Inositol 1,4,5-Trisphosphate and Calcium Regulate the Calcium Channel Function of the Hepatic Inositol 1,4,5-Trisphosphate Receptor*

(Received for publication, September 17, 1996, and in revised form, November 8, 1996)

Jean-François Dufour Dagger , Irwin M. Arias and Timothy J. Turner

From the Department of Cellular and Molecular Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The regulation of the inositol 1,4,5-trisphosphate (IP3) receptor in liver was analyzed using a novel superfusion method. Hepatic microsomes were loaded with 45Ca2+, and superfused at high flow rates to provide precise control over IP3 and Ca2+ concentrations ([Ca2+]) and to isolate 45Ca2+ release from reuptake. 45Ca2+ release was dependent on both [Ca2+] and IP3. The initial rate of 45Ca2+ release was a biphasic function of [Ca2+], increasing as [Ca2+] approached 3 µM but decreasing at higher concentrations, suggesting that the hepatic IP3 receptor is regulated by [Ca2+] at two sites, a high affinity potentiation site and a low affinity inhibitory site. The relationship between initial rates and IP3 concentration was steep (Hill coefficient of 3.4), suggesting that activation of the calcium channel requires binding of at least 3 IP3 molecules. IP3 concentrations above 10 µM produced rapid decay of release rates, suggesting receptor inactivation. Superfusion with 10 µM IP3 under conditions that minimize calcium release ([Ca2+] < 1 nM) inhibited 45Ca2+ release in response to subsequent stimulation (400 nM Ca2+). These data suggest sequential positive and negative regulation of the hepatic IP3 receptor by cytosolic calcium and by IP3, which may underlie hepatocellular propagation of regenerative, oscillatory calcium signals.


INTRODUCTION

In many cells including hepatocytes, binding of certain hormones to cell surface receptors results in the activation of phospholipase C, thereby generating the intracellular second messenger inositol 1,4,5-trisphosphate (IP3).1 The intracellular receptor for IP3 is a calcium channel that permits efflux of Ca2+ sequestered in the endoplasmic reticulum out into the cytoplasm. Calcium diffuses through the cytoplasm, and under certain conditions, kinematic waves of elevated Ca2+ concentration can be observed (1). The amplitude of these waves is roughly constant, but the frequency of the waves correlates with extracellular receptor agonist concentration. Thus, calcium waves couple the activation of cell surface receptors to activation of calcium-dependent intracellular processes in a frequency-encoded manner (2). The importance and complexity of changes in Ca2+ concentration in transmitting information implies that calcium signaling is tightly regulated.

The IP3 receptor occupies a central position among the multitude of molecules involved in intracellular calcium metabolism (1, 2). It possesses regulatory properties that in principle can account for the spatio-temporal complexity of calcium signals (1, 2). In many tissues, cytoplasmic Ca2+ concentrations modulate IP3-dependent calcium release in a biphasic manner (3-7). At submicromolar concentrations, Ca2+ activates the release process, whereas, at higher concentrations, Ca2+ is inhibitory. This regulation by calcium has been proposed to be a means of controlling calcium oscillations (1, 2). The role of IP3 in regulating the activity of its receptor is less clear. Results that support (8) as well as undermine (5, 9, 10) cooperative receptor activation have been reported. Recently, an inhibitory role for IP3 has been proposed (11).

The aim of this study was to characterize the regulation of the hepatic IP3 receptor by both Ca2+ and IP3. A superfusion assay, which was developed to study IP3-dependent calcium release from brain microsomes (5), was adapted for use with liver microsomes. This assay achieves subsecond time resolution, allows for precise control of extravesicular Ca2+ and IP3 concentrations, and isolates the release of 45Ca2+ from its reuptake. We show that both extravesicular Ca2+ and IP3 can activate and inhibit the calcium channel function of the IP3 receptor.


EXPERIMENTAL PROCEDURES

Materials

D-myo-Inositol 1,4,5-trisphosphate (hexapotassium salt) was purchased from LC Laboratories (Woburn, MA). Magnesium ATP, MOPS, dithiothreitol (DTT), EDTA, EGTA, HEPES, Percoll, phenylmethylsulfonyl fluoride, antipain, leupeptin, pepstatin, and other chemicals were obtained from Sigma. Hanks' buffer saline solution was from Life Technologies, Inc. Fura 2 was from Molecular Probes (Eugene, OR). 45CaCl2 was purchased from DuPont NEN.

Preparation of Microsomes

A low-sedimentation low-density microsomal fraction (12) was prepared from sodium pentobarbital-anaesthetized male Sprague-Dawley rats (200-300 g), perfused in situ with 100 ml of Hanks' buffer saline solution containing 0.5 mM EGTA, 1 mM DTT, and protease inhibitors (phenylmethylsulfonyl fluoride, 100 µg/ml, leupetin, 2 µg/ml, pepstatin, 1 µg/ml, and antipain, 2 µg/ml, pH 7.3). The tissue was minced in 40 ml of ice-cold sucrose buffer (250 mM sucrose, 5 mM HEPES, 5 mM KOH, 1 mM EDTA, 1 mM DTT, and protease inhibitors, pH adjusted with KOH to 7.4), homogenized with a glass tissue grinder (BellCo) (10 loose strokes, 10 tight strokes), and centrifuged for 10 min at 500 × g. The supernatant was centrifuged for 10 min at 2,000 × g and the resulting supernatant was further centrifuged for 40 min at 36,000 × g to sediment the microsomes (low sedimentation). The pellet was resuspended in sucrose buffer containing 35% (v/v) Percoll and fractionated by centrifugation at 36,000 × g for 30 min. The superior low density fraction was washed twice in 10 volumes of MK buffer (120 mM KCl, 20 mM MOPS, pH 7.2), with 1 mM DTT and protease inhibitors. The final pellet was resuspended at 2-5 mg/ml in MK buffer with protease inhibitors and 0.7 M glycerol, divided into 50-µl portions, and stored at -70 °C.

45Ca2+ Release Assay

Microsomes were thawed on ice and loaded with 45Ca2+ for 10 min at room temperature in 2.5 volumes of loading buffer (MK buffer containing 5 mM MgATP, 50 µM EGTA, 45CaCl2 (free 45Ca2+ 10 µM), 1 mM DTT, and protease inhibitors, pH 7.2). Loaded microsomes were then superfused as described previously (13). Briefly, the microsomes were washed in 16 volumes of basal buffer (100 mM KCl, 20 mM MOPS, 1 mM EGTA, 5 mM MgCl2, free Ca2+ less than 1 nM), applied to a filter support, and mounted in a superfusion chamber accessed by three solenoid-driven valves that were operated by computer. Each valve controlled the delivery of a separate pressurized solution to the chamber. Microsomes were superfused at 40 p.s.i. (flow rate ~ 2 ml/s) for 1.05 s with basal buffer to establish a baseline rate of release. Release was evoked at t = 0, by switching to a second stimulation buffer (100 mM KCl, 20 mM MOPS, 4 mM EDTA, 5 mM MgCl2, sufficient CaCl2 to obtain the desired free calcium concentration, and IP3 at the concentration specified for each experiment). The superfusate containing released 45Ca2+ was collected with a rotating fraction collector, which divided the effluent stream into 70-ms fractions. Radioactivity in each fraction and that remaining on the filter was measured by liquid scintillation counting (Beckman LS 6800) after addition of 2 ml of Biosafe II (Research Products International, Mount Prospect, IL) to each fraction. Experiments were performed in groups of 6, 3 with the desired concentrations of IP3 and 3 with no IP3 to control for calcium-dependent release, in a randomized sequence to minimize any time-dependent changes in the preparation.

Calculations

45Ca2+ release rates were calculated by dividing the amount of radioactivity in each fraction by the total amount remaining on the filter, expressed as % of total. In all experiments, the baseline calcium release rates were adjusted to the mean of 45Ca2+ release during the 3 fractions preceding the switch to the stimulating buffer. This adjustment was generally on the order of 5-10% of the baseline rate of release. Net IP3-dependent calcium release was calculated by subtracting the release rates observed in the absence of IP3 from the rates in the presence of IP3 for each concentration of Ca2+ (5). Cumulative IP3-dependent calcium release was obtained by stepwise addition of IP3-dependent calcium release rates. The peak rate of release was defined as the largest IP3-dependent 45Ca2+ release rate in a single fraction, expressed in percent per s. The initial rate of release was defined as the rate of 45Ca2+ release (expressed in percent per s) derived from the cumulative release between t = 0 and the fraction with maximum rate of release.

Calcium Buffers

Free Ca2+ concentrations in the loading and stimulation buffers were calculated according to Fabiato and Fabiato (14), assuming a nominal Ca2+ concentration of 3 µM in distilled water. These calculations agreed with measurements of free Ca2+ obtained with Fura 2 (for free Ca2+ concentrations less than 1 µM) or with an ion-selective electrode (for free Ca2+ concentrations above 1 µM).


RESULTS

The Kinetics of IP3-dependent Calcium Release Were Complex

Superfusion of 45Ca2+ loaded hepatic microsomes with a stimulation buffer containing 400 nM Ca2+ and no IP3 resulted in a small but sustained increase in the rate of 45Ca2+ efflux (Fig. 1A). When the stimulus buffer contained 400 nM Ca2+ and 10 µM IP3, the release event reached a maximum within two fractions (~140 ms), and subsequently decayed toward a plateau rate that was significantly higher than that observed with 400 nM Ca2+ alone. IP3-dependent release was blocked by heparin (100 µg/ml) (Fig. 1B), and was not due to calcium-dependent 45Ca2+ release (such as that mediated by the ryanodine receptor), since superfusion with 100 µM Ca2+ in the absence of IP3 produced the same amount of calcium release as did superfusion with 400 nM Ca2+ alone (not shown). Nonetheless, the small amount of calcium-dependent 45Ca2+ release was subtracted from the total release rates to arrive at the net IP3-dependent release rates (see "Experimental Procedures"). The net IP3-dependent release event (Fig. 1C) could be well described as the sum of two exponential decay phases, a fast phase that decayed with a time constant tau 1 = 130 ms, and a slower component that decayed slowly (tau 2 = 0.73 s). The cumulative 45Ca2+ release event could be reconstructed by sequentially summing the release rates (Fig. 1D).


Fig. 1. Kinetic and pharmacological characteristics of IP3-dependent 45Ca2+ release from hepatic microsomes. A, superfusion with 400 nM Ca2+ and 10 µM IP3 induced a rapid transient 45Ca2+ release. Hepatic microsomes were superfused with basal buffer to establish a baseline release rate. At t = 0, the superfusion buffer was switched to a stimulating buffer that contained 400 nM Ca2+ (bullet , n = 9) or 400 nM Ca2+ and 10 µM IP3 (black-triangle, n = 9). The rate of Ca2+ release reached a maximum within two fractions (~140 ms) and decayed monotonically thereafter. B, inhibition of IP3-dependent Ca2+ release by heparin. Superfusion with 10 µM IP3 at 400 nM Ca2+ with (diamond , n = 6) or without (black-diamond  n = 6) heparin (100 µg/ml). C, the net IP3-dependent release was calculated by subtracting release measured in the absence of IP3 from that in its presence (black-diamond ). The smooth curve describes the net release fit to the sum of two exponentials: y = A1e-k1t + A2e-k2t + C, where A1 = 0.287% s-1, A2 = 0.045% s-1, k1 = 7.71 k2 = 1.37 s-1, and C = 0.011% s-1. D, the cumulative release obtained by summing net IP3-dependent release rate as a function of time. Smooth curve is the integral of the fit in C.
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Characterization of the Effects of Ca2+ on the IP3 Receptor

The effects of extravesicular Ca2+ concentration on IP3 receptor activity were examined using a standard IP3 concentration of 10 µM, and measuring the net IP3-dependent 45Ca2+ release rates. Increasing the Ca2+ concentrations from nanomolar levels up to 3 µM resulted in marked changes in the kinetics of 45Ca2+ release; the maximal rate of release was greater and occurred more rapidly, and the decay of the fast component of release was accelerated (Fig. 2A). However, with increases in Ca2+ concentrations above 3 µM, the maximal rate of release and the cumulative release of 45Ca2+ decreased. Analysis of the relationship between the maximal rate of release or the initial rate of release and the extravesicular Ca2+ concentration was biphasic (Fig. 2B) as has been shown for the IP3-dependent 45Ca2+ release in rat brain (3, 5). Significantly, when we analyzed the relationship between cumulative release and extravesicular Ca2+ concentration, we observed stimulation of release with Ca2+ concentrations less than 1 µM, but the concentration-response relationship was shifted to lower Ca2+ concentrations. This observation emphasized the need to make measurements on a subsecond time scale in order to study the aspects of regulation of the transient properties of the IP3 receptor.


Fig. 2. Concentration-response relationship the effect of extravesicular Ca2+ on IP3-dependent 45Ca2+ efflux. A, IP3-dependent Ca2+ release with 10 µM IP3 and the following concentrations of extravesicular Ca2+: 1 nM (square ), 20 nM (black-square), 100 nM (open circle ), 200 nM (bullet ), 400 nM (diamond ), 1 µM (black-diamond ), 3 µM (triangle ), 10 µM (+), and 100 µM (×). Each curve is the mean of two experiments in triplicate. B, analysis of the dependence of net 45Ca2+ release on Ca2+ concentration. The initial rate of Ca2+ release (black-diamond ) and the cumulative amount of Ca2+ release (diamond ) were plotted as a function of the extravesicular Ca2+ concentration. Results are the mean of three experiments performed in triplicate.
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Characterization of the Effects of IP3 on the IP3 Receptor

To investigate the potency of IP3 in this system, the IP3 concentration of the stimulation buffer was varied while holding the free Ca2+ concentration constant (400 nM). As the IP3 concentration in the stimulation buffer was increased from 0.3 to 50 µM, the maximal rate of release increased and occurred faster (Fig. 3A). The fast component of calcium release was not detectable at IP3 concentrations of 300 nM or less, but could be observed as the IP3 concentration was increased into the micromolar range. The amplitude of the slow component of release was similar at all IP3 concentrations, but the peak rate was reached more rapidly as the concentration of the agonist was increased. Decay of the fast component increased with the IP3 concentration, but the decay was not solely dependent on depletion of intravesicular calcium. Because of the increased rate of decay at IP3 concentrations above 10 µM, the intravesicular 45Ca2+ content at the end of the fast phase was greater than for lower concentrations of IP3 where decay of release rates was slower.


Fig. 3. Concentration-response relationship for IP3-dependent 45Ca2+ efflux. A, net IP3-dependent Ca2+ release at 400 nM Ca2+ with 0.3 (square ), 1 (black-square), 3 (open circle ), 5 (bullet ), 10 (diamond ), 30 (black-diamond ), or 50 (triangle ) µM IP3. Each curve is the mean of two experiments in triplicate. B, cumulative IP3-dependent Ca2+ release at 400 nM Ca2+ calculated from A. C, Hill analysis of IP3 concentration-response data, using the initial rates of Ca2+ release (diamond ) and the cumulative amount of Ca2+ released (bullet ). The initial rate of 45Ca2+ release had a sigmoidal relationship to the IP3 concentration (nH = 3.43, IC50 = 4.5 µM), whereas the cumulative release had a normal hyperbolic relationship to agonist concentration (nH = 1.15, IC50 = 0.93 µM).
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Inspection of the cumulative IP3-dependent 45Ca2+ release clearly showed sigmoidal kinetics, most demonstrably at lower IP3 concentrations (Fig. 3B). Concentration-response relationships were plotted for initial release rates and cumulative release after 2.55 s of stimulation (Fig. 3C). The initial rate data were fit by non-linear least squares regression, yielding the following parameters; the Hill coefficient (nH) was 3.4, the Vmax value was 4.4% per s, and the apparent Km value for IP3 was 4.5 µM. Significantly, analysis of the relationship between IP3 concentration and the cumulative release measured after 2.55 s of stimulation was best described by a normal hyperbolic fit, i.e. the Hill coefficient of these data is 1.2, Vmax value was 1.7% per s, and the apparent Km value for IP3 was 0.9 µM. This analysis demonstrates that the sigmoidicity of the concentration-response analysis is best observed when measuring the initial release events.

IP3-dependent Inhibition of the IP3 Receptor

To test the possibility that IP3 itself inhibited IP3-dependent calcium release, we took advantage of the fact that the IP3 receptor requires both calcium and IP3 for its activity. This permitted us to pretreat the microsomes with IP3 and no added Ca2+ ([Ca2+] < 1 nM) for various intervals, conditions that produced only small increases in the rate of 45Ca2+ release. Thus, the IP3 receptor can be occupied by ligand without depleting intravesicular 45Ca2+, which could confound attempts to measure receptor inactivation. The vesicles were superfused with basal buffer as usual, and at t = -1.50 s, the superfusion buffer was switched to a prestimulus solution that contained either 1 nM Ca2+ (bullet , black-diamond ) or 1 nM Ca2+, 10 µM IP3 (diamond ) (Fig. 4A). At t = 0, the solution was again switched to a test buffer containing 400 nM Ca2+, 10 µM IP3 (black-diamond , diamond ), or 400 nM Ca2+ only (bullet ). Prestimulation with 10 µM IP3, 1 nM Ca2+ produced a small amount of 45Ca2+ release (Fig. 4, B and C), but not enough to significantly reduce the amount of 45Ca2+ contained in the vesicles (less than 0.2% of total). However, prestimulation with IP3 significantly inhibited the response to the test buffer. The initial rate of 45Ca2+ release was diminished by 42% by prestimulation with 10 µM IP3. When the peak rate of release observed after prestimulation was scaled to the peak rate of release in the control experiment (×), it was apparent that there was no qualitative change in the rate of decay or the relative amplitudes of the two components, consistent with agonist-dependent inactivation of the IP3 receptor, leading to a diminished response to the test buffer due to a decrease in the number of receptors available to conduct Ca2+ efflux.


Fig. 4. Inhibition of IP3-dependent 45Ca2+ release by IP3. A, microsomes were pretreated with 1 nM Ca2+ with (diamond ) or without (bullet , black-diamond ) 10 µM IP3 for 1.5 s prior to the test pulse. At t = 0, a test pulse containing 400 nM Ca2+ with (diamond , black-diamond ) or without (bullet ) 10 µM IP3 was delivered. Results are the mean of two experiments performed in duplicate. B, net difference between the baseline run (1 nM Ca2+ followed by 400 nM Ca2+ test pulse) and the two IP3 runs shows the inhibition produced by prestimulation with 10 µM IP3 (diamond ). The peak rate of release in the prestimulation run was scaled to the peak rate in the control run (×), demonstrating that pre-exposure to IP3 inhibited the amplitude of the response to the test pulse, did not significantly alter the kinetics of decay or the relative contributions of the fast and slow components.
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DISCUSSION

The aim of this study was to examine the regulation of the IP3 receptor by two second messengers implicated in its function, IP3 and Ca2+. We used a subsecond kinetic approach to measure 45Ca2+ release from microsomes prepared from rat liver. IP3-dependent 45Ca2+ release from hepatic microsomes was rapid and transient when strong stimulation was used. The decay of this release event was fit by two exponentials, representing an initial fast phase superimposed on a slower phase of Ca2+ release, as noted previously (9, 15). The Ca2+ release specifically represents IP3-dependent 45Ca2+ release since it was abolished by heparin, which interferes with the binding of IP3 to its receptor (16). The activity was regulated by both Ca2+ and IP3. Furthermore, pre-exposure of the receptor to IP3 (at [Ca2+] < 1 nM) produced a decrease in the activity of the receptor in response to subsequent test solutions. Our results are consistent with a model for the IP3 receptor where either Ca2+ or IP3 are sufficient to promote receptor inactivation, but both are necessary to promote receptor activation.

The superfusion method used in this study possesses unique advantages. Classical filtration studies rely on the calculation of the IP3-dependent calcium release by measuring the 45Ca2+ remaining in the stores of different experimental samples before and after partial discharge by IP3. The superfusion method directly and continuously measures release rates. The high flow rate (2 ml/s) relative to the small volume of the superfusion chamber (~30 µl) allows rapid solution changes (tau  = 40 ms) and precise control of the extravesicular concentrations of IP3 and calcium. Continuous superfusion of the membranes alleviates possible agonist depletion or degradation. The resulting rapid chelation of released 45Ca2+ by EDTA isolates the efflux of Ca2+ from its reuptake and minimizes accumulation of Ca2+ near the channel pore which might influence its activity. This contrasts with studies of permeabilized hepatocytes, in which changes in fluorescence of the calcium-sensitive dye Fura 2 were measured. This experimental protocol is limited, due to membrane permeabilization (17), the inability to isolate Ca2+ release from Ca2+ reuptake, and poor control over changes in IP3 and Ca2+ concentrations in the vicinity of the IP3 receptor.

Many cells have multiple mechanisms in place to produce calcium release activity. In addition to the IP3 receptor, hepatocytes contains a ryanodine receptor (18), which is structurally and functional related to the IP3 receptor. The ryanodine receptor is thought to be responsible for Ca2+-induced Ca2+ release from the stores, and can be distinguished from the IP3 receptor by functional criteria. In addition to the being a ligand-gated receptor, the IP3 receptor is relatively insensitive to Mg2+ (5, 19). Any ryanodine receptor activity in this preparation would be blocked by the high Mg2+ concentration (5 mM) used in the superfusion buffers. Superfusion with Ca2+ at high concentrations induced only a minimal Ca2+ release, an observation consistent with low levels of ryanodine receptor activity. In any case, IP3-dependent Ca2+ release was always corrected, using the control experiment performed at the same free Ca2+ concentration in the absence of IP3.

The initial rate of calcium release was a steep function of IP3 concentration. The apparent Hill coefficient of 3.4 suggests that binding of at least 3 IP3 molecules to the channel is required for activation of the ion channel activity, as suggested previously (8). Our results differ from those reported by Finch et al. (5) who used the same assay with synaptosomes-derived microsomal vesicles. Their experiments were performed at 10 µM Ca2+, which could explain a loss in apparent cooperativity. Combettes (20), using permeabilized hepatocytes, reported significantly reduced apparent cooperativity at a Ca2+ concentration of 10 µM compared to 100 nM. In contrast, the relationship between IP3 concentration and cumulative Ca2+ release (after 2.55 s of stimulation) had a Hill coefficient of ~1.2. The apparent potency of IP3, when based on cumulative release (0.9 µM), was somewhat higher than that based on the initial rate of release (4.5 µM). Using a stopped-flow method with permeabilized hepatocytes, Champeil et al. (9) determined comparable apparent affinities (0.2 and 1 µM respectively). At 10 µM free Ca2+ concentration, i.e. at saturating concentrations of Ca2+ required for the coagonist effect, Finch et al. (5) found that the initial release rate and the cumulative release had the same dependence on IP3 concentration, suggesting that IP3-dependent Ca2+ release was transient even at low IP3 concentrations. Combettes (19) reported that, at low concentrations of both Ca2+ and IP3, the release event is sustained, which is consistent with our data. This was subsequently confirmed by Finch (21); at 10 and 300 nM free Ca2+ concentrations, well below the range of extravesicular Ca2+ concentrations that inhibit release, the apparent potency of IP3 was higher when cumulative measurements of Ca2+ release over 2 s were used rather than the maximum rate of 45Ca2+ release.

IP3-dependent inactivation of IP3 receptor has been reported previously (11). In these experiments, the Ca2+ channel of the IP3 receptor function was assessed by measuring the quench of Fura 2 fluorescence by Mn2+ entry into the stores. This approach assumes that Mn2+ moves inward through the IP3 receptor from the cytoplasm of permeabilized hepatocytes into empty Ca2+ stores, and mimics the flux of Ca2+ through the Ca2+ channel. Our design allows preincubation with IP3 without significant movement of Ca2+ and measures directly Ca2+ efflux from microsomes with a better time resolution. Preincubation with IP3 inhibited release in a time dependent manner. It appears that inhibition of the Ca2+ channel by IP3 is faster than previously thought and might be rapid enough to explain the transient nature of the IP3-dependent Ca2+ release observed at constant extravesicular Ca2+.

A biphasic effect of cytosolic Ca2+ on IP3-dependent Ca2+ release has been described in Xenopus oocytes (3), smooth muscle (4), brain (5, 6), and hepatocytes (7). This property is important to our understanding of the mechanisms of Ca2+ waves (2, 22, 23), but has been recently suggested to be an effect produced by the metal-free Ca2+ chelators used to buffer calcium (19, 24). EDTA has similar affinities for Ca2+ and Mg2+, and in the presence of excess of Mg2+, nearly all of the Ca2+-free EDTA is complexed with Mg2+. Under these conditions where metal-free EDTA concentrations are minimal, Combettes (19) found that the cumulative amount and initial rate of IP3-dependent Ca2+ release had only weak dependence on Ca2+ concentrations between 30 nM and 2 µM. Measuring IP3-dependent Ca2+ release from permeabilized hepatocytes after 5 s, the same investigators reported that increasing the free Ca2+ concentration from 1 nM to 1 µM in this EDTA buffer did not stimulate IP3-dependent Ca2+ releases at high IP3 doses (20). We exposed hepatic microsomes to IP3 (10 µM) and the desired free Ca2+ concentration using EDTA as a calcium buffer. We found that when measured on a subsecond time scale, cytosolic Ca2+ at submicromolar concentration activated the initial rate of IP3-dependent 45Ca2+ release, consistent with the role of Ca2+ as a coagonist of the IP3 receptor. Our results do not exclude the possibility that metal-free chelators are inhibitors of the IP3 receptor, but they confirm that at submaximal IP3 concentration, Ca2+ activates IP3-dependent calcium release during the first 100-200 ms of stimulation.

The transient nature of the IP3-dependent calcium release observed in these experiments is potentially an important property of the IP3 receptor. Repetitive oscillatory calcium signals that diffuse as kinematic waves can only occur if mechanisms exist to terminate calcium release. Many cellular mechanisms exist which could explain the transient nature of the IP3 induced calcium release from hepatic microsomes. For example, sequential increases and decreases in IP3 concentration, local transients of Ca2+ concentration near the mouth of the pore, or depletion of intravesicular Ca2+ all could lead to transient release patterns. These possibilities seem unlikely in our system, since agonist concentrations were held constant during the experiment, and the vesicles were superfused at high flow rates with a strong Ca2+ buffer to clamp the extravesicular Ca2+ concentration. As for depletion, the vesicles were always loaded to the same extent, and therefore to the same initial intravesicular Ca2+ concentration. At higher IP3 concentrations, the fast phase of release decayed more rapidly, resulting in higher luminal Ca2+ content at the end of the release event (Fig. 3b), arguing that the decay is not solely due to depletion of intravesicular Ca2+.

The simplest model to account for our observations incorporates IP3-dependent as well as calcium-dependent inactivation of the receptor. By mass action, increases in IP3 concentrations lead to an increase in the fraction of receptors in the fully liganded conformation (Fig. 5). By analogy with cell surface ionotropic receptors such as the nicotinic acetylycholine receptor (25) and N-methyl-D-aspartate receptors (26), this conformation is in equilibrium with two states, the open state that conducts calcium, and an inactive conformation that does not. As the calcium concentration is increased into the micromolar range, the equilibrium between fully-liganded receptor and the active ion channel is shifted to favor the activated state, allowing for calcium efflux. At nanomolar calcium concentrations, the fully liganded receptor enters the inactive conformation without going through the active state. Thus, at low calcium concentrations, application of IP3 leads to a significant fraction of receptors that are occupied by IP3, but the open state of the receptor is bypassed, resulting in accumulation of the receptor in the inactivated state. Numerical simulations (27) using this model faithfully reproduce a number of important features of the empirical results, including the kinetics of the release event, the biphasic effect of Ca2+ on the response, and the effect of pre-exposure to IP3 at subnanomolar [Ca2+]. Repetitive propagation of kinematic Ca2+ waves within hepatocytes can only occur with limited autocatalytic mechanisms capable of terminating and reinitiating Ca2+ signals. Our data, supported by this quantitative model, show that sequential positive and negative feedback regulation of the channel by both cytosolic Ca2+ and IP3 may provide such mechanisms.


Fig. 5. Numerical simulation of IP3 receptor kinetics. The kinetic behavior of the IP3 receptor was simulated using a numerical method (27). The model assumed that the receptor consists of a tetramer, and that each monomer must be occupied by ligand (IP3) for the channel to be activated. The fully-occupied receptor exists in equilibrium with two distinct states, the activated state (R4*) that conducts ion flux, and an absorbing inactivated state (R4x) that does not conduct ion flux.
<UP>R</UP><SUB>0</SUB> <LIM><OP><ARROW>&rlarr2;</ARROW></OP><LL>k<SUB>−1</SUB></LL><UL>4k<SUB>1</SUB></UL></LIM> <UP> R</UP><SUB>1 </SUB><LIM><OP><ARROW>&rlarr2;</ARROW></OP><LL>2k<SUB>−1</SUB></LL><UL>3k<SUB>1</SUB></UL></LIM>  <UP>R</UP>  <LIM><OP><ARROW>&rlarr2;</ARROW></OP><LL>3k<SUB>−1</SUB></LL><UL>2k<SUB>1</SUB></UL></LIM>  <UP>R</UP><SUB>3 </SUB><LIM><OP><ARROW>&rlarr2;</ARROW></OP><LL>4k<SUB>−1</SUB></LL><UL>k<SUB>1</SUB></UL></LIM>  <UP>R</UP><SUB>4 </SUB><LIM><OP><ARROW>&rlarr2;</ARROW></OP><LL>  &bgr;  </LL><UL>  &agr;  </UL></LIM>  <UP>R</UP><SUB>4</SUB><SUP>*</SUP>
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The following constants were found to optimize the fit of the simulation to the empirical data: k1 = 4.9 × 106 M-1 s-1; k-1 = 0.147 s-1; kx = 1.5 × 105 s-1; k-x = 0.18 s-1; alpha  = 1.5 + 83* {1/1+(1.02 × 10-6 M/[Ca2+])}; beta  = 18 + 760 * {1/1+(3.3 × 10-5 M/[Ca2+])}. A, simulated receptor activation kinetics (at a fixed calcium concentration of 400 nM) as a function of [IP3]: 0.3 µM (black-diamond ); 1.0 µM (triangle ); 3.0 µM (bullet ); 10 µM (diamond ); 30 µM (black-square). B, simulated receptor kinetics (at an [IP3] of 10 µM) as a function of extravesicular [Ca2+]: 1 nM (black-square); 20 nM (square ); 0.1 µM (black-diamond ); 0.4 µM (diamond ); 1.0 µM (black-triangle); 3.0 µM (triangle ); 10 µM (bullet ); 100 µM (open circle ).
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In conclusion, we have conducted experiments designed to obtain quantitative data regarding the regulation of the IP3 receptor by the two second messengers, Ca2+ and IP3. We have provided substantive evidence that both IP3 and Ca2+ alter the calcium channel activity of the receptor in a similar fashion, such that either ligand promotes receptor inactivation, but both coagonists are required to maximize calcium release activity. The coincidence properties of the IP3 receptor are likely to have profound implications in the maintenance of intracellular calcium waves, and in regulation of cellular activity.


FOOTNOTES

*   This work was supported in part by National Institutes of Health Grants DK35652 and DK34928 (to I. M. A.) and NS28815 (to T. J. T.). 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. Section 1734 solely to indicate this fact.
Dagger    Recipient of the Advanced Research Training Award from the American Gastroenterology Association. To whom correspondence should be addressed: Dept. of Physiology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Tel.: 617-636-6739; Fax: 617-636-0445; E-mail: JDUFOUR@OPAL.TUFTS.EDU.
1    The abbreviations used are: IP3, D-myo-inositol 1,4,5-trisphosphate; DTT, 2,3-dihydroxybutane-1,4-dithiol; MOPS, 3-[N-morpholino]propansulfonic acid.

REFERENCES

  1. Rooney, T. A., and Thomas, A. P. (1993) Cell Calcium 14, 674-690 [Medline] [Order article via Infotrieve]
  2. Clapham, D. E., and Sneyd, J. (1995) Adv. Second Messanger Phosphoprotein Res. 30, 1-24 [Medline] [Order article via Infotrieve]
  3. Parker, I., and Ivorra, I. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 260-264 [Abstract]
  4. Iino, M., and Endo, M. (1992) Nature 360, 76-78 [CrossRef][Medline] [Order article via Infotrieve]
  5. Finch, E. A., Turner, T. J., and Goldin, S. M. (1991) Science 252, 443-445 [Medline] [Order article via Infotrieve]
  6. Bezprozvanny, I., Watras, J., and Ehrlich, B. E. (1991) Nature 351, 751-754 [CrossRef][Medline] [Order article via Infotrieve]
  7. Marshall, I. C. B., and Taylor, C. W. (1993) J. Biol. Chem. 268, 13214-13220 [Abstract/Free Full Text]
  8. Meyer, T., Holowka, D., and Stryer, L. (1988) Science 240, 653-656 [Medline] [Order article via Infotrieve]
  9. Champeil, P., Combettes, L., Berthon, B., Doucet, E., Orlowski, S., and Claret, M. (1989) J. Biol. Chem. 264, 17665-17673 [Abstract/Free Full Text]
  10. Watras, J., Bezprozvanny, I., and Ehrlich, B. E. (1991) J. Neurosci. 11, 3239-3245 [Abstract]
  11. Hajnoczky, G., and Thomas, A. P. (1994) Nature 370, 474-477 [CrossRef][Medline] [Order article via Infotrieve]
  12. Rossier, M. F., Bird, G. J., and Putney, J. W. (1991) Biochem. J. 274, 643-650 [Medline] [Order article via Infotrieve]
  13. Turner, T. J., Pearce, L. B., and Goldin, S. M. (1989) Anal. Biochem. 178, 8-16 [CrossRef][Medline] [Order article via Infotrieve]
  14. Fabiato, A., and Fabiato, F. (1979) J. Physiol. 75, 463-505
  15. Hirose, K., and Iino, M. (1994) Nature 372, 791-794 [CrossRef][Medline] [Order article via Infotrieve]
  16. Ghosh, T. K., Eis, P. S., Mullaney, J. M., Ebert, C. L., and Gill, D. L. (1988) J. Biol. Chem. 263, 11075-11079 [Abstract/Free Full Text]
  17. Renard-Rooney, D. C., Hajnoczky, G., Seitz, M. B., Schneider, T. G., and Thomas, A. P. (1993) J. Biol. Chem. 268, 23601-23610 [Abstract/Free Full Text]
  18. Shoshan-Barmatz, V., Zhang, G. H., Garretson, L., and Kraus-Friedmann, N. (1990) Biochem. J. 268, 699-705 [Medline] [Order article via Infotrieve]
  19. Combettes, L., Hannaert-Merah, Z., Coquil, J.-F., Rousseau, C., Claret, M., Swillens, S., and Champeil, P. (1994) J. Biol. Chem. 269, 17561-17571 [Abstract/Free Full Text]
  20. Combettes, L., Claret, M., and Champeil, P. (1993) Cell Calcium 14, 279-292 [Medline] [Order article via Infotrieve]
  21. Finch, E. A., and Goldin, S. M. (1994) Science 265, 813-815 [Medline] [Order article via Infotrieve]
  22. Atri, A., Amundson, J., Clapham, D., and Sneyd, J. (1993) Biophys. J. 65, 1727-1739 [Abstract]
  23. Jafri, M. S., and Keizer, J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9485-9489 [Abstract/Free Full Text]
  24. Combettes, L., and Champeil, P. (1994) Science 265, 813-815 [Medline] [Order article via Infotrieve]
  25. Edmonds, B., Gibb, A. J., and Colquhoun, D. (1995) Annu. Rev. Physiol. 57, 469-493 [CrossRef][Medline] [Order article via Infotrieve]
  26. Edmonds, B., Gibb, A. J., and Colquhoun, D. (1995) Annu. Rev. Physiol. 57, 495-519 [CrossRef][Medline] [Order article via Infotrieve]
  27. Benveniste, M., Clements, J., Vyklicky, L., and Mayer, M. L. (1990) J. Physiol. 428, 333-357 [Abstract]

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