effect on channel
activity. However, IP3 addition to
sea urchin microsomes actively loaded with
Ca2+ caused significant
Ca2+ release, indicating that the
microsomes described in this study have release pathways responsive to
IP3. Accordingly, a
straightforward interpretation of the lack of effect of
IP3 on channel activity is that
the IP3-gated channels of sea
urchin eggs (not described so far in bilayers) are different from the
Ca2+ channels activated by cADPR
studied in this work. It is pertinent to mention in this regard that
there is significant evidence indicating that the cADPR- and
IP3-sensitive
Ca2+ release pathways of sea
urchin eggs are different. 1)
Ruthenium red and procaine, two antagonists of
Ca2+-induced
Ca2+ release in SR, inhibit
cADPR-sensitive Ca2+ release in
egg homogenates without affecting
IP3-sensitive release (12, 13).
2) Even high concentrations of
IP3 do not alter the
Ca2+-releasing activity induced by
cADPR (12). 3) cADPR-induced Ca2+ release is insensitive to
heparin (13, 24), a competitive inhibitor of
IP3 binding to its receptor.
Furthermore, the lack of effect of heparin on
Ca2+ release is in agreement with
the negligible effect of heparin on channel activity found in this
work, even at heparin concentrations that completely inhibit
IP3-sensitive
Ca2+ release.
Ca2+
dependence of channel activity in the presence of cADPR.
Sea urchin egg Ca2+ channels were
moderately activated by increasing cis
[Ca2+] from 0.7 to 30 µM, but in the presence of micromolar concentrations of cADPR,
channel activation by increasing cis
[Ca2+] in this range
became more prominent. These results agree with a recent report
describing significant enhancement of cADPR-gated Ca2+ release rates in sea urchin
eggs by increasing
[Ca2+] (16).
In the standard experimental conditions used to record channel
activity, 1 µM cADPR did not activate the channels at
0.7 µM
Ca2+. In contrast, we found that 1 µM cADPR was effective in eliciting Ca2+ release from microsomes
actively loaded with Ca2+ and
bathed in solutions containing 0.3-0.4 µM
Ca2+. These results are in
agreement with a previous report showing that cADPR induces
Ca2+ release from sea urchin egg
homogenates at submicromolar
[Ca2+] (5).
Nonetheless, it is important to note in this regard that
Ca2+ release from microsomal
vesicles actively loaded with Ca2+
is measured under experimental conditions quite different from those
prevailing in bilayer experiments. In release experiments, 1) the vesicles have actively
accumulated significant luminal Ca2+,
2) they are bathed in solutions
containing millimolar Mg-ATP, and 3)
other vesicular proteins are present that may or may not fuse with the
channels into the bilayers. Each one of these factors might in
principle enhance the effect of cADPR at submicromolar [Ca2+], since all of
them regulate the activity of mammalian RyR-channels (8, 18, 31).
However, even in the presence of 1 mM
Mg2+ channels were not
consistently activated by cADPR in submicromolar [Ca2+], making
unlikely Mg2+ by itself as the
cause of the different Ca2+
sensitivity. Whether the other factors mentioned above are responsible for the differences between bilayer and vesicular release experiments should be investigated. In particular, the effects of luminal Ca2+ should be studied, since we
have preliminary results (not shown) indicating that cADPR was more
effective in stimulating channel activity when the
trans compartment contained 50 µM
Ca2+ in addition to 50 mM
Cs+. Additionally, other factors
that enhance the effects of cADPR on
Ca2+ release from sea urchin egg
homogenates, such as the oxidation state of critical SH groups (13, 28)
or the presence of palmitoyl-CoA (6), may enhance the channel response
to cADPR at low
[Ca2+].
Effects of
Mg2+ on channel
activity.
It has been reported that 1 mM
MgCl2 is required for optimal
activation of Ca2+ release by
cADPR (12-15) and that cADPR-activated
Ca2+ release from sea urchin egg
homogenates is inhibited by millimolar concentrations of
Mg2+ (6, 15). In agreement with
these reports, our results indicate that
Mg2+ presented a dual effect over
the activity of cADPR-stimulated channels. Thus 1 mM
Mg2+ increased markedly the
stimulatory effect of cADPR on channel activity, but at higher
concentrations it was less effective and eventually became inhibitory.
Furthermore, in the presence of 1 mM
Mg2+, the sea urchin egg channels
displayed two types of responses toward activation by cADPR. Why
channels presented these two different responses is not clear and
should be the subject of future investigation.
Conclusions.
The results of this work indicate that sea urchin egg microsomes
possess Ca2+ channels that share
similar properties with vertebrate RyR channels, such as activation by
Ca2+, ATP, and caffeine and
inhibition by ruthenium red. Because activation of oocyte channels by
cADPR was enhanced by Ca2+, we
propose that these Ca2+ channels
of sea urchin eggs constitute an intracellular
Ca2+-induced
Ca2+ release pathway in these
cells.
 |
ACKNOWLEDGEMENTS |
We thank Dr. M. T. Nuñez for helpful criticism of the
manuscript.
 |
FOOTNOTES |
This study was supported by Fondo Nacional de Ciencia y
Tecnología Grants 2950037, 1940369, and
1970914, by European Community Grant CI1CT940129, and by
institutional support to the Centro de Estudios Científicos de
Santiago from a group of Chilean companies.
Address for reprint requests: C. Hidalgo, Centro de Estudios
Científicos de Santiago, Casilla 16443, Santiago 9, Chile.
Received 16 June 1997; accepted in final form 20 October 1997.
 |
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