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Biophys J, July 2000, p. 271-278, Vol. 79, No. 1
and
*Department of Physiology, University of Connecticut Health Center,
Farmington, Connecticut 06030, Departments of
Pharmacology and Cellular and Molecular Physiology, Yale
University, New Haven, Connecticut 06520 USA
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Many hormones and neurotransmitters raise intracellular
calcium (Ca2+) by generating InsP3 and
activating the inositol 1,4,5-trisphosphate receptor
(InsP3R). Multiple isoforms with distinct InsP3
binding properties (Cardy et al., 1997
) have been identified (Morgan et al., 1996). The type III InsP3R lacks
Ca2+-dependent inhibition, a property that makes it ideal
for signal initiation (Hagar et al., 1998). Regulation of the type III
InsP3R by InsP3 and ATP was explored in detail
using planar lipid bilayers. In comparison to the type I
InsP3R, the type III InsP3R required a higher
concentration of InsP3 to reach maximal channel activity (EC50 of 3.2 µM versus 0.5 µM for the types III and I
InsP3R, respectively). However, the type III
InsP3R did reach a 2.5-fold higher level of activity.
Although activation by InsP3 was isoform-specific, regulation by ATP was similar for both isoforms. In the presence of 2 µM InsP3, low ATP concentrations (<6 mM) increased the
open probability and mean open time. High ATP concentrations (>6 mM) decreased channel activity. These results illustrate the complex nature
of type III InsP3R regulation. Enhanced channel activity in
the presence of high InsP3 may be important during periods of prolonged stimulation, whereas allosteric modulation by ATP may help
to modulate intracellular Ca2+ signaling.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The inositol 1,4,5-trisphosphate receptor
(InsP3R) is an important component of
intracellular Ca2+ signaling in most cells
(Berridge, 1993; Clapham, 1995). Activation of the
InsP3R opens a
Ca2+-permeable channel that leads to an increase
in cytoplasmic Ca2+ by releasing
Ca2+ from the endoplasmic reticulum.
InsP3-mediated Ca2+ release
regulates many cellular processes, such as the expression of
transcription factors (Negulescu et al., 1994), the formation of the
fertilization envelope during egg activation (Nuccitelli et al., 1993),
stimulus-contraction coupling in smooth muscle (Walker et al., 1987),
and the development of long-term depression (Finch and Augustine, 1998;
Inoue et al., 1998; Khodakhah and Armstrong, 1997).
The InsP3R exists as a tetramer in which each
subunit is 260 kD. Three isoforms have been cloned (Blondel et al.,
1993; Furuichi et al., 1989; Maranto, 1994; Mignery et al., 1989;
Morgan et al., 1996; Sudhof et al., 1991). Both the expressed isoform
and the extent of expression depend upon the cell type. Cerebellar
Purkinje cells, for example, express almost exclusively the type I
InsP3R. Pancreatic acinar cells express types II
and III InsP3R, whereas several epithelia express
all three isoforms (Bush et al., 1994; Nathanson et al., 1994;
Wojcikiewicz, 1995). Additional receptor diversity is achieved through
the formation of heterotetramers due to the association of different
isoforms (Joseph et al., 1995; Monkawa et al., 1995).
Ca2+ release by the type I
InsP3R is regulated by a variety of cofactors and
cellular processes, including cytosolic and intraluminal free
Ca2+, phosphorylation of the
InsP3R, and intracellular pH (Ferris et al.,
1991a,b; Finch et al., 1991; Iino, 1990; Missiaen et al., 1992;
Supattapone et al., 1988). Regulation by Ca2+ is
bell-shaped with maximal channel activity occurring at 300 nM free
Ca2+ (Bezprozvanny et al., 1991). ATP has been
shown to enhance the gating of the type I InsP3R
by allosteric regulation (Bezprozvanny and Ehrlich, 1993; Mak et al.,
1999). In Xenopus oocyte nuclei, ATP was shown to alter the
Ca2+ sensitivity of
Ca2+-activation sites on the
InsP3R (Mak et al., 1999). This type of
regulation allows ATP to shape the extent and duration of cytoplasmic Ca2+ signals depending upon stimulus intensity
and a cell's metabolic state.
The InsP3R contains a large regulatory domain
between the InsP3 binding site (at the
NH2-terminal end) and the pore-forming region (the COOH-terminal end,
which contains six membrane-spanning regions) where these modulators
are able to exert their effects. Additional cytoplasmic factors and
associated proteins (MacKrill, 1999; Thrower et al., 1998) contribute
important regulatory functions that permit the wide range of responses
that are seen for InsP3-induced Ca2+ release. Pancreatic cells, for example,
display markedly different Ca2+ responses
depending on the agonist (such as acetylcholine and cholecystokinin)
that is used to stimulate the cells (Lawrie et al., 1993; Osipchuk et
al., 1990; Petersen et al., 1991; Thorn et al., 1993; Yule et al.,
1991). These agonists can produce different Ca2+
oscillation patterns by affecting the phosphorylation state of the
InsP3R (LeBeau et al., 1999).
Recently, the single-channel properties of the types II and III
InsP3R have been determined in planar lipid
bilayers. The types II and III InsP3R do not show
the same bell-shaped Ca2+-dependence curve as the
type I InsP3R (Hagar et al., 1998; Ramos-Franco et al., 1998). Despite this difference in Ca2+
regulation, ion permeation and channel gating properties for the type
III InsP3R are similar to the type I
InsP3R (Mak et al., 2000). The types II and III
InsP3R, however, are ideal for signal initiation
because they lack Ca2+-dependent negative
feedback
a characteristic that supports the generation of
Ca2+ oscillations. Differences in the
Ca2+-dependence of the three isoforms may
underlie the spatial and temporal patterns of cytosolic
Ca2+ signals that are important for many cellular
responses. Further, the subcellular distribution of the different
isoforms may help to coordinate Ca2+ signals
within the cell.
The types II and III InsP3R have an overall
sequence homology with the type I InsP3R of 69 and 64%, respectively (Blondel et al., 1993; Sudhof et al., 1991).
Despite their similarity, the three isoforms possess different
affinities for InsP3: the relative order of
affinity is type II > type I > type III (Maranto, 1994). A
specific binding site for ATP on each subunit of the InsP3R has been identified through biochemical
studies (Maeda et al., 1991). For the type I
InsP3R, low concentrations of ATP (<4 mM)
increase channel open probability (Bezprozvanny and Ehrlich, 1993).
Concentrations of ATP above 4 mM cause a decrease in channel activity.
In the current study, the effects of both InsP3 and ATP on the type III InsP3R were investigated. Channel activity and mean open time were measured over a range of InsP3 concentrations. The type III InsP3R required a 10-fold higher InsP3 concentration (with respect to the type I InsP3R) to be maximally activated. As found for the type I InsP3R, low concentrations of ATP (<6 mM) increased the open probability of the type III InsP3R, and high concentrations of ATP (>6 mM) inhibited channel activity. Elevated InsP3 levels restored channel activity inhibited by ATP, suggesting that ATP can displace InsP3 from the InsP3 binding site. Thus, the effects of ATP on the types I and III InsP3R are similar, whereas the activation by InsP3 is isoform-specific.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Isolation of microsomes
Endoplasmic reticulum microsomes were isolated from RIN-m5F
cells using the gradient centrifugation protocol for cerebellum as
described previously (Bezprozvanny and Ehrlich, 1994). All solutions
used for the isolation and storage of microsomes included a protease
cocktail (5 µg/ml leupeptin, 2 µg/ml aprotinin, 1 µg/ml pepstatin
A, 10 µg/ml trypsin inhibitor, and 1 mg/ml Pefabloc Plus) to minimize proteolysis.
Single-channel recordings
Microsomal vesicles were fused into planar lipid bilayers
composed of the synthetic lipids phosphatidylethanolamine and
phosphatidylserine (3:1, wt:wt; Avanti Polar Lipids, Alabaster, AL)
dissolved in decane (20 mg/ml) so that the cis and
trans chambers corresponded to the cytosol and lumen of the
endoplasmic reticulum, respectively. Cytoplasmic bilayer solutions
contained 110 mM TRIS and 250 mM HEPES (pH 7.35), and lumenal solutions
contained 53 mM Ba(OH)2 and 250 mM HEPES (pH
7.35). The trans chamber was held at virtual ground, and the
transmembrane voltage was maintained at 0 mV. Single-channel currents
were recorded under voltage clamp conditions using a bilayer clamp
amplifier (BC-525B, Warner Instruments Corp., Hamden, CT) and stored on
VHS tape (Instrutech Corp., Great Neck, NY). Data were filtered at 1 kHz and digitized at 4 kHz for computer analysis using pClamp 6.0.3 (Axon Instruments, Foster City, CA). Additions of
InsP3 (Calbiochem, La Jolla, CA), ATP (disodium
salt, Sigma, St. Louis, MO), and GTP (disodium salt, Sigma) were made to the cis chamber to obtain the desired concentrations of
each compound. The cytosolic Ca2+ concentration
was fixed at 160 nM (pCa = 6.8) using calibrated amounts of
CaCl2 and EGTA in the cis chamber
(Fabiato, 1988). At least 3 min of continuous recording was analyzed to
determine mean open times, mean closed times, open probability, and
current amplitude. The number of active channels in each bilayer was
estimated using a statistical model that is dependent on the maximum
number of channels that are observed simultaneously during the course of an experiment (Horn, 1991).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Open probability of the type III InsP3R as a function of InsP3 concentration
After incorporation of RIN-m5F endoplasmic reticulum microsomes
with planar lipid bilayers, the type III InsP3R
was activated with InsP3 concentrations ranging
from 0.1 to 200 µM (Fig. 1, A and B). In these experiments, 0.5 mM ATP and 2 µM ruthenium red were present. The ATP was added to act as a cofactor
in the activation of the channel, and ruthenium red was added to
inhibit any ryanodine receptors that may have co-incorporated into the bilayer. A 10-fold increase in InsP3
concentration (2 to 20 µM) caused a threefold increase in channel
open probability (2.2 to 7.6%). To fit the data with the Hill
equation, a value of 1.9 was used for the Hill coefficient, suggesting
that at least 2 molecules of InsP3 are needed to
bind to the receptor complex before the channel will open. Over this
range of InsP3 concentrations, the mean open time
was unchanged (Fig. 1 C) for the type III
InsP3R. The open probability was maximal once the
InsP3 concentration was raised to 20 µM (Fig.
1, A and B, circles and solid
line). In contrast, the open probability for the type I
InsP3R was maximal with 2 µM
InsP3 (Fig. 1 B, dashed
line; data from Moraru et al., 1999). As shown in Fig.
1 B, the EC50 for the types I and III InsP3R were 0.5 and 3.2 µM, respectively.
Finally, the type III InsP3R remained very active
at 10 nM free cytoplasmic Ca2+ in the presence of
20 µM InsP3 (see Fig. 4 D; open
probability was 6.8 ± 0.8%, n = 3). This high
level of activity at high InsP3 and low
Ca2+ was predicted by a recent model that
considers the effect of Ca2+ on
InsP3 binding for the type III
InsP3R (LeBeau et al., 1999).
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Activation of the type III InsP3R by low concentrations of ATP
In the presence of 2 µM InsP3 and 2 µM
ruthenium red, the channel open probability was observed with
increasing amounts of ATP (Fig. 2,
A and C). Variation in the
single-channel open probability in separate experiments was minimized
by normalizing the single-channel open probabilities in each experiment
to the value obtained at 6 mM. The data were then fit using a modified
Hill equation:
![<UP>P</UP>(x)=<UP>P</UP>(o)+[<UP>P</UP><SUB><UP>max</UP></SUB> ∗ x<SUP><UP>n</UP></SUP>]/[k<SUP><UP>n</UP></SUP>+x<SUP><UP>n</UP></SUP>],](/content/vol79/issue1/fulltext/271/img001.gif)
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(1) |
).
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As the ATP concentration was raised from 0.5 to 6 mM, the mean open
time increased (Fig.
3 A),
whereas mean closed time decreased (Fig. 3 B). Over this
range of ATP concentrations, the current amplitude was unchanged (Fig.
3 C). A similar result was obtained for the type I
InsP3R in Xenopus oocyte nuclei; ATP
activated gating by stabilizing the open state and destabilizing the
closed state (Mak et al., 1999). As a result, it is likely that ATP
activates the type III InsP3R by increasing both
the duration of channel openings (mean open time) and the frequency of
the openings (by decreasing the mean closed time).
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Inactivation of the type III InsP3R by high concentrations of ATP
High ATP concentrations (>6 mM) inhibited the type III
InsP3R (Fig. 4,
A and C). The single-channel
open probability in each experiment was normalized to the channel open
probability at 6 mM ATP. The steep inhibitory effect of high ATP
concentrations suggests cooperative binding of ATP to a low affinity
site as found previously for the type I InsP3R
(k = 10.6 mM, n = 5; Bezprozvanny and
Ehrlich 1993). Although channel activity was completely inhibited by
these high levels of ATP, activity was restored by raising the
InsP3 concentration (Fig. 4 B). This
finding supports previous studies that have found a competitive
interaction between InsP3 and ATP (Guillemette et
al., 1987; Iino, 1991; Maeda et al., 1991; Spat et al., 1987).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In this paper, two properties of the type III
InsP3R were examined: the effects of different
InsP3 concentrations and regulation by ATP. The
channel open probability increased threefold as the InsP3 concentration was increased from 2 to 20 µM, and open probability was maximal at 20 µM
InsP3 (Fig. 1, A and B).
The type III InsP3R reached a maximal level of
channel activity that was ~2.5-fold higher than the maximal level for
the type I InsP3R (Fig. 1 B, dashed line; data from Moraru et al., 1999). Although both
isoforms required the binding of at least two molecules of
InsP3 to open the channel, the type I
InsP3R activated at lower
InsP3 concentrations (the
EC50 was 0.5 µM for the type I
InsP3R and 3.2 µM for the type III
InsP3R). For these two reasons, activation by
InsP3 is an isoform-specific characteristic.
The EC50 for the type III
InsP3R (3.2 µM) is much higher than the
InsP3 binding affinity of the type III
InsP3R (Kd = 151 nM) (Maranto, 1994). However, this value for the
EC50 is consistent with the
InsP3 concentration that is necessary for
half-maximal release in both pancreatic islet cells (2.5 µM) (Wolf et
al., 1985) and insulinoma microsomes (3.0 µM) (Prentki et al., 1984). In addition, InsP3 concentrations needed to
induce half-maximal Ca2+ release are often in the
µM range (Berridge, 1987) even though InsP3
binding affinities in a variety of tissues are in the low nM range
(Taylor and Richardson, 1991). This discrepancy is likely due to the
very different conditions under which InsP3
binding and Ca2+ release measurements are
generally performed. Frequently, InsP3 binding is
done on ice, in the absence of ATP, and at pH 8.5, whereas
Ca2+ mobilization is done at 37°C, in the
presence of ATP and at physiological pH, 7.4. When conditions for
binding and release were matched, similar values for the
Kd and EC50 were obtained
(Mauger et al., 1989; Nunn and Taylor, 1990).
Calcium release from rat insulinoma microsomes (containing
predominately the type III InsP3R) showed a
similar threefold increase in open probability when the
InsP3 concentration was raised from 2 to 20 µM
(Prentki et al., 1984). Interestingly, a second addition of
InsP3 did not elicit a subsequent release of
Ca2+. In light of the fact that the type III
InsP3R can deplete intracellular stores in
RIN-m5F cells (Hagar et al., 1998), it is likely that activation of the
type III InsP3R allowed depletion of the
microsomal Ca2+ stores during the first
stimulation so that a second response did not occur.
Despite the change in open probability as the
InsP3 concentration was increased from 0.1 to 200 µM, the mean open time did not change (Fig. 1 C). The
increase in channel activity is unlikely to involve other cellular
processes that have been implicated in the regulation of
InsP3R (Cameron et al., 1995). Phosphorylation of
the channel by protein kinase C, for example, could not occur because
the experimental protocol uses Na-ATP and does not include a kinase. A
low affinity InsP3 binding site
(Kd = 10 µM) has recently been
identified for the type I InsP3R (Kaftan et al.,
1997) and explains persistent channel activity at high
Ca2+ and InsP3 levels. An
alternate explanation is that an elevated InsP3
concentration relieves Ca2+ inhibition by
decreasing the affinity of Ca2+ for a
Ca2+ inhibitory site on the
InsP3R (Mak et al., 1998). It is presently unknown whether the type III InsP3R also
possesses the low affinity InsP3 binding site, or
if elevated InsP3 relieves Ca2+ inhibition.
Calmodulin, a Ca2+-dependent regulatory protein
with four Ca2+-binding sites (Means and Dedman,
1980), has recently been shown to mediate
Ca2+-dependent inactivation of the type I
InsP3R (Michikawa et al., 1999). Although RIN-m5F
cells contain high levels of calmodulin, which are not affected by
glucose stimulation (Nelson et al., 1983), the type III
InsP3R is unique among the
InsP3R isoforms because it does not bind
calmodulin (Yamada et al., 1995).
InsP3 binding to the type III
InsP3R is enhanced by an increase in cytoplasmic
Ca2+ (Cardy et al., 1997; Yoneshima et al.,
1997). Modeling that considers the effect of Ca2+
on InsP3 binding predicts a leftward shift of the
steady-state open probability curve for the type III
InsP3R as the InsP3
concentration is increased (LeBeau et al., 1999). Indeed, a high level
of channel activity exists in the presence of 10 nM
Ca2+ and 20 µM InsP3
(Fig. 1 D). Under these conditions, the type I
InsP3R is not active (Kaftan et al., 1997).
Likewise, open probability for the type III
InsP3R is very low at this
Ca2+ concentration when the
InsP3 concentration is 2 µM (Hagar et al.,
1998). Consequently, high InsP3 concentrations
can strongly activate the type III InsP3R even when cytoplasmic
Ca2+ levels are low. High concentrations of
InsP3 have been found in a variety of cell types
under both basal (0.1-3 µM InsP3) and agonist-stimulated (1-20 µM) conditions (Putney, 1990).
Two putative ATP binding sites are located in the region between the
InsP3 binding site and the transmembrane region
in the primary sequence of the InsP3R (Furuichi
et al., 1989; Mignery et al., 1990; Mignery and Sudhof, 1990). Both
high- and low-affinity sites are present and mediate the effects of
ATP. For the type I InsP3R, the high affinity
site provides allosteric regulation of channel activity (Iino, 1991;
Maeda et al., 1991; Smith et al., 1985); ATP concentrations (<4 mM)
increase the intrinsic efficacy of InsP3
(Bezprozvanny and Ehrlich, 1993). Low concentrations of ATP are also
able to increase channel open probability for the type III
InsP3R (Fig. 2, A and C).
The increase in channel activity reflects an increase in the mean open
time and a decrease in mean closed time with no change in current
amplitude (Fig. 3, A-C) as found for the type I
InsP3R of dog cerebellum (Bezprozvanny and
Ehrlich, 1993) and Xenopus oocyte nuclei (Mak et al., 1999). In addition, this effect is specific for ATP; GTP is unable to increase
channel activity (Fig. 2, B and C,
squares).
High ATP concentrations inhibit InsP3-induced
Ca2+ release from permeabilized smooth muscles if
low InsP3 concentrations are used (Iino, 1991).
Other studies also suggest a competitive interaction between
InsP3 and ATP (Guillemette et al., 1987; Maeda et
al., 1991; Spat et al., 1987) and imply that the low affinity site is
the InsP3 binding site. High ATP concentrations
inhibit channel activity for both the type I (Bezprozvanny and Ehrlich,
1993) and type III (Fig. 4, A and C)
InsP3R. In both cases, raising the
InsP3 concentration restores activity in the
presence of an inhibitory amount of ATP (Fig. 4 B).
In summary, InsP3 and ATP both regulate the
activity of the type III InsP3R in important
ways. High InsP3 concentrations (10-100 µM)
maintain channel activity and may allow a cell to raise cytoplasmic Ca2+ levels during periods of prolonged
stimulation. Regulation of the types I and III
InsP3R by ATP is similar: both isoforms are activated by low concentrations of ATP (<6 mM) and inhibited by high
concentrations of ATP (>6 mM). From a mechanistic point of view, it is
likely that ATP allosterically regulates each isoform at low
concentrations and competes for the InsP3 binding
site at high concentrations. Allosteric regulation of the type III InsP3R by ATP may be important for the modulation
of intracellular Ca2+ signaling (Ferris et al.,
1990) and the maintenance of cell viability during conditions of energy
starvation (Katz, 1992).
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FOOTNOTES |
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Received for publication 18 January 2000 and in final form 4 April 2000.
Address reprint requests to Robert Hagar, Department of Pharmacology, Yale University, 333 Cedar St., New Haven CT 06520. Tel.: 203-737-1158; Fax: 203-785-7670; E-mail: rob{at}hermen.med.yale.edu.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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an intracellular calcium receptor.
Nature.
285:73-77[Medline].
Biophys J, July 2000, p. 271-278, Vol. 79, No. 1
© 2000 by the Biophysical Society 0006-3495/00/07/271/08 $2.00
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