ADP-ribose gates the fertilization channel in ascidian
oocytes
Martin
Wilding1,
Gian Luigi
Russo2,
Anthony
Galione3,
Marcella
Marino1, and
Brian
Dale1
1 Stazione Zoologica "Anton
Dohrn," 80121 Naples;
2 Institute of Food Science and
Technology, 83100 Avellino, Italy; and
3 University Department
of Pharmacology, Mansfield Road, Oxford OX1 3QT, United Kingdom
 |
ABSTRACT |
We report an ion
channel in the plasma membrane of unfertilized oocytes of the ascidian
Ciona intestinalis that is directly gated by the second messenger ADP-ribose. The ion channel is permeable to Ca2+ and
Na+ and is
characterized by a reversal potential between 0 and +20 mV and a
unitary conductance of 140 pS. Preinjection of the
Ca2+ chelator
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) or antagonists of intracellular
Ca2+ release channels into oocytes
did not inhibit the ADP-ribose current, demonstrating that the channel
is activated in a Ca2+-independent
manner. Both the fertilization current and the current induced by the
injection of nicotinamide nucleotides are blocked by nicotinamide,
suggesting that the ADP-ribose channel is activated at fertilization in
a nicotinamide-sensitive manner. These data suggest that ascidian sperm
trigger the hydrolysis of nicotinamide nucleotides in the oocyte to
ADP-ribose and that this mechanism is responsible for the production of
the fertilization current.
nicotinamide nucleotides; electrophysiology
 |
INTRODUCTION |
TWO OF THE FIRST EVENTS of fertilization are the
generation of an ion current across the oocyte plasma membrane (20) and a transient increase in intracellular
Ca2+ (9, 29). In many species
including echinoderms, amphibians, and mammals, the fertilization
current is triggered by the release of intracellular
Ca2+ (7, 16, 21). A notable
exception, however, is the ascidian, where the fertilization current is
Ca2+ independent (4, 5, 26). The
ion channel responsible for the ascidian fertilization current is a
large, nonspecific ion channel with a reversal potential of about +20
mV (5). Although the physical properties of this channel are well
characterized, the physiological trigger of the channel is not known
(4, 26).
One of the early events stimulated at fertilization is the metabolism
of nicotinamide nucleotides (10, 28). One such metabolite, cyclic
ADP-ribose (cADPR), has been shown to behave as a potent Ca2+-mobilizing enzyme in some
systems (12, 18). A second metabolite, ADP-ribose, is much less well
known as a second messenger and is thought to be involved uniquely in
signaling through nonenzymatic ADP ribosylation (25). In this
manuscript, we show that the plasma membrane of Ciona
intestinalis oocytes contains an ion channel that is
gated by ADP-ribose. Furthermore, our data suggest that this channel is
the previously characterized "fertilization channel" (5). An
inhibitor of nicotinamide nucleotide breakdown blocks both currents
induced by nicotinamide nucleotide injection and the fertilization
current, suggesting that ascidian sperm induce the fertilization
current by stimulating the breakdown of nicotinamide nucleotides to
ADP-ribose.
 |
MATERIALS AND METHODS |
Collection and preparation of oocytes.
Oocytes were dissected from the ascidian C. intestinalis collected from the Bay of Naples and kept
in tanks with running seawater until use. Oocytes were manually
dechorionated using steel needles and placed in an injection chamber
containing 2 ml of natural filtered seawater from the Bay of Naples.
Fragments were prepared by cutting ascidian oocytes with steel needles
on extrusion through the chorion. The sizes of fragments were measured,
and fragments with a diameter of 20 µm were used for the experiments.
Ca2+-free seawater (0 Ca2+) contained (in mM) 500 NaCl, 10 KCl, 50 MgSO4, 2.5 NaHCO3, and 10 EGTA, pH 8.0. Constituents of low-Na+ seawater
(0 Na+) were (in mM) 5 NaCl, 495 choline chloride, 10 KCl, 10 CaCl2, 25 MgSO4, 25 MgCl2, 2.5 NaHCO3, and 10 HEPES, pH 8.0. High-Ca2+ seawater contained (in
mM) 370 NaCl, 27 MgCl2, 28 MgSO4, 2.5 NaHCO3, 100 CaCl2, 10 KCl, and 1 EDTA, pH
8.0.
Microinjection and electrophysiological
techniques. Standard patch pipettes of 2 µm diameter
and 10 M
resistance were used for both microinjection and
electrophysiology. Pipettes were backfilled with reagents dissolved in
an intracellular solution (ICS) containing (in mM) 200 K2SO4,
20 NaCl, and 10 HEPES, pH 7.5, unless otherwise stated. After formation
of a gigaohm seal, the patch was ruptured and reagents injected by
pressure using an Eppendorf Transjector 5246 (the pressure injection
system is required to introduce reagents into large cells such as
ascidian oocytes). Injection volumes were estimated by estimating the
size of the pulse in the oocyte, measured by the displacement of
cytoplasm after an injection at a controlled pressure. Control
injections of up to 10% of oocyte volume of ICS did not affect
oocytes. Membrane potentials were held at
80 mV for all
experiments except where stated. Currents were recorded with a List
L/M-EPC7 patch-clamp amplifier in the whole cell voltage-clamp
configuration and stored on a microcomputer with a Bio-Rad CRS-400
electrophysiology/ion measurement system. cADPR, ADP-ribose, cyclic
aristeromycin diphosphate ribose, and 8-NH2-cADPR analogs were supplied
by Dr. Anthony Galione. Heat-inactivated cADPR was produced by boiling
cADPR for 45 min. This compound had no activity as a
Ca2+-mobilizing agent in sea
urchin homogenates, confirming the inactivation of cADPR to ADP-ribose
(data not shown). All other reagents were obtained from Sigma except
where stated.
For single-channel recording, cADPR (5 µM pipette concentration) and
ADP-ribose (10 nM pipette concentration) were introduced in a
continuous flow into an oocyte through an electrode in the whole cell
configuration. The same electrode was used to clamp the membrane
potential. A second electrode containing ICS was used in the
cell-attached patch configuration. The single-channel electrode was
held at 0 mV. Excised patch recordings in the outside-out configuration
were prepared by clamping an unfertilized oocyte in whole cell
configuration with a pipette containing 10 nM ADP-ribose and then
gently pulling the pipette off the oocyte membrane. Currents were
recorded with a List L/M-EPC7 patch-clamp amplifier and stored on
videotape for subsequent analysis.
Estimates of channel density were made by dividing the saturating
current obtained after injection of ADP-ribose into ascidian oocyte
fragments by the peak single-channel currents. The data were obtained
using a clamped membrane potential of
80 mV.
 |
RESULTS |
An inward current is triggered by nicotinamide
nucleotides in ascidian oocytes. Pressure injection of
nicotinamide nucleotides and nicotinamide nucleotide metabolites into
C. intestinalis oocytes through a
micropipette in the whole cell voltage-clamp configuration generated
inward currents that varied in amplitude in a
dose-dependent manner (Fig.
1A).
The most potent compound was ADP-ribose, which induced an inward
current at intracellular concentrations as low as 10 nM. Microinjection
of ADP did not induce plasma membrane currents, even at a concentration
of 100 µM in the oocyte (Table 1), suggesting that the inward currents
observed after ADP-ribose injection are due to an effect of this
molecule and not due to its metabolism to ADP. cADPR is hydrolyzed to
ADP-ribose by cADPR hydrolase (reviewed in Refs. 12 and 18). Cyclic
aristeromycin disphosphate ribose, a poorly hydrolyzable analog of
cADPR (1), did not trigger inward currents (Table 1). In contrast, heat inactivation of cADPR (which produces ADP-ribose) caused a dramatic increase in peak current after injection of an equivalent concentration of cADPR (see Fig. 1A and Table 1).
These data suggest that cADPR is hydrolyzed to ADP-ribose before the
current is induced and therefore imply that ADP-ribose is the unique
trigger that gates the current.

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Fig. 1.
A: plasma membrane currents gated by
nicotinamide nucleotide and their metabolites.
Top: raw data from a typical
experiment. Arrow, time of injection. Peak current is recorded as
maximum inward current triggered by microinjection of reagent.
Bottom: dose-response curve for
microinjection of nicotinamide nucleotide indicated. Symbols represent
mean with SE as bars. L, ligand concentration;
I, current; ADPR, ADP-ribose;
HI-cADPR, heat-inactivated cyclic ADP-ribose. See Table 1 for
statistics. B: timing of development
of plasma membrane ion currents after injection of labeled reagents.
Arrow, time of injection. See Table 1 for statistics.
C: nicotinamide nucleotides and
ADP-ribose gate currents through same plasma membrane mechanism.
ADP-ribose was injected into 20-µm-diameter fragments of ascidian
oocytes to give a saturating current. Current does not increase when
ADP-ribose is coinjected with any of the 4 nicotinamide nucleotides.
ADP-ribose was injected to 1 µM (n = 13). ADP-ribose (1 µM) was then coinjected with 500 µM
NAD+
(n = 6), 50 µM NADH
(n = 7), 100 µM
NADP+
(n = 5), or 200 µM NADPH
(n = 5). Data are shown as mean with
error bars representing SE. D:
nicotinamide inhibits membrane current induced by microinjection of
nicotinamide nucleotides. Solid bars, controls (nicotinamide absent).
Open bars, oocytes bathed in 20 mM nicotinamide. Bars represent mean,
with error bars representing SE. Levels of significance according to
Student's t-test: * 95%,
** 99%.
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Microinjection of nicotinamide nucleotides triggered inward currents
(Fig. 1A and Table 1), but with a
0.2- to 0.5-s latency (Fig. 1B and
Table 1). The latency for the currents triggered by nicotinamide
nucleotide microinjection suggests that enzymatic modification of these
nucleotides takes place before the current is gated. We tested whether
nicotinamide nucleotide precursors gated inward currents through the
same mechanism as ADP-ribose by coinjecting a saturating concentration
of ADP-ribose and a nicotinamide nucleotide into 20-µm diameter
ascidian oocyte fragments (fragments are required to determine a
saturating concentration of ADP-ribose without saturating the
patch-clamp amplifier). The peak current did not increase in size (Fig.
1C), suggesting that nicotinamide
nucleotide breakdown forms ADP-ribose. The breakdown of
NAD+ to cADPR by ADP-ribosyl
cyclase and to ADP-ribose by NADase can be blocked through metabolic
end-product inhibition by the addition of nicotinamide (2, 24). In our
system, 20 mM nicotinamide significantly inhibited the currents
triggered by microinjection of nicotinamide nucleotides, without
significantly affecting the currents triggered by ADP-ribose or cADPR
microinjection (Fig. 1D and Table 1).
The ADP-ribose channel is a large, nonspecific ion
channel. The peak inward current generated by injection
of ADP-ribose was attenuated but not totally abolished when oocytes
were bathed in Ca2+-free seawater
(Fig. 2). In contrast, the peak current
increased in amplitude in
high-Ca2+ seawater (Fig. 2).
Replacement of external Na+ by
choline also reduced the peak ADP-ribose current (Fig. 2). These
experiments suggest that the ADP-ribose channel is permeable to both
Ca2+ and
Na+. Both whole cell and
single-channel current-voltage curves for currents induced by
ADP-ribose or cADPR show a reversal potential of between
+15 and +20 mV (Fig. 3,
A and
B; Table 2), again
suggesting that the channel is nonspecific. The single-channel data
give an estimate of unitary conductance of 140 pS for both cADPR and ADP-ribose (Fig. 3C; Table 2). From
these data, and the data in Fig. 1C,
we estimate the channel density to be 46 µm
2 (see
MATERIALS AND METHODS). Furthermore,
apart from the range
20 to +20 mV, where single-channel currents
were immeasurable, the data indicate that single-channel open
probability is voltage independent (Fig.
3D), suggesting that opening of the
ADP-ribose channel is not affected by membrane potential.

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Fig. 2.
Properties of plasma membrane current induced by ADP-ribose
microinjection. ADP-ribose was injected to 10 or 200 nM. Bars represent
mean ± SE. Control, injection of intracellular solution (ICS) to
1% in natural filtered seawater (NSW); 0 Ca2+ and 0 Na+ are 0 Ca2+ and
low-Na+ seawaters, respectively.
Hi Ca2+, seawater containing 100 mM Ca2+. ** Significance at
99% level, Student's
t-test.
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Fig. 3.
Electrophysiological properties of the cADPR/ADP-ribose-sensitive
channel. A: current-voltage
(I-V) graph of peak currents
recorded at different clamped membrane potentials. cADPR ( ) and
ADP-ribose ( ) were used at 5 µM and 10 nM intracellular
concentration, respectively. Data represent mean with bars representing
SE (n = 6 for both reagents).
B: single-channel currents recorded
with ADP-ribose and cADPR. cADPR and ADP-ribose intracellular
concentrations were as in A. Control,
single-channel trace before addition of reagent. ADP-ribose excised
patch represents a recording in outside-out configuration after
excision of a patch of membrane from the oocyte. ADP-ribose was 100 nM
in the pipette (n = 3 for all
experiments). C: single-channel
reversal potential
(Em) for
currents triggered by nicotinamide nucleotide metabolites. Data
represent mean and SE (n = 3 for both
reagents); , cADPR; , ADP-ribose.
D: analysis of single-channel
currents. Top: open/close times for
ADP-ribose channel. Data from a single-channel recording at 80
mV were analyzed. Solid bars, open time; open bars, close time. Bins
are of 10-ms intervals for open time and 50-ms intervals for close
time. Bottom: probability of
ADP-ribose channel opening. Data in
top were used to calculate open
probability of the ADP-ribose channel at diverse membrane potentials.
Apart from range 20 to +20 mV, open probability remained equal.
Dip in probability between 20 and +20 mV could be due to the
inability to measure currents at these potentials.
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We ruled out the contribution of
Ca2+ in gating the current,
because the peak inward current gated by either cADPR or ADP-ribose was
augmented, not diminished, after injection of
Ca2+ chelators to buffer
cytoplasmic Ca2+ (Fig.
4). Furthermore, neither eight-substituted
analogs of cADPR, which competitively inhibit cADPR-induced
Ca2+ release (27), nor the
ryanodine receptor agonist ryanodine or antagonist ruthenium red (11)
had any detectable effect on channel activity gated by cADPR (Fig. 4).
These data suggest that the channel is not gated by local
Ca2+ increases triggered by cADPR
or through ryanodine receptors.

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Fig. 4.
ADP-ribose channel is not gated by
Ca2+. Data represent mean with
bars as SE. Currents triggered by 5 µM cADPR and 10 nM ADP-ribose are
taken as controls. In presence of BAPTA, 10 mM intracellular
concentration, there is a significant rise in the peak current
triggered by 5 µM cADPR or 10 nM ADP-ribose.
8-NH2-cADPR represents an
experiment where oocytes were preloaded with the cADPR inhibitor
8-NH2-cADPR to 100 µM, and the
currents triggered by 5 µM cADPR were measured. 8-Br-cADPR represents
an experiment using a similar cADPR inhibitor. Intracellular
concentration of 8-Br-cADPR was 200 µM. Ryanodine was injected alone
to an intracellular concentration of up to 10 µM. In final
experiment, oocytes were preinjected with Ca2+-induced
Ca2+ release antagonist ruthenium red (Ruth. Red) to 500 µM. ** Significance at 99% level, Student's
t-test.
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The ADP-ribose channel is gated by sperm at
fertilization. The reversal potential, conductance, and
Ca2+-independent properties of the
ADP-ribose channel closely resemble the properties of a previously
reported ion channel gated by ascidian sperm at fertilization (4, 5).
This suggests that sperm trigger the hydrolysis of nicotinamide
nucleotides at fertilization to ADP-ribose and that this triggers the
fertilization current. We tested this hypothesis by measuring the
current induced by ascidian sperm in the presence of nicotinamide. When
50 mM nicotinamide was added to the bath, the peak current induced at
fertilization was strongly inhibited (Fig.
5A and
Table 3). The ascidian fertilization current includes both
Ca2+-dependent and
Ca2+-independent components
(unpublished observations). The fertilization current observed when
cytoplasmic Ca2+ is buffered by
Ca2+ chelators (26) is also
blocked by nicotinamide (Fig. 5 and Table 3), strongly suggesting that
this current is triggered by a mechanism involving nicotinamide
nucleotide metabolism and is not
Ca2+ dependent. Single-channel
data for the sperm-induced current demonstrated a unitary conductance
of 140 pS and reversal potential of +15 mV (Fig.
5B and Table 2), strongly suggesting
that the ADP-ribose channel and the fertilization channel are
equivalent.

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Fig. 5.
A: effect of nicotinamide on membrane
current at fertilization. Top trace,
control fertilization current [natural filtered seawater
(NSW)]. Second trace
(nicotinamide), currents observed at fertilization when oocytes were
bathed in 50 mM nicotinamide dissolved in NSW. Third
trace (BAPTA), an experiment where BAPTA was
microinjected to 10 mM intracellular concentration, and oocyte
fertilized in NSW. Fourth trace (BAPTA + nicotinamide), an oocyte microinjected with BAPTA to 10 mM
intracellular concentration, incubated in 50 mM nicotinamide in NSW and
fertilized. Scale bars represent time and current.
B: single-channel trace of ion channel
opened at fertilization. Scale bar represents time against peak
single-channel current. Current has an equivalent unitary conductance
to ADP-ribose channel. See Table 2 for statistics.
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 |
DISCUSSION |
In this paper, we have shown that microinjection of nicotinamide
nucleotides and nicotinamide nucleotide metabolites triggers an inward
current in ascidian oocytes by opening a specific ion channel. Our data
point strongly to ADP-ribose as the physiological trigger for the ion
channel. The channel has a unitary conductance of 140 pS and
furthermore appears to be nonselective. The presence of large,
nonselective ion channels in the oocyte plasma membrane appears very
unusual. However, such ion channels do exist in other species; for
example, Ca2+-gated nonspecific
ion channels can be found in the plasma membrane of the sea urchin (8).
The unique property of the ion channel characterized in the present
paper is the fact that a second messenger, ADP-ribose, gates the
channel. We believe that similar channels have not been previously
reported in any system. In fact, although ADP-ribose is a
well-characterized mediator in several cell processes (25), it is not
generally thought to be a second messenger.
ADP-ribose and related compounds are produced through the metabolism of
nicotinamide nucleotides (14, 15, 18). Generally, NAD+ is the precursor nucleotide,
at least in systems where cADPR is the second messenger (17, 23). In
the present data, NAD+ was the
least sensitive nucleotide precursor in terms of peak current produced.
It has previously been noted that all forms of adenine dinucleotide can
be metabolized into active forms, in terms of
Ca2+ release (3). These data
suggest either that NAD+ is not
exclusively metabolized by the nicotinamide nucleotide metabolic
pathway or that enzymatic conversion between different forms of
nicotinamide nucleotide occurs before metabolism of the active form
occurs. The enzymatic constituents of the C. intestinalis nicotinamide nucleotide metabolic pathway
are currently being examined by our laboratory and our collaborators.
Nicotinamide nucleotides are known to be metabolized at fertilization
(10, 27). This suggests that nicotinamide nucleotide metabolites are
important second messengers at fertilization. Although doubts remain as
to the function of cADPR in the sea urchin oocyte at fertilization (19,
22), our data strongly suggest that nicotinamide nucleotide metabolites
play an active role at fertilization. The fact that nicotinamide blocks
both the fertilization current and the current induced by the injection of nicotinamide nucleotides suggests that the pathway of nicotinamide nucleotide metabolism to ADP-ribose is present in ascidians and that
sperm sensitize this pathway at fertilization.
The mechanism of activation of the nicotinamide nucleotide metabolic
pathway in other systems appears to involve the production of nitric
oxide (13, 24, 32). We have recently shown that nitric oxide induces an
inward current in ascidian oocytes through nicotinamide nucleotide
metabolism (13), suggesting that nitric oxide may be a mediator in the
generation of the ascidian fertilization current. Interestingly,
soluble sperm extracts do not contain molecules capable of gating the
fertilization current in ascidians (30, 31). Possibly, direct injection
of nitric oxide by the sperm contributes to this process.
 |
ACKNOWLEDGEMENTS |
We are grateful to Elisabetta Tosti, Vincenzo Monfrecola, and
Giuseppe Gargiulo for their valuable contributions.
 |
FOOTNOTES |
This work was supported by European Economic Community Human Capital
and Mobility Network Grant CHRX-CT94-0646.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: B. Dale, Stazione Zoologica "Anton
Dohrn," Villa Comunale 1, 80121 Naples, Italy.
Received 3 March 1998; accepted in final form 13 July 1998.
 |
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