Atp-Dependent Adenophostin Activation of Inositol 1,4,5-Trisphosphate Receptor Channel Gating

Abstract

The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) is a ligand-gated intracellular Ca2+ release channel that plays a central role in modulating cytoplasmic free Ca2+ concentration ([Ca2+]i). The fungal metabolite adenophostin A (AdA) is a potent agonist of the InsP3R that is structurally different from InsP3 and elicits distinct calcium signals in cells. We have investigated the effects of AdA and its analogues on single-channel activities of the InsP3R in the outer membrane of isolated Xenopus laevis oocyte nuclei. InsP3R activated by either AdA or InsP3 have identical channel conductance properties. Furthermore, AdA, like InsP3, activates the channel by tuning Ca2+ inhibition of gating. However, gating of the AdA-liganded InsP3R has a critical dependence on cytoplasmic ATP free acid concentration not observed for InsP3-liganded channels. Channel gating activated by AdA is indistinguishable from that elicited by InsP3 in the presence of 0.5 mM ATP, although the functional affinity of the channel is 60-fold higher for AdA. However, in the absence of ATP, gating kinetics of AdA-liganded InsP3R were very different. Channel open time was reduced by 50%, resulting in substantially lower maximum open probability than channels activated by AdA in the presence of ATP, or by InsP3 in the presence or absence of ATP. Also, the higher functional affinity of InsP3R for AdA than for InsP3 is nearly abolished in the absence of ATP. Low affinity AdA analogues furanophostin and ribophostin activated InsP3R channels with gating properties similar to those of AdA. These results provide novel insights for interpretations of observed effects of AdA on calcium signaling, including the mechanisms that determine the durations of elementary Ca2+ release events in cells. Comparisons of single-channel gating kinetics of the InsP3R activated by InsP3, AdA, and its analogues also identify molecular elements in InsP3R ligands that contribute to binding and activation of channel gating.

Full text

299
J. Gen. Physiol.
© The Rockefeller University Press
•
0022-1295/2001/04/299/16 $5.00
Volume 117 April 2001 299–314
http://www.jgp.org/cgi/content/full/117/4/299
ATP-dependent Adenophostin Activation of Inositol 1,4,5-Trisphosphate
Receptor Channel Gating
Kinetic Implications for the Durations of Calcium Puffs in Cells
Don-On Daniel Mak, Sean McBride, and J. Kevin Foskett
From the Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104
abstract
The inositol 1,4,5-trisphosphate (InsP
3
) receptor (InsP
3
R) is a ligand-gated intracellular Ca
2
⫹
release
channel that plays a central role in modulating cytoplasmic free Ca
2
⫹
concentration ([Ca
2
⫹
]
i
). The fungal metab-
olite adenophostin A (AdA) is a potent agonist of the InsP
3
R that is structurally different from InsP
3
and elicits dis-
tinct calcium signals in cells. We have investigated the effects of AdA and its analogues on single-channel activities
of the InsP
3
R in the outer membrane of isolated
Xenopus laevis
oocyte nuclei. InsP
3
R activated by either AdA or
InsP
3
have identical channel conductance properties. Furthermore, AdA, like InsP
3
, activates the channel by tun-
ing Ca
2
⫹
inhibition of gating. However, gating of the AdA-liganded InsP
3
R has a critical dependence on cytoplas-
mic ATP free acid concentration not observed for InsP
3
-liganded channels. Channel gating activated by AdA is in-
distinguishable from that elicited by InsP
3
in the presence of 0.5 mM ATP, although the functional affinity of the
channel is 60-fold higher for AdA. However, in the absence of ATP, gating kinetics of AdA-liganded InsP
3
R were
very different. Channel open time was reduced by 50%, resulting in substantially lower maximum open probabil-
ity than channels activated by AdA in the presence of ATP, or by InsP
3
in the presence or absence of ATP. Also, the
higher functional affinity of InsP
3
R for AdA than for InsP
3
is nearly abolished in the absence of ATP. Low affinity
AdA analogues furanophostin and ribophostin activated InsP
3
R channels with gating properties similar to those of
AdA. These results provide novel insights for interpretations of observed effects of AdA on calcium signaling, in-
cluding the mechanisms that determine the durations of elementary Ca
2
⫹
release events in cells. Comparisons of
single-channel gating kinetics of the InsP
3
R activated by InsP
3
, AdA, and its analogues also identify molecular ele-
ments in InsP
3
R ligands that contribute to binding and activation of channel gating.
key words:
patch-clamp •
Xenopus
oocyte • single-channel electrophysiology • intracellular calcium signaling
• calcium release channel
INTRODUCTION
The inositol 1,4,5-trisphosphate receptor (InsP
3
R)
1
is
an intracellular Ca
2
⫹
release channel that is localized to
the endoplasmic reticulum. It plays a central role in the
modulation of free cytoplasmic Ca
2
⫹
concentration
([Ca
2
⫹
]
i
) by a ubiquitous cellular signaling system in-
volving activation of phospholipase C. Binding of extra-
cellular ligands to plasma membrane receptors gener-
ates InsP
3
, which diffuses through the cytoplasm to
bind and activate the InsP
3
R, releasing Ca
2
⫹
from the
endoplasmic reticulum lumen into the cytoplasm to
raise [Ca
2
⫹
]
i
. Complex InsP
3
-mediated calcium signals
in the form of repetitive spikes, oscillations, and propa-
gating waves initiated from specific locations in the cell
have been observed in many cell types (Bootman and
Berridge, 1995; Toescu, 1995). The molecular bases of
these spatially and temporally complex calcium signals
include cytoplasmic and organellar Ca
2
⫹
buffering sys-
tems, location of intracellular Ca
2
⫹
stores and, most im-
portantly, the properties of the InsP
3
R. The InsP
3
R
Ca
2
⫹
release channel is highly regulated by complex
mechanisms that are still only poorly understood, in-
cluding cooperative activation by InsP
3
(Meyer et al.,
1988; Finch et al., 1991; Mak et al., 1998) and bipha-
sic concentration-dependent feedback from the per-
meant Ca
2
⫹
ion (Iino, 1990; Bezprozvanny et al., 1991;
Finch et al., 1991; Mak et al., 1998). Three isoforms of
InsP
3
R (types 1, 2, and 3) as products of different genes
with alternatively spliced isoforms have been identified
and sequenced (Mignery et al., 1989; Mikoshiba, 1993).
The InsP
3
R isoforms all have
ⵑ
2,700 amino acid re-
sidues contained in three (InsP
3
-binding, regulatory
[modulatory], and transmembrane channel-forming)
domains (Mignery et al., 1989; Mikoshiba, 1993). The
sequences of the regulatory domains of all InsP
3
R iso-
forms include putative ATP-binding site(s) (Mikoshiba,
1993). ATP has been shown to bind to the InsP
3
R
Address correspondence to Dr. J. Kevin Foskett, Department of Phys-
iology, B400 Richards Building, University of Pennsylvania, Philadel-
phia, PA 19104-6085. Fax: (215) 573-6808;
E-mail: foskett@mail.med.upenn.edu
1
Abbreviations used in this paper:
AdA, adenophostin A; [Ca
2
⫹
]
i
, cyto-
plasmic free Ca
2
⫹
concentration; Fur, furanophostin; InsP
3
, inositol
1,4,5-trisphosphate; InsP
3
R, InsP
3
receptor; pdf, probability density
function; Rib, ribophostin.

300
Adenophostin Activation of InsP
3
Receptor Gating
(Maeda et al., 1991) and regulate InsP
3
R-mediated
Ca
2
⫹
release in permeabilized cells (Ferris et al., 1990;
Iino, 1991; Bezprozvanny and Ehrlich, 1993; Missiaen
et al., 1997; Landolfi et al., 1998; Mak et al., 1999; Meas
et al., 2000). At the single-channel level, ATP activates
InsP
3
-dependent InsP
3
R gating (Bezprozvanny and
Ehrlich, 1993; Mak et al., 1999; Hagar and Ehrlich,
2000). Activation of the
Xenopus
type 1 InsP
3
R channel
by ATP is accomplished by allosteric tuning of the affin-
ity of the Ca
2
⫹
activation sites, enabling InsP
3
-depen-
dent channel gating to be more sensitive to activation
by cytoplasmic Ca
2
⫹
(Mak et al., 1999).
Adenophostin A (AdA), a fungal glyconucleotide me-
tabolite (Takahashi et al., 1994), and its analogues
(Marchant et al., 1997; Shuto et al., 1998; Beecroft et
al., 1999) were recently discovered as potent agonists of
the InsP
3
R. Although their molecular structures are sig-
nificantly different from those of InsP
3
and its ana-
logues (Irvine et al., 1984; Fig. 1), they activate the
channel by interactions with the InsP
3
binding site
(Glouchankova et al., 2000). AdA is 10–80-fold more
potent than InsP
3
in binding to the InsP
3
R and stimu-
lating InsP
3
R-mediated Ca
2
⫹
release, and it is metaboli-
cally stable (Takahashi et al., 1994; Hirota et al., 1995;
Murphy et al., 1997). AdA has been applied in studies
of the InsP
3
R and its regulation (Missiaen et al., 1998;
He et al., 1999; Adkins et al., 2000; Jellerette et al.,
2000; Kashiwayanagi et al, 2000; Vanlingen et al., 2000),
Ca
2
⫹
release mediated by InsP
3
R (Marchant and
Parker, 1998; Bird et al., 1999), and Ca
2
⫹
entry due to
depletion of intracellular Ca
2
⫹
stores (DeLisle et al.,
1997; Hartzell et al., 1997; Huang et al., 1998; Broad et
al., 1999; Gregory et al., 1999; Machaca and Hartzell,
1999). Compared with InsP
3
, AdA induced temporally
and spatially different calcium signals (Marchant and
Parker, 1998; Bird et al., 1999) and Ca
2
⫹
-dependent Cl
⫺
currents (Hartzell et al., 1997; Machaca and Hartzell,
1999) in
Xenopus
oocytes. Furthermore, AdA, but not
InsP
3
, activated Ca
2
⫹
entry with an apparent lack of
Ca
2
⫹
release from stores (DeLisle et al., 1997). These
observations suggested that the effects of AdA on cal-
cium signaling were different from those expected if it
was simply a more potent equivalent of InsP
3
. However,
there have been no direct examinations of the single-
channel properties of InsP
3
R activated by AdA.
Whereas AdA has a significantly higher affinity for
binding to the InsP
3
R and a higher potency to activate
the channel than InsP
3
, ribophostin (Rib) and fura-
nophostin (Fur) (Marchant et al., 1997; Shuto et al.,
1998), structural analogues of AdA (Fig. 1), have bind-
ing affinities and activating potencies that are compara-
ble to that of InsP
3
. The relationships between the dis-
tinct binding affinities of these various ligands and the
detailed gating properties of the InsP
3
R channel they
elicit are unknown. The molecular structural feature
that is unique to AdA and not shared by its less potent
analogues is the adenine moiety linked to the ribose
ring, which has structural resemblance to that of ATP
(Fig. 1). Nevertheless, it has been reported that AdA
does not bind to glutathione-S-transferase fusion poly-
peptides containing the putative ATP binding se-
quences of the type 1 InsP
3
R (Maes et al., 1999). Thus,
the molecular determinants involved in the high affin-
ity interaction of AdA with the InsP
3
R are still unclear.
The unique cytoplasmic calcium signals elicited by ac-
tivation of the InsP
3
R with different ligands suggest that
a detailed understanding of the mechanisms of action
of AdA and its analogues could provide important novel
insights into the molecular basis for the linkage be-
tween ligand binding and activation of the InsP
3
R chan-
nel. Here, we have performed a systematic investigation
of the effects of AdA and its analogues on the single-
channel activities of the InsP
3
R. We have previously ap-
plied the patch-clamp technique to the outer mem-
brane of isolated
Xenopus laevis
oocyte nuclei to study
extensively the single-channel properties of the endoge-
nous type 1 InsP
3
R in its native membrane environment
under rigorously controlled experimental conditions
(Mak and Foskett, 1994, 1997, 1998; Mak et al., 1998,
1999). Here we characterize the conduction and chan-
nel gating properties of single InsP
3
R channels activated
by AdA and its analogues, Rib and Fur, in the presence
of a wide range of cytoplasmic Ca
2
⫹
, ligand, and ATP
concentrations. Our studies demonstrate that the chan-
nel conductance properties are identical for InsP
3
R
channels activated by either AdA or InsP
3
. However, gat-
ing of the InsP
3
R activated by AdA or its analogues has a
Figure 1. Molecular structures of various InsP3R-binding ligands.

301
Mak et al.
critical dependence on cytoplasmic ATP free acid con-
centration that is not observed for InsP
3
-liganded chan-
nels. Channel gating activated by AdA is indistinguish-
able from that elicited by InsP
3
in the presence of 0.5
mM cytoplasmic free ATP, although the functional affin-
ity of the channel is
ⵑ
60-fold higher for AdA. However,
the AdA-liganded channel exhibits very different chan-
nel gating kinetics in the absence of cytoplasmic free
ATP. The channel open time is reduced by nearly 50%
when the channel is activated by AdA in the absence of
ATP, resulting in a substantially lower maximum open
probability than channels activated by AdA in the pres-
ence of ATP, or by InsP
3
in the presence or absence of
ATP. Furthermore, the higher functional affinity of AdA
compared with InsP
3
is nearly abolished in the absence
of ATP. The low affinity AdA analogues Fur and Rib acti-
vated channels with gating properties similar to those of
AdA in either the presence or absence of ATP. Our
study reveals a prominent role of ATP as an allosteric
regulator of the InsP
3
R channel, and it provides novel
insights for interpretations of observed effects of AdA
on intracellular calcium signaling. In particular, the ef-
fects of AdA on the kinetics of channel gating suggest
novel mechanisms that determine the durations of ele-
mentary Ca
2
⫹
release events in cells. Comparisons of the
single-channel gating kinetics of the InsP
3
R activated by
InsP
3
, AdA, and its analogues have also enabled identi-
fication of molecular structural elements in InsP
3
R
ligands that contribute to their ability to bind and acti-
vate channel gating.
MATERIALS AND METHODS
Patch-clamping the Oocyte Nucleus
Patch-clamp experiments were performed using isolated
Xenopus
oocyte nuclei as described previously (Mak and Foskett, 1994,
1997, 1998; Mak et al., 1998). In brief, stage V or VI oocytes,
which express only a single InsP3R isoform (type 1) and lacks
other (e.g., ryanodine receptor) Ca2⫹ release channels (Kume et
al., 1993), were opened mechanically just before use. The nu-
cleus was separated from the cytoplasm and transferred to a dish
on the stage of a microscope for patch-clamping. Experiments
were performed in the “on-nucleus” configuration, with the solu-
tion in the perinuclear lumen between the outer and inner nu-
clear membranes in apparent equilibrium with the bath solution
(Mak and Foskett, 1994), and the cytoplasmic aspect of the
InsP3R channel facing into the patch pipet. Experiments were
performed at room temperature with the pipet electrode at ⫹20
mV relative to the reference bath electrode.
Data Acquisition and Analysis
Single-channel currents were amplified by an Axopatch-1D am-
plifier (Axon Instruments, Inc.) with antialiasing filtering at 1
kHz, digitized at 5 kHz, and recorded by Pulse⫹PulseFit software
(HEKA Elektronik). The patch-clamped Xenopus InsP3R inacti-
vates with a time constant of ⵑ30 s after its activation by InsP3
(Mak and Foskett, 1997). Similar inactivation was observed in
this study when the channels were activated by AdA. As inactiva-
tion was generally abrupt with no detectable change in channel
kinetics up to the disappearance of channel activity (Mak and
Foskett, 1997), current traces obtained during the entire period
the channels were active were analyzed (Mak and Foskett, 1998;
Mak et al., 1998). Channel opening and closing events were iden-
tified with a 50% threshold, and channel open probability Po and
dwell time distribution evaluated using TAC software (Bruxton).
Current traces exhibiting one InsP3R channel, or two InsP3R
channels determined to be identical and independently gated
(Mak and Foskett, 1997), were used for Po evaluation, whereas
only current traces with a single InsP3R channel were used for
dwell time analyses. Each set of open and closed dwell time histo-
grams was derived from one patch-clamp current record of a sin-
gle active InsP3R channel. The probability density function (pdf)
was fitted to the histograms by the maximum likelihood method
(Sigworth and Sine, 1987).
The number of channels in the membrane patch was assumed
to be the maximum number of open channel current levels ob-
served throughout the current record. Assuming there are n
identical and independent channels in the membrane patch,
and each channel is Markovian with open probability of Po and
open duration distribution characterized by a single exponential
component of time constant ␶o, the mean dwell time of highest
channel current level is ␶o/n. If T is the minimum duration of an
open event that is detectable in the experimental system, i.e.,
only events with duration ⬎T will have amplitudes greater than
the 50% threshold after filtering, then the rate of detection of
the highest current level:
(1)
In our patch-clamp set up, T was empirically determined to be
ⵑ0.2 ms using test pulses of variable duration. ␶o of InsP3R chan-
nels is ⵑ3–15 ms over the range of experimental conditions used
(Mak et al., 1998). In experimental conditions with Po ⬎ 0.1, only
current records with longer than 10 s of InsP3R channel activities
were used. Because 10 s Ⰷ 1/R3, there is little uncertainty in the
number of channels in the current traces used. In experimental
conditions with Po ⬍ 0.1, only current records exhibiting one
open channel current level with record duration ⬎ 5/R2 were
used, to ensure that they were truly single-channel records.
Each data point shown is the mean of results from at least four
separate patch-clamp experiments performed under the same
conditions. Error bars indicate the SEM. Theoretical Hill equa-
tion curves were fitted to experimental Po data using IgorPro
(WaveMetrics).
Solutions for Patch-clamp Experiments
All patch-clamp experiments were performed with solutions con-
taining 140 mM KCl and 10 mM HEPES, with pH adjusted to 7.1
using KOH. By using K⫹ as the current carrier and appropriate
quantities of the high affinity Ca2⫹ chelator, BAPTA (1,2-bis[O-
aminophenoxy] ethane-N,N,N
⬘
,N
⬘
-tetraacetic acid; 100–1,000
␮M; from Molecular Probes), or the low affinity Ca2⫹ chelator,
5,5
⬘
-dibromo BAPTA (100–400 ␮M; Molecular Probes), or ATP
(0.5 mM) alone to buffer Ca2⫹ in the experimental solutions,
free Ca2⫹ concentrations in our experimental solutions were
tightly controlled. Total Ca2⫹ content (5–330 ␮M) in the solu-
tions was determined by induction-coupled plasma mass spec-
trometry (Mayo Medical Laboratory). Free [Ca2⫹] was calculated
using the Maxchelator software (C. Patton, Stanford University,
Stanford, CA). The free [Ca2⫹] of the solutions was verified by
measurements using Ca2⫹-selective minielectrodes (Baudet et al.,
1994) and found to agree with the calculated [Ca2⫹] to within
the accuracy of the electrode measurement (10%). The bath so-
lutions used in all experiments had 140 mM KCl, 10 mM Hepes,
Rn
nP
o
()
n
␶
o
----------------nT
␶
o
-------


–exp .=

302 Adenophostin Activation of InsP3 Receptor Gating
380 ␮M CaCl2, 500 ␮M BAPTA ([Ca2⫹] ⫽ 500 nM), and pH 7.1.
Pipet solutions, to which the cytoplasmic aspects of the channels
were exposed, contained either 0 or 0.5 mM of Na2ATP (Sigma-
Aldrich), various concentration of InsP3 (Molecular Probes),
AdA, Rib, or Fur (Calbiochem), as stated. All reagents were used
without further purification. Because Mg2⫹ was absent from the
experimental solutions, ATP mostly existed in free acid forms
(ATP4⫺, ATP3⫺). We previously demonstrated that ATP free acid,
not MgATP complex, was responsible for ATP regulation of
InsP3R gating (Mak et al., 1999).
RESULTS
Properties of AdA-liganded InsP3R Channels in the Presence of
0.5 mM Cytoplasmic Free ATP
To compare the single-channel conductance and gating
properties of the Xenopus type 1 InsP3R (X-InsP3R-1) ac-
tivated by AdA with those activated by InsP3, we per-
formed patch-clamp experiments on the outer mem-
brane of nuclei isolated from Xenopus oocytes using the
same experimental conditions employed in our previ-
ous studies (Mak and Foskett, 1994, 1997; Mak et al.,
1998), but with AdA instead of InsP3 as the agonist.
With cytoplasmic ATP free acid concentration ([ATP])
of 0.5 mM, the conductance properties and gating ki-
netics of the X-InsP3R-1 channel activated by saturating
concentrations of either AdA (100 nM) or InsP3 (10
␮M) were indistinguishable in all cytoplasmic free Ca2⫹
concentrations ([Ca2⫹]i) examined (Fig. 2 A). In addi-
tion, all AdA-liganded X-InsP3R-1 channels observed in
our experiments inactivated despite the continuous
presence of AdA in the pipet solution, with durations of
channel activity that were comparable to those observed
for InsP3-liganded channels (Mak and Foskett, 1997).
The open probability (Po) of the InsP3-liganded chan-
nel varies with [Ca2⫹]i in a biphasic manner (Mak et al.,
1998). To determine the [Ca2⫹]i dependence of the gat-
ing of AdA-liganded channels, a saturating concentra-
tion (100 nM) of AdA was used as the ligand in the pres-
ence of various [Ca2⫹]i. The Po of the channel activated
by 100 nM AdA also varied with [Ca2⫹]i in a biphasic
manner (Fig. 3). At [Ca2⫹]i ⬍ 1 ␮M, increases in [Ca2⫹]i
enhanced the channel Po. Between 1 and 20 ␮M [Ca2⫹]i,
the channel Po remained high (ⵑ0.8). As [Ca2⫹]i in-
creased beyond 20 ␮M, Po decreased precipitously. This
[Ca2⫹]i dependence of the AdA-liganded X-InsP3R-1 was
essentially identical to that of the channel activated by
saturating concentrations of InsP3 (Mak et al., 1998).
The results were well fitted by a biphasic Hill equation:
(2)
The Hill equation parameters—maximum Po (Pmax),
half-maximal activating [Ca2⫹]i (Kact), activation Hill co-
efficient (Hact), half-maximal inhibitory [Ca2⫹]i (Kinh),
and inhibition Hill coefficient (Hinh)—for the AdA-
liganded channel were all very similar to those for the
InsP3-liganded channel (Table I, A and B). The identical
Pmax indicates that InsP3 and AdA have similar efficacy in
gating the channel in the presence of 0.5 mM free ATP.
PoPmax 1Kact Ca2⫹
[]
i
⁄()
Hact
+[]
1–
1Ca2⫹
[]
iKinh
⁄()
Hinh
+[]
1–.
=
Figure 2. Typical single-channel current traces of X-InsP3R-1 in
v
arious [Ca2⫹]i, activated by InsP3 or AdA as indicated. Arrows in-
dicate the closed channel current levels. (A) In the presence of 0.5
mM ATP. (B) In the absence of ATP.
TABLE I
Hill Equation Parameters of X-InsP3R-1
Ligand
concentration Pmax Kact Hact Kinh Hinh
nM
␮
M
A 100 nM AdA 0.81 ⫾ 0.03 200 ⫾ 50 1.8 ⫾ 0.3 45 ⫾ 5 3.5 ⫾ 0.4
B 10 ␮M InsP30.81 ⫾ 0.03 190 ⫾ 20 1.9 ⫾ 0.3 54 ⫾ 6 3.9 ⫾ 0.7
C 0.5 nM AdA 0.84 ⫾ 0.03 250 ⫾ 50 1.8 ⫾ 0.3 8.9 ⫾ 0.5 4.3 ⫾ 0.7
D 33 nM InsP30.81 ⫾ 0.03 190 ⫾ 20 1.9 ⫾ 0.3 11.0 ⫾ 1.5 3.9 ⫾ 0.7
E 20 nM InsP30.81 ⫾ 0.03 190 ⫾ 20 1.9 ⫾ 0.3 0.21 ⫾ 0.04 3.9 ⫾ 0.7
F 10 ␮M InsP30.79 ⫾ 0.02 420 ⫾ 40 2.2 ⫾ 0.3 110 ⫾ 10 4.0 ⫾ 0.7
G 33 nM InsP30.80 ⫾ 0.05 540 ⫾ 70 2.0 ⫾ 0.3 1.4 ⫾ 0.2 3.5 ⫾ 0.7
H 100 nM AdA 0.43 ⫾ 0.03 400 ⫾ 50 2.4 ⫾ 0.3 130 ⫾ 10 4.0 ⫾ 0.7
I 20 nM AdA 0.44 ⫾ 0.03 440 ⫾ 40 2.6 ⫾ 0.6 9 ⫾ 2 4.0 ⫾ 0.7
Parameters for the biphasic Hill equations (Eq. 2) that fit the [Ca2⫹]i dependence
of the Po of X-InsP3R-1 channel under various experimental conditions.

303 Mak et al.
The broad biphasic Po versus [Ca2⫹]i curve of the
AdA-liganded X-InsP3R-1 channel remained the same
when the concentration of AdA was reduced from 100
to 5 nM (data not shown). However, when the concen-
tration of AdA was further decreased to 0.5 nM, the
channel exhibited a higher sensitivity to Ca2⫹ inhibition,
with Kinh reduced, but Hinh unaltered (Table I C). The
[Ca2⫹]i dependence of the activation of the channel and
the Pmax were not significantly affected by the concentra-
tion of AdA (Fig. 3). AdA appears to activate the InsP3R
channel by reducing the affinity of the Ca2⫹ inhibition
site, which is reminiscent of the tuning of Ca2⫹ inhibi-
tion of the channel by InsP3 (Mak et al., 1998). Thus,
the mechanism by which ligand binding activates the
channel (elevation of Kinh) is similar for both AdA and
InsP3. The value of Kinh of the channel activated by 0.5
nM AdA lies between those activated by 20 and 33 nM
InsP3 (Table I, D–E; Mak et al., 1998). Thus, in the pres-
ence of 0.5 mM ATP, AdA activates the X-InsP3R-1 chan-
nel in the same manner with a similar efficacy as InsP3,
but AdA is ⵑ60 times more potent than InsP3.
InsP3-liganded X-InsP3R-1 Channel
Gating in the Absence of ATP
Part of the molecular structure of AdA is analogous to
that of InsP3: AdA has a glucose moiety with a 3⬘⬘,4⬘⬘-bis-
phosphate/2⬘⬘-hydroxyl motif that is structurally similar
to the 4,5-bisphosphate/6-hydroxyl motif of InsP3 (Ho-
toda et al., 1999), and the 2⬘-phosphoryl group in the ri-
bose ring of AdA is probably in an analogous position as
the 1-phosphoryl group in InsP3 (Wilcox et al., 1995).
However, the rest of the AdA molecular structure is very
different from that of InsP3 (Fig. 1). In particular, AdA
has an adenosine 2⬘-phosphate moiety not present in
InsP3. An interaction between the adenine structure in
AdA and unknown site(s) in the InsP3R has been sug-
gested to contribute to the high potency of AdA as an
agonist of the InsP3R (Hotoda et al., 1999).
The sequences of the regulatory domains of all
InsP3R isoforms include putative ATP-binding site(s)
(Mikoshiba, 1993). ATP was shown to bind to the
InsP3R and regulate InsP3R-mediated Ca2⫹ release and
InsP3R single-channel gating (see introduction). Be-
cause ATP and AdA share a common adenine moiety
(Fig. 1), we reasoned that an ATP binding site(s) in the
InsP3R structure might interact with the adenine moi-
ety in AdA to promote high affinity binding of AdA to
the channel. Such a mechanism suggests that ATP
might function as an antagonist, competing with AdA
for the same binding site in the InsP3R. A prediction
from this model is that the affinity of the channel for
AdA would be increased in the absence of cytoplasmic
free ATP. To test this hypothesis, we examined the activ-
ities of the channel in the absence of ATP, using either
AdA or InsP3 to stimulate gating.
We first examined the effects of InsP3. In the absence
of cytoplasmic free ATP, the channel conductance and
gating properties activated by a saturating concentra-
tion of InsP3 (10 ␮M) were identical to those of the
channel activated in the presence of 0.5 mM ATP (Fig.
2 B). The [Ca2⫹]i dependence of the channel Po (Fig.
4) remained well characterized by a biphasic Hill equa-
tion (Eq. 2). The channel was fully activated in 2 ␮M ⬍
[Ca2⫹]i ⬍ 50 ␮M with a Pmax of 0.8. Whereas Hact and
Hinh were similar in either the presence or absence of
free ATP, the InsP3-liganded channel in 0 ATP was less
sensitive to Ca2⫹ activation and to Ca2⫹ inhibition (Fig.
5 A), with twofold increases in both Kact and Kinh (com-
paring Table I, F and B).
The biphasic [Ca2⫹]i dependence of InsP3-liganded
channel gating in the absence of ATP remained un-
changed when the concentration of InsP3 was de-
creased from 10 ␮M to 100 nM (data not shown). A fur-
ther reduction of the concentration of InsP3 to 33 nM
Figure 3. [Ca2⫹]i dependence of the Po of the X-InsP3R-1 chan-
nel in the presence of 0.5 mM ATP, activated by AdA or InsP3 (Mak
and Foskett, 1998). The solid and dashed curves are theoretical fits
by the Hill equation (Eq. 2) of the Po data from [AdA] ⫽ 100 nM
and 0.5 nM, respectively.
Figure 4. [Ca2⫹]i dependence of the Po of the X-InsP3R-1 channel
in the absence of ATP, activated by saturating (10 ␮M) or subsatu-
rating (33 nM) concentrations of InsP3. The solid and dashed
curves are theoretical fits by the Hill equation (Eq. 2) of the Po data.

304 Adenophostin Activation of InsP3 Receptor Gating
caused the channel to exhibit a higher sensitivity to
Ca2⫹ inhibition (Fig. 4). The [Ca2⫹]i dependence of
the channel Po activated by 33 nM InsP3 in the absence
of ATP was fitted by the biphasic Hill equation (Eq. 2)
with Kinh reduced from 110 to 1.4 ␮M, while the other
parameters Hinh, Kact, Hact, and Pmax remained essen-
tially unchanged (Table I G). Thus, InsP3 regulation of
X-InsP3R-1 channel gating was similar in the presence
or absence of ATP, with comparable efficacy and func-
tional affinity in both cases.
AdA-liganded X-InsP3R-1 Channel
Gating in the Absence of ATP
We next examined the effects of AdA. The conduc-
tance properties of the X-InsP3R-1 channel activated by
a saturating concentration (100 nM) of AdA were indis-
tinguishable in either the presence or absence of ATP
(Fig. 2). In contrast, gating of the AdA-liganded chan-
nel in the absence of ATP was dramatically different
from that of the InsP3-liganded channel. Whereas the
InsP3-liganded channel exhibited Pmax ⬇ 0.8 at [Ca2⫹]i
⬎ 2 ␮M, the Pmax of the AdA-liganded channel was only
0.4 (Figs. 5 B and 6). Instead of staying open most of
the time with only brief closings like the InsP3-liganded
channel, the AdA-liganded channel had substantially
shorter channel openings (Fig. 2 B). Similar channel
gating characterized by short openings (Fig. 2 B) and
Pmax of ⵑ0.4 (Fig. 6) was also observed in suprasaturat-
ing concentrations (500 nM) of AdA. Therefore, the
altered gating kinetics of the AdA-liganded channel
observed in the absence of ATP was not due to insuffi-
cient channel activation by subsaturating concentra-
tions of AdA.
The [Ca2⫹]i dependence of the AdA-liganded chan-
nel Po in the absence of ATP (Figs. 5 A and 6) was well
fitted by the biphasic Hill equation (Eq. 2) with Kact,
Hact, Kinh, and Hinh comparable with those for the chan-
nel activated by InsP3, but with a Pmax decreased to ⵑ0.4
(Table I H). Therefore, in the absence of ATP, the af-
finities (Kact and Kinh) of the activating and inhibitory
Ca2⫹-binding sites and their levels of cooperativity (Hact
Figure 5. [Ca2⫹]i dependence of the Po of the X-InsP3R-1 chan-
nel activated by saturating concentrations of ligands in the pres-
ence or absence of ATP. (A) 10 ␮M InsP3; (B) 100 nM AdA. The
solid and dashed curves are theoretical fits by the Hill equation
(Eq. 2) of the data in 0 or 0.5 mM of ATP, respectively.
Figure 6. [Ca2⫹]i dependence of the Po of the X-InsP3R-1 channel
activated by AdA or InsP3 in the absence of ATP. The solid and
dashed curves are theoretical fits by the Hill equation (Eq. 2) of the
Po data from [InsP3] ⫽ 10 ␮M and [AdA] ⫽ 100 nM, respectively.
Figure 7. [Ca2⫹]i dependence of the Po of the X-InsP3R-1 chan-
nel in the absence of ATP, activated by saturating (100 nM) or sub-
saturating (20 nM) concentrations of AdA. The solid and dashed
curves are theoretical fits by the Hill equation (Eq. 2) of the Po
data. Note that the scale of the Po axis is different from that in the
previous Po versus [Ca2⫹]i graphs.

305 Mak et al.
and Hinh) of the AdA and InsP3-liganded X-InsP3R-1
channels were similar, but the maximal level of channel
activity induced by AdA in the absence of ATP was only
about half that activated by InsP3 at all [Ca2⫹]i. In other
words, in the absence of free ATP, AdA was less effica-
cious than InsP3 in activating channel gating, acting in-
stead as a partial agonist.
When the concentration of AdA was reduced from
100 to 20 nM in the absence of ATP, the channel be-
came more sensitive to Ca2⫹ inhibition (Fig. 7), with
only Kinh reduced while the other Hill equation param-
eters remained similar to those observed in 100 nM
AdA (Table I). Thus, despite the lower Pmax value ob-
served for the channel activated by AdA in the absence
of ATP, the channel was still activated by ligand tuning
of its sensitivity to Ca2⫹ inhibition, as it was when it was
activated by InsP3 (Mak et al., 1998; Fig. 4), or by AdA
in 0.5 mM ATP (Fig. 3).
Of note, the value of Kinh for the channel activated by
20 nM AdA in the absence of ATP lay between those val-
ues for channels activated by 33 and 100 nM InsP3. Thus,
in the absence of ATP, AdA was only 1.5–5 times more po-
tent than InsP3 in activating the channel, whereas it was
ⵑ60 times more potent in the presence of 0.5 mM ATP.
In summary, the affinities of the activating and inhibi-
tory Ca2⫹-binding sites (Kact and Kinh) of the InsP3-
liganded channels were the only parameters affected by
the presence or absence of ATP (Fig. 5 A). In contrast,
ATP regulates not only the affinities of the Ca2⫹-bind-
ing sites, but also the level of maximum activity of the
AdA-liganded channel (Fig. 5 B) as well as the potency
of AdA to activate the channel. Thus, the presence or
absence of ATP affects all regulation parameters of the
AdA-liganded channel except the level of cooperativity
of the Ca2⫹-binding sites. In addition, these results dem-
onstrate that the high affinity of AdA is not conferred
by its interaction with ATP-binding sites in the channel
sequence, in contrast to our working hypothesis.
Properties of the X-InsP3R-1 Channel
Activated by Rib and Fur
Because AdA and InsP3 had distinct effects on the gat-
ing properties of the InsP3R when the channel was stim-
ulated in the absence of ATP, we speculated that the
distinct molecular structures of the two ligands con-
ferred unique ATP-dependent gating properties. To de-
termine the molecular structural determinants in the
activating ligand that influence the gating properties of
the channel, we investigated the effects of Rib and Fur,
structural analogues of AdA that lack the adenine moi-
ety found in AdA (Fig. 1). In previous studies, these an-
alogues of AdA were found to stimulate InsP3-mediated
Ca2⫹ release with an apparent affinity that was signifi-
cantly lower than that of AdA but similar to that of
InsP3 (Marchant et al., 1997; Shuto et al., 1998).
In optimal conditions, with [Ca2⫹]i between 4.4 and
6.2 ␮M and in the presence of saturating concentra-
tions (10 ␮M) of Rib or Fur, the channels exhibited in-
activation kinetics and conductance and gating proper-
ties (Fig. 8) that were indistinguishable from those ob-
served when the channels were activated by AdA, in
either the absence or presence (0.5 mM) of ATP (Fig.
2). Whereas InsP3-liganded channels exhibited Pmax of
ⵑ0.8 in both 0 and 0.5 mM ATP, channels activated by
AdA, Fur, or Rib only exhibited this high Pmax in the
presence of 0.5 mM ATP. In the absence of ATP, the
X-InsP3R-1 channel activated by AdA, Fur, or Rib had a
significantly (P ⬍ 0.01) lower Pmax ⬇ 0.4 (Fig. 9). Thus,
the responses of the channel to saturating concentra-
tions of Fur or Rib were clearly similar to that for AdA
and different from those for InsP3.
Closed Channel Dwell Time Distributions of X-InsP3R-1
Channel Activated by Various Ligands
To elucidate the kinetic features associated with the
regulation of X-InsP3R-1 channel gating, we studied in
detail the mean open and closed channel durations
(〈␶o〉 and 〈␶c〉, respectively) under various experimental
conditions in the presence of different ligands. Fur-
thermore, dwell time histogram analyses were per-
formed on single-channel current records of channels
activated by saturating concentrations of AdA (100 nM)
Figure 8. Typical single-channel current traces of the X-InsP3R-1
activated by 10 ␮M Fur or Rib. In 0.5 mM ATP (⫹ ATP), [Ca2⫹]i ⫽
5.0 ␮M. In the absence of ATP (0 ATP), [Ca2⫹]i ⫽ 6.2 ␮M. The ar-
rows indicate the closed channel current levels.
Figure 9. Po of the X-InsP3R-1 channel in optimal [Ca2⫹]i (4.4–6.2
␮M) and saturating concentrations of various ligands in 0 (white
bars) and 0.5 mM (shaded bars) ATP.

306 Adenophostin Activation of InsP3 Receptor Gating
or InsP3 (10 ␮M), in the presence or absence of ATP,
and in various [Ca2⫹]i, except when such analyses were
precluded by Ca2⫹ inhibition at high [Ca2⫹]i and chan-
nel inactivation (Mak and Foskett, 1997).
In general, under all conditions examined (activation
by AdA or InsP3, in the presence or absence of ATP), the
[Ca2⫹]i dependence of the channel Po mainly resided in
a [Ca2⫹]i dependence of 〈␶c〉 (Mak et al., 1998; Fig. 10).
The increase in Po due to Ca2⫹ activation in the low
[Ca2⫹]i range (⬍1 or 2 ␮M, in the presence or absence
of ATP, respectively) was mostly caused by a decrease in
〈␶c〉 with increases in [Ca2⫹]i. 〈␶c〉 stayed within a narrow
range (1 to 5 ms) when Po remained at maximum level
in higher, optimal [Ca2⫹]i. The precipitous decrease in
Po at higher [Ca2⫹]i due to Ca2⫹ inhibition was mostly
the result of a dramatic rise in 〈␶c〉 as [Ca2⫹]i increased.
The increase in the sensitivity of the channel to Ca2⫹ in-
hibition observed in the presence of subsaturating con-
centrations of either ligand (AdA or InsP3) was reflected
in an onset of the rise in 〈␶c〉 at lower [Ca2⫹]i.
The closed dwell time histograms of the X-InsP3R-1
channel revealed that it had at least four distinguishable
closed kinetic states with time constants ␶c ⬎100 ms, 20–
Figure 10. [Ca2⫹]i dependencies of the mean open (〈␶o〉) and
closed (〈␶c〉) dwell times of the X-InsP3R-1 channel. In the 〈␶c〉
graphs, data points from the same experimental conditions are
connected with solid or dashed lines for clarity. (A) Channel acti-
v
ated by AdA or InsP3 (Mak and Foskett, 1998), in 0.5 mM ATP.
(B) Channel activated by saturating (10 ␮M) or subsaturating (33
nM) concentrations of InsP3 in the absence of ATP. (C) Channel
activated by saturating (100 nM) or subsaturating (20 nM) concen-
trations of AdA in the absence of ATP.
Figure 11. Open and closed dwell time histograms of the
X-InsP3R-1 channel in 0.5 mM ATP and various [Ca2⫹]i, activated
by saturating concentrations of InsP3 (10 ␮M) or AdA (100 nM).
The smooth curves are the pdf. The time constant and relative
w
eight of each exponential component of the pdf are tabulated
next to the corresponding peak in the curves. [Ca2⫹]i used in each
of the analyzed experiments and its Po are tabulated next to the
corresponding graphs.

307 Mak et al.
60 ms, 2–10 ms, and ⬍1 ms, respectively (Figs. 11 and
12). The decrease in 〈␶c〉 associated with Ca2⫹ activation
of InsP3-liganded channels in 0.5 mM ATP was caused by
sequential destabilization and, therefore, reduction of
the relative weights, of the three longer closed kinetic
states, until the shortest closed kinetic state with ␶c ⬍ 1
ms became dominant in [Ca2⫹]i ⬎ 1 ␮M (Fig. 11, A–E).
Reduction of ␶c of the longer closed kinetic states also
contributed, to a lesser extent, to the decrease in 〈␶c〉.
As suggested by their essentially identical [Ca2⫹]i de-
pendencies of the Po (Fig. 3) and 〈␶c〉 (Fig. 10 A) of
channels activated in 0.5 mM ATP by either AdA or
InsP3, the closed channel dwell time distributions of
the channels activated by either ligand in 0.5 mM ATP
were very similar (Fig. 11). Although the sensitivity of
the channels to Ca2⫹ activation was diminished in the
absence of ATP, the closed dwell time distributions of
InsP3-liganded channels in the absence of ATP resem-
bled those in the presence of 0.5 mM ATP when com-
pared at [Ca2⫹]i that gave comparable channel Po
(compare Fig. 12, A–C, with Fig. 11, C–E).
Interestingly, although the gating kinetics of the
InsP3R channel activated by AdA in the absence of ATP
were very different from those of channels activated by
AdA in 0.5 mM ATP or activated by InsP3 (Fig. 2), Ca2⫹
activation of the AdA-liganded InsP3R in the absence of
ATP was still caused by destabilization of the longer
closed kinetic states (Fig. 12, D–F), although the closed
channel time constants were generally longer than in
other conditions.
Open Channel Dwell Time Distributions of X-InsP3R-1
Channels Activated by Various Ligands
The mean open channel duration (〈␶o〉) of the X-InsP3R-1
channel activated by saturating concentrations of InsP3
in 0.5 mM ATP remained within a narrow range, be-
tween 5 and 15 ms, over a wide range of [Ca2⫹]i (50
nM–50 ␮M; Fig. 10 A). 〈␶o〉 dropped below 5 ms at very
low or very high [Ca2⫹]i. In subsaturating concentra-
tions of InsP3, 〈␶o〉 dropped below 5 ms at lower [Ca2⫹]i
(Mak et al., 1998). This [Ca2⫹]i dependence of 〈␶o〉 was
mirrored in AdA-liganded channels in 0.5 mM ATP
(Fig. 10 A). A similar [Ca2⫹]i dependence of 〈␶o〉 was
also observed in InsP3-liganded channels in 0 ATP, ex-
cept that 〈␶o〉 was ⬎5 ms for [Ca2⫹]i between 300 nM
and 100 ␮M in saturating concentrations of InsP3 be-
cause of the change in [Ca2⫹]i sensitivity of the channel
in the absence of ATP (Fig. 10 B).
In contrast, a very different [Ca2⫹]i dependence was
observed for 〈␶o〉 of the AdA-liganded channel in 0 ATP.
〈␶o〉 never rose above 5 ms over the entire wide range of
[Ca2⫹]i examined (Fig. 10 C). This reduced 〈␶o〉 ac-
counted for the distinct channel gating kinetics of the
InsP3R activated by AdA in 0 ATP. Thus, a typical open-
ing event of the channel activated by AdA in the absence
of ATP was significantly shorter than a typical opening
event of the channel under other activating conditions
examined. This was the major factor contributing to the
low value of Pmax for the AdA-liganded channel in 0 ATP.
Open dwell time histograms of the fully activated
X-InsP3R-1 channel generally contained two exponen-
tial components, corresponding to at least two distin-
guishable open kinetic states (Figs. 11 and 12). Over
most [Ca2⫹]i in which the channel 〈␶o〉 remained high,
the long open kinetic state was the dominant one. At
very low [Ca2⫹]i, 〈␶o〉 of the channel was shorter because
of either the sharp reduction in the relative weight of
the long open kinetic state in favor of the short one (for
InsP3-liganded channel in 0.5 mM ATP; Fig. 11, A and B;
and AdA-liganded channel in 0 ATP; Fig. 12 D), or the
Figure 12. Open and closed dwell time histograms of the
X-InsP3R-1 channel in the absence of ATP and various [Ca2⫹]i, ac-
tivated by saturating concentrations of InsP3 (10 ␮M) or AdA (100
nM). The smooth curves are the pdf. The time constant and rela-
tive weight of each exponential component of the pdf are tabu-
lated next to the corresponding peak in the curves. [Ca2⫹]i used in
each of the analyzed experiments and its Po are tabulated next to
the corresponding graphs.

308 Adenophostin Activation of InsP3 Receptor Gating
reduction of the time constant of the long open kinetic
state (for InsP3-liganded channel in 0 ATP; Fig. 12 A).
The time constant ␶o of the dominating long open ki-
netic state was 5–8 ms for all experimental conditions in
which the channel had Pmax of 0.8: channels in 0.5 mM
ATP activated by InsP3 (Fig. 11, C–E) or AdA (Fig. 11, F
and G), and InsP3-liganded channels in 0 ATP (Fig. 12,
B and C). In contrast, ␶o of the dominating long open
kinetic state was only ⵑ2 ms for AdA-liganded channel
in 0 ATP with Pmax of 0.4 (Fig. 12, E and F).
Comparison of 〈␶o〉 and 〈␶c〉 of the InsP3R channel in
saturating concentrations of various ligands (Fig. 13)
clearly indicated that the channel optimally activated
by Rib or Fur exhibited the same gating kinetics as
AdA-liganded channels, characterized by having a sig-
nificantly shorter 〈␶o〉 and a longer 〈␶c〉 in the absence
of ATP than in the presence of ATP. In contrast, InsP3-
liganded channels exhibited the same 〈␶o〉 and 〈␶c〉 un-
der both conditions.
DISCUSSION
Since its discovery as a potent, metabolically stable ago-
nist of the InsP3R, AdA has been used in studies of the
InsP3R and its regulation and in studies that examined in-
tracellular Ca2⫹ release in cells (see introduction). Our
study represents the first investigation of the single-chan-
nel properties of the InsP3R channel in its native mem-
brane environment activated by AdA and its analogues.
ATP-dependent Differences in InsP3R
Gating Activated by InsP3 and AdA
The major finding in our study is that AdA activates the
InsP3R channel with distinct properties depending on
the presence or absence of ATP. In the presence of 0.5
mM cytoplasmic free ATP, the endogenous Xenopus
type 1 InsP3R channel activated by AdA was indistin-
guishable from the InsP3-liganded channel. The con-
ductance properties, channel gating properties, bipha-
sic Ca2⫹ activation and inhibition, and tuning of the
sensitivity to Ca2⫹ inhibition by the agonist concentra-
tion were identical for InsP3- and AdA-liganded InsP3R.
The efficacy of the two ligands (i.e., Pmax of the channel
that the two ligands can elicit) was also comparable.
However, the potency of AdA as an agonist to reduce
the sensitivity of the channel to Ca2⫹ inhibition (i.e., in-
creasing Kinh) was ⵑ60 times that of InsP3. This figure
agrees well with the affinity of the channel for AdA de-
termined by binding and Ca2⫹ release assays (Taka-
hashi et al., 1994; Hirota et al., 1995; Murphy et al.,
1997). Thus, in the presence of ATP, the sole distin-
guishing feature between channels activated by the two
ligands is the higher functional affinity of the channel
for AdA compared with InsP3. Therefore, the liganded
channel in the presence of ATP must attain compara-
ble structural conformations that result in kinetically
indistinguishable gating and regulatory behaviors, in-
dependent of the nature of the specific ligand.
On the other hand, the nature of the ligand was criti-
cally important in determining the kinetic and regula-
tory properties of the channel when ATP was absent.
ATP has been previously shown to stimulate the activities
of the InsP3-liganded type 1 InsP3R channels (Ferris et
al., 1990; Iino, 1991; Bezprozvanny and Ehrlich, 1993;
Missiaen et al., 1997; Landolfi et al., 1998). In a detailed
study that used the same experimental conditions as
those employed in the present study, ATP was demon-
strated to enhance the sensitivity (lowering Kact) of the
type 1 InsP3R channels to Ca2⫹ activation (Mak et al.,
1999). New data obtained in this study indicates that
ATP also increases the sensitivity of the channel to Ca2⫹
inhibition (lowering Kinh). However, the Pmax and the
gating kinetics (〈␶o〉 and 〈␶c〉) of optimally activated InsP3-
liganded channels are not affected by ATP (Mak et al.,
1999; and this study). Furthermore, the affinity of the
channel for InsP3 is also not substantially affected by ATP
(up to 0.5 mM; this study).
In marked contrast, when the channels were activated
by AdA, the presence or absence of ATP (0 vs. 0.5 mM)
profoundly affected the Pmax of the channel and the gat-
ing kinetics, as well as the potency of AdA to activate the
channel. Although ATP enhanced the sensitivities of the
AdA-liganded X-InsP3R-1 channel to both Ca2⫹ activa-
tion and inhibition to the same extent as for InsP3-
liganded channels, channels activated by AdA in the ab-
sence of ATP had a decreased Pmax, altered gating kinet-
ics (mainly decreased 〈␶o〉), and diminished functional
affinity for AdA. Thus, in the absence of ATP, several fea-
tures distinguish channels activated by either InsP3 or
AdA. Both the efficacy and apparent affinity of AdA be-
come significantly reduced in the absence of ATP.
Whereas AdA is a full agonist in the presence of ATP, it is
only a partial agonist in its absence. InsP3, on the other
hand, is a full agonist in either the presence or absence
of free ATP. Therefore, the InsP3-liganded and AdA-
Figure 13. 〈␶c〉 and 〈␶o〉 of X-InsP3R-1 channels in optimal [Ca2⫹]i
(4.4–6.2 ␮M) and saturating concentrations of various ligands in 0
(white bars) and 0.5 mM (shaded bars) ATP.

309 Mak et al.
liganded channels in the absence of ATP must attain dis-
tinct structural conformations that result in kinetically
distinguishable gating and regulatory behaviors.
Molecular Structural Basis of Interactions between
the InsP3R and Its Agonists
Based on comparisons of the molecular structures of
analogues of InsP3 (Irvine et al., 1984) and AdA (Taka-
hashi et al., 1994; Wilcox et al., 1995; Marchant et al.,
1997; Shuto et al., 1998; Beecroft et al., 1999; Hotoda et
al., 1999) that activate Ca2⫹ release through the InsP3R
channel, and on our study of the single-channel activi-
ties of InsP3R activated by AdA and its analogues under
various conditions, three structural elements can be
identified that contribute to the interactions between
the channel and its agonists.
First, AdA and most of its structural analogues that ac-
tivate the InsP3R with high potency have a glucose moi-
ety with a 3⬘⬘,4⬘⬘-bisphosphate/2⬘⬘-hydroxyl motif (Fig.
1) that is structurally similar to the 4,5-bisphosphate/6-
hydroxyl motif of InsP3 and its analogues that activate
the InsP3R. Therefore, interactions between this struc-
tural element and the InsP3 binding site of the InsP3R
are necessary for activation of InsP3R channel activity.
Second, although many structural analogues of AdA
also bind and activate the InsP3R, their binding affini-
ties (1/Kd) and functional potencies (1/EC50) for the
channel are all significantly lower than those of AdA.
AdA has an adenosine 2⬘-phosphate moiety (Fig. 1) not
present in any of its analogues. Thus, it has been pro-
posed that interactions between this second structural
element and the InsP3R enhance the affinity of the
channel for AdA (Hotoda et al., 1999). As AdA and
ATP share a common adenine moiety in their molecu-
lar structures (Fig. 1), we initially considered that some
interaction of AdA with an ATP binding site(s) in the
InsP3R might contribute to high affinity binding. How-
ever, our single-channel results provide no evidence for
ATP being an antagonist competing with AdA for the
same binding site(s) in the InsP3R, as the presence of
cytoplasmic free ATP enhanced rather than reduced
the functional potency of AdA to activate channel gat-
ing (Figs. 3 and 7). Therefore, we conclude that ATP
and AdA must bind to distinct sites in the InsP3R. In
support of this conclusion, peptides containing puta-
tive ATP-binding sequences in the InsP3R bind ATP but
not AdA (Maes et al., 1999), and the NH2-terminal
ligand-binding domain of the InsP3R itself contains the
site(s) responsible for high affinity binding of AdA to
the receptor (Glouchankova et al., 2000).
The third structural element contributing to AdA in-
teraction with the InsP3R is the 2⬘-phosphoryl group in
the ribose ring of AdA, Rib, and Fur. This element is
probably in an analogous position as the 1-phosphoryl
group in InsP3 (Wilcox et al., 1995), although it has a
different physical location relative to the 3⬘⬘,4⬘⬘-bisphos-
phate/2⬘⬘-hydroxyl motif in AdA and its analogues
compared with the 1-phosphoryl group in InsP3 relative
to the 4,5-bisphosphate/6-hydroxyl motif (Hotoda et
al., 1999). Interaction between this element and the
InsP3R is necessary for the activation of the InsP3R by
its agonists (Irvine et al., 1984)
Molecular Model for Allosteric Effects of
ATP on Ligand Gating of InsP3R
How can we account for the dramatic effects of ATP on
the functional interaction of AdA with the channel?
How is it that, in the presence of ATP, AdA elicited
identical channel activation and gating as InsP3 but
with a much higher potency, whereas in the absence of
ATP, AdA had only approximately twofold higher po-
tency than InsP3 and could only activate the InsP3R half
as efficaciously as InsP3?
As discussed above, there is no evidence for a direct
interaction of AdA and ATP with the same sites in the
InsP3R. Therefore, the effects of ATP on the functional
interaction of AdA with the receptor are likely mediated
by allosteric interactions. We suggest that ATP binds to a
site in the InsP3R different from the NH2-terminal
ligand-binding site, likely within the regulatory domain
that links the ligand-binding domain to the channel do-
main. Binding of ATP to this site produces an allosteric
conformational change in the ligand-binding site that
enhances the binding of AdA to the channel, as illus-
trated in Fig. 14. The model shown in Fig. 14 assumes
that this enhanced binding of AdA to InsP3R is caused
by interaction between the receptor and the adenine
moiety in AdA (Hotoda et al., 1999), but it is possible
that the enhanced functional affinity of AdA to the
InsP3R in the presence of ATP is due to the adenine
structure in AdA positioning the 2⬘-phosphoryl group
in the ribose ring of AdA for a more optimal interaction
with the receptor (Hotoda et al., 1999).
As shown in Fig. 14, the 1-phosphoryl group in InsP3
interacts equally well with the conformations of the
ligand-binding site of the InsP3R in either the presence
or absence of ATP. This interaction elicits the same chan-
nel gating kinetics independent of ATP (Fig. 2). In the
presence of ATP, the 2⬘-phosphoryl group in AdA, Rib,
or Fur can bind to the same phosphoryl group-binding
site in the receptor that InsP3 interacts with, so that the
channel gating kinetics evoked by AdA, Fur, and Rib are
indistinguishable from those evoked by InsP3 (Figs. 2 A
and 8). Under these conditions, AdA has equal effica-
ciousness as a full agonist as InsP3. In contrast, in the dis-
tinct conformation that the InsP3R assumes in the ab-
sence of ATP, the 2⬘-phosphoryl group in AdA, Fur, or
Rib has a different interaction with the InsP3R ligand-
binding site (possibly through an alternate phosphoryl
group interacting site). When AdA is bound to the chan-

310 Adenophostin Activation of InsP3 Receptor Gating
nel in this conformation, the channel gates differently
(Figs. 2 B and 8) because the interaction is less able to
stabilize the channel open state as when the channel is
bound to InsP3 or AdA in the presence of ATP. There-
fore, AdA gates the channel less efficaciously, behaving
as a partial agonist. The interaction between this ele-
ment and the InsP3R is not only necessary for the activa-
tion of the InsP3R by its agonists, but also determines the
gating kinetics of the activated channel.
The regulatory region of the InsP3R, where ATP
likely binds, has been regarded as a transduction re-
gion which links the NH2-terminal ligand-binding do-
main to the gating machinery associated with the
COOH-terminal channel pore region. Our results sug-
gest that the regulatory region influences the proper-
ties of the ligand-binding domain as well as the cou-
pling between ligand binding and channel gating.
Binding of either ligand, InsP3 or AdA, activates chan-
nel gating by destabilizing channel closed states (Mak
et al., 1998). ATP activates the liganded channel also by
destabilizing closed states, tuning the Ca2⫹ sensitivity of
distinct activating Ca2⫹-binding sites (Mak et al., 1999).
Regulation of open channel states has not been previ-
ously implicated in the mechanisms by which InsP3R
channel gating is regulated by ligands, Ca2⫹, and ATP
(Mak et al., 1998; Mak et al., 1999; and this study). The
present study has identified distinct channel open
times as the major kinetic feature that accounts for the
significant reduction in the efficacy of AdA as an ago-
nist. This result now suggests that ligand binding plays
a role in stabilizing channel open states, in addition to
destabilizing closed kinetic states.
Relationship of Channel Gating to Kinetics of
AdA-induced Ca2
⫹
Release through the
InsP3R Observed in Xenopus Oocytes
Previous studies of AdA-induced intracellular Ca2⫹ re-
lease in Xenopus oocytes by confocal imaging (Marchant
and Parker, 1998) or measurements of plasma mem-
brane Ca2⫹-activated Cl⫺ currents (DeLisle et al., 1997;
Hartzell et al., 1997; Machaca and Hartzell, 1999) indi-
cated that Ca2⫹ release through InsP3R activated by AdA
was qualitatively different from that activated by InsP3.
Our results demonstrate that the properties of the
X-InsP3R-1 channel activated by AdA are indistinguish-
able from those activated by InsP3 in the presence of 0.5
mM cytoplasmic ATP. The profound effects of ATP on
the AdA-liganded channels observed in this study were
due to free ATP, as Mg2⫹ was not present. Thus, the dis-
tinct cytoplasmic calcium signals measured in oocytes
activated by AdA may suggest that the level of free ATP
in Xenopus oocyte cytoplasm was lower than 0.5 mM in
Figure 14. Schematic dia-
grams representing interac-
tions between the X-InsP3R-1
molecule and various ligands.
(A) Interaction between
X-InsP3R-1 and InsP3 in the
presence of ATP. (B) Interac-
tion between X-InsP3R-1 and
AdA in the presence of ATP.
(C) Interaction of X-InsP3R-1
and InsP3 in the absence of
ATP. (D) Interaction of
X-InsP3R-1 and AdA in the
absence of ATP.

311 Mak et al.
those studies. Total ATP content in cells is 4–8 mM,
most of which is complexed with Mg2⫹ (Flatman, 1991).
With 4 mM each of ATP and Mg2⫹, free ATP is pre-
dicted to be ⵑ0.35 mM; with 8 mM of each, free ATP is
predicted to be ⵑ0.5 mM. Thus, free ATP concentra-
tions in the oocyte cytoplasm may realistically be ex-
pected to be ⬍0.5 mM, as our results predict.
Nevertheless, some features of the calcium signals
evoked by AdA in oocytes are consistent with a high af-
finity of the InsP3R for AdA, which our results suggest is
dependent on the presence of ATP. The slower rate of
propagation of calcium waves activated by AdA (Bird et
al., 1999; Machaca and Hartzell, 1999) and the more
spatially restricted calcium signals observed in the pres-
ence of AdA (Bird et al., 1999) were both interpreted to
reflect a substantially reduced diffusion coefficient of
AdA in the oocyte cytoplasm because of high affinity
binding to the InsP3R (Machaca and Hartzell, 1999).
Our study suggests that the affinity of the InsP3R for
AdA is significantly higher than that for InsP3 only in
the presence of cytoplasmic free ATP (Figs. 3 and 7).
Thus, the oocyte cytoplasm, although having a free ATP
concentration ⬍0.5 mM, must nevertheless contain a fi-
nite concentration of free ATP, as expected. Therefore,
we conclude that the Xenopus oocytes have cytoplasmic
free ATP concentrations between 0 and 0.5 mM.
Our results demonstrate that AdA may or may not
elicit a similar response from the InsP3R as InsP3, de-
pending on the concentration of cytoplasmic free ATP.
Without knowledge or control of the cytoplasmic free
ATP concentration in experiments using AdA to inves-
tigate intracellular calcium signaling, AdA cannot be
regarded simply as a nonmetabolizable, more potent
substitute for InsP3 as an agonist of the InsP3R.
With this in mind, the single-channel gating kinetics
of the X-InsP3R-1 activated by AdA observed in our nu-
clear patch-clamp experiments can reasonably account
for results obtained in the in vivo Ca2⫹ release studies.
First, the rate of Ca2⫹ release in oocytes stimulated with
a high concentration of AdA (ⵑ2 ␮M) was only half of
that stimulated by high concentrations of InsP3 (ⵑ20
␮M; Machaca and Hartzell, 1999). This can be ex-
plained by our observation that Pmax of the InsP3R chan-
nel activated by AdA was substantially lower than that ac-
tivated by InsP3 at the suboptimal ATP concentrations
in the oocyte cytoplasm. Second, 5 nM AdA elicited a
significantly slower rate of Ca2⫹ release than 2 ␮M AdA
(Machaca and Hartzell, 1999), although both concen-
trations would have been predicted to be saturating for
binding to the channel (Takahashi et al., 1994; Hirota et
al., 1995; Murphy et al., 1997; and this study). Our
patch-clamp experiments revealed that at suboptimal
free ATP concentrations in the oocyte cytoplasm, the af-
finity of AdA for the channel is likely reduced, such that
5 nM AdA may become subsaturating and, therefore,
elicit a significantly lower rate of Ca2⫹ release than 2 ␮M
AdA, as observed. Third, whereas InsP3 invariably stimu-
lated the Ca2⫹-activated Cl⫺ current ICl1-S (Hartzell,
1996; Hartzell et al., 1997; Kuruma and Hartzell, 1999;
Machaca and Hartzell, 1999), AdA was insufficient to
stimulate ICl1-S but still generate store-operated Ca2⫹ in-
flux (DeLisle et al., 1997; Hartzell et al., 1997; Machaca
and Hartzell, 1999). Our experimental results offer a
likely explanation. The rate of Ca2⫹ release activated by
InsP3 was invariably high enough to activate ICl1-S be-
cause of the high Pmax of InsP3-liganded InsP3R chan-
nels. In contrast, in suboptimal cytoplasmic-free ATP
concentrations, the reduced Pmax of the AdA-liganded
channels gave rise to a slower rate of Ca2⫹ release that
was insufficient to stimulate ICl1-S. This slow rate of re-
lease activated by AdA was nevertheless sufficient over
time to deplete the InsP3 sensitive Ca2⫹ stores, thereby
generating store-operated Ca2⫹ influx.
Of considerable interest is the possibility to correlate
distinct single-channel properties of the InsP3R acti-
vated by either InsP3 or AdA with the distinct kinetics of
elementary Ca2⫹ release events (puffs) triggered by
these ligands in Xenopus oocytes. Puffs mediated by the
X-InsP3R-1 have been imaged in oocytes activated se-
quentially by InsP3 and AdA (Marchant and Parker,
1998). These spatially restricted puffs reflect the activa-
tion of several InsP3R channels within a cluster of chan-
nels. The variable amplitudes of puffs can be under-
stood as reflecting variable numbers of InsP3R channels
within clusters (Mak and Foskett, 1997; Sun et al., 1998;
Thomas et al., 1998) and the stochastic nature of the
channel gating (Mak and Foskett, 1997). Puffs have
been characterized by quantitative determinations of
the peak change in fluorescence, as a measure of the
peak rate of Ca2⫹ liberation; the duration and the rise
time, both reflecting the length of time the channels
were open to release Ca2⫹; and signal mass, represent-
ing the total amount of Ca2⫹ released (Marchant and
Parker, 1998; Sun et al., 1998). A major unresolved
question in calcium signaling is the nature of the mech-
anisms that regulate the duration of Ca2⫹ release dur-
ing a puff. Puffs elicited by activation with low concen-
trations of AdA had similar peak rates of Ca2⫹ libera-
tion but were temporally shorter (faster rise time and
shorter duration) and released less total Ca2⫹ com-
pared with those activated by InsP3 (Marchant and
Parker, 1998). The principle conclusion from these re-
sults was that the duration of a Ca2⫹ puff bears no sim-
ple relationship to the affinity of the agonist, ruling out
agonist dissociation as the mechanism which delimits
the period of Ca2⫹ flux through the InsP3R channels
during a puff (Marchant and Parker, 1998). Therefore,
it was speculated (Marchant and Parker, 1998) that
Ca2⫹-mediated inhibition (Parker and Ivorra, 1990;
Finch et al., 1991) and InsP3-induced channel inactiva-

312 Adenophostin Activation of InsP3 Receptor Gating
tion (Hajnóczky and Thomas, 1994; Mak and Foskett,
1997) may be involved. Our results now suggest an-
other possible mechanism. Our study of the single-
channel properties of the X-InsP3R-1 has revealed that
in suboptimal ATP concentrations, the main difference
between InsP3R channels activated by AdA and InsP3 is
the significantly shorter 〈␶o〉 of the AdA-liganded chan-
nels (Fig. 10, B and C). Thus, there is a correlation be-
tween 〈␶o〉 of the single InsP3R channel and the rise
time, duration, and total amount of Ca2⫹ released of a
Ca2⫹ puff. Therefore, we suggest that a major determi-
nant of the duration of Ca2⫹ release, and of the amount
of Ca2⫹ released during a puff, is the ligand-dependent
〈␶o〉, rather than Ca2⫹-mediated inhibition or ligand-
induced channel inactivation. It is interesting to note
that Ca2⫹ sparks mediated by ryanodine receptor Ca2⫹
release channels in frog skeletal muscle fibers have
faster rise times and reduced total Ca2⫹ released when
〈␶o〉 of the channels is prematurely shortened by mem-
brane repolarization (Lacampagne et al., 2000). Thus,
〈␶o〉 can be a major determinant of the duration of ele-
mentary Ca2⫹ release events mediated by both major
families of intracellular Ca2⫹ release channels.
Based on our studies of the regulation of the single-
channel activities of X-InsP3R-1, we consider the follow-
ing scenario as one that can account for the correlation
between 〈␶o〉 and the duration of a puff. It is generally
believed that each Ca2⫹ puff is initiated by the stochas-
tic opening of one of the InsP3R channels clustered to-
gether in the Ca2⫹ release site (Yao et al., 1995). The
Ca2⫹ released by one channel can diffuse to neighbor-
ing channels, increasing the local [Ca2⫹]i in the vicinity
of those channels so that they too open, by Ca2⫹ activa-
tion (Ca2⫹ induced Ca2⫹ release). Other mechanisms
may serve to couple the channels to affect a concerted
opening of several of them (Mak and Foskett, 1997;
Marx et al., 1998). This rapid concerted activation of
the channels in a cluster generates the Ca2⫹ puffs (Yao
et al., 1995; Mak and Foskett, 1997; Sun et al., 1998).
The locally high [Ca2⫹]i near the channels as a result of
this liberation can, in turn, feed back to inhibit them
(Figs. 3, 4, and 7). Indeed, because puffs are generated
under conditions of low agonist concentration (Yao et
al., 1995; Berridge, 1997; Marchant and Parker, 1998),
the channel has a high sensitivity to Ca2⫹ inhibition
(Mak et al., 1998; and this study). Nevertheless, a criti-
cal observation is that 〈␶o〉 has very little dependence on
[Ca2⫹]i (Fig. 10). This lack of sensitivity of the open
channel to Ca2⫹ inhibition implies that, once a channel
has opened, it will stay open for a duration approxi-
mately equal to 〈␶o〉, independent of the local [Ca2⫹]i in
the vicinity of the channel. Only after the channel
closes can cytoplasmic Ca2⫹ feed back to inhibit it from
reopening, as Ca2⫹ inhibition of gating operates by sta-
bilizing the channel closed state (Fig. 10). Thus, once a
channel has opened during a puff, it will stay open for
a duration approximately equal to 〈␶o〉 and then close.
At that time, it is possible that the high local [Ca2⫹]i,
contributed by Ca2⫹ released from the channel itself as
well as from its neighbors, will prevent it from reopen-
ing within the duration of the puff. Therefore, during a
single puff, each channel in the Ca2⫹ release site likely
opens at most once. As the conductance properties of
the AdA- and InsP3-liganded channels are indistin-
guishable (Fig. 2), the mean amount of Ca2⫹ released
in an opening of each InsP3R channel in a cluster is
therefore predicted to be directly proportional to 〈␶o〉.
This model also predicts that the mean duration of the
puff will be proportional to 〈␶o〉.
Alternatively, the puff could terminate as a result of
the stochastic nature of channel gating without invok-
ing Ca2⫹ inhibition of reopening. When all the acti-
vated channels in the cluster become closed at the
same time, simply as a result of the nonzero probability
that the stochastic closed times of all the activated
channels will coincide, the Ca2⫹ release needed for
stimulation of further openings will be eliminated,
thereby extinguishing the puff (Niggli, 1999; Stern et
al., 1999). This model is similar to the stochastic attri-
tion model developed by Stern (1992) to help account
for termination of Ca2⫹ release through clusters of ry-
anodine receptors. Analytical derivation of the time
constant for the puff duration in the stochastic attrition
model showed it to be directly proportional to the
channel mean open duration (Stern et al., 1999). Thus,
models using Ca2⫹ inhibition or stochastic attrition
both suggest that the difference between the duration
of puffs and the quantities of Ca2⫹ released by puffs ac-
tivated by InsP3 and AdA (Marchant and Parker, 1998)
is a consequence of the difference in the mostly Ca2⫹-
independent 〈␶o〉 of the InsP3- and AdA-liganded
InsP3R channels. Importantly, our results demonstrate
that this difference is a function of the cytoplasmic free
ATP concentration, suggesting that free ATP concen-
tration helps to shape the properties of elementary
Ca2⫹ release signals generated by AdA.
This work was supported by grants to J.K. Foskett from the Na-
tional Institutes of Health (MH59937 and GM56328) and to
D.-O.D. Mak from the American Heart Association (9906220U).
Submitted: 21 December 2000
Revised: 12 February 2001
Accepted: 13 February 2001
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