HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mak, D.-O. D. Articles by Foskett, J. K. Search for Related Content PubMed PubMed Citation Articles by Mak, D.-O. D. Articles by Foskett, J. K. Social Bookmarking                      What's this?

J Biol Chem, Vol. 274, Issue 32, 22231-22237, August 6, 1999

ATP Regulation of Type 1 Inositol 1,4,5-Trisphosphate Receptor Channel Gating by Allosteric Tuning of Ca²⁺ Activation^*

Don-On Daniel Mak, Sean McBride, and J. Kevin Foskett^§¶

From the  Department of Physiology and ^§ Institute for Human Gene Therapy, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6100

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Inositol 1,4,5-trisphosphate (InsP₃) mobilizes intracellular Ca²⁺ by binding to its receptor (InsP₃R), an endoplasmic reticulum-localized Ca²⁺ release channel. Patch clamp electrophysiology of Xenopus oocyte nuclei was used to study the effects of cytoplasmic ATP concentration on the cytoplasmic Ca²⁺ ([Ca²⁺]_i) dependence of single type 1 InsP₃R channels in native endoplasmic reticulum membrane. Cytoplasmic ATP free-acid ([ATP]_i), but not the MgATP complex, activated gating of the InsP₃-liganded InsP₃R, by stabilizing open channel state(s) and destabilizing the closed state(s). Activation was associated with a reduction of the half-maximal activating [Ca²⁺]_i from 500 ± 50 nM in 0 [ATP]_i to 29 ± 4 nM in 9.5 mM [ATP]_i, with apparent ATP affinity = 0.27 ± 0.04 mM, similar to in vivo concentrations. In contrast, ATP was without effect on maximum open probability or the Hill coefficient for Ca²⁺ activation. Thus, ATP enhances gating of the InsP₃R by allosteric regulation of the Ca²⁺ sensitivity of the Ca²⁺ activation sites of the channel. By regulating the Ca²⁺-induced Ca²⁺ release properties of the InsP₃R, ATP may play an important role in shaping cytoplasmic Ca²⁺ signals, possibly linking cell metabolic state to important Ca²⁺-dependent processes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Modulation of free cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) is a ubiquitous cellular signaling system. In many cell types, binding of ligands to plasma membrane receptors activates the hydrolysis of phosphatidylinositol 4,5-bisphosphate by membrane-bound phospholipase C, generating inositol 1,4,5-trisphosphate (InsP₃).¹ InsP₃ causes the release of Ca²⁺ from the endoplasmic reticulum (ER) by binding to its receptor (InsP₃R), which itself is a Ca²⁺ channel (1-3). Complex control of Ca²⁺ release through the InsP₃R by various intracellular factors, including cooperative activation by InsP₃ (4-8) and biphasic feedback from the permeant Ca²⁺ ion (6, 8-11) generates intricate [Ca²⁺]_i signals that can be manifested temporally as repetitive spikes or oscillations, with frequencies often tuned to the level of stimulation, and spatially as propagating waves or highly localized events (2, 12, 14) and display properties of "adaptation" and "quantal release," which are poorly understood (15). Several types of InsP₃R as products of different genes with alternatively spliced isoforms have been identified and sequenced (16, 17). The InsP₃Rs have about 2700 amino acid residues in InsP₃ binding, regulatory (modulatory) and transmembrane channel domains (16-18). The sequences of the regulatory domains of all InsP₃R isoforms include putative ATP-binding site(s) (17). ATP was shown to bind to the InsP₃R (19) and regulate InsP₃R-mediated Ca²⁺ release (20-24), although the detailed mechanisms of this regulation remain unclear.

Several studies have demonstrated that mitochondria and the ER are in close physical and functional proximity in many cell types, including neurons (24-27). [Ca²⁺]_i signals generated by InsP₃R-mediated Ca²⁺ release from the ER appear to be rapidly and efficiently transmitted to mitochondria (28-30), acutely affecting mitochondrial functions (31-33), including ATP synthesis (34). It is unknown, however, whether the communication between these two organelles is reciprocal. ATP release from mitochondria, globally into the cytoplasm and locally into the vicinity of the InsP₃R channels that are in close apposition, may provide a signaling pathway for communication from the mitochondria back to the ER. Thus, regulation of the InsP₃R by ATP could have considerable significance for intracellular signaling, particularly if the channel is sensitive to ATP levels in normal physiological as well as pathological conditions, including ischemia.

Most previous studies of ATP regulation of the InsP₃R have been limited to indirect measurements, i.e. Ca²⁺ fluxes or concentrations, to infer InsP₃R channel activity, because the intracellular location of the Ca²⁺ release channel has limited its accessibility to electrophysiological approaches. Furthermore, only a limited range of [Ca²⁺]_i was examined in previous studies, despite the fact that the InsP₃R is intricately regulated by [Ca²⁺]_i (6, 8-11) and that the primary known regulator of the channel, InsP₃, mediates its effects by modulating the [Ca²⁺]_i dependence of channel gating (8). Therefore, in the present study, we have systematically investigated the effects of cytoplasmic ATP concentration on the [Ca²⁺]_i response of single InsP₃R channels. We applied the patch clamp technique to isolated Xenopus oocyte nuclei (35-37) to study the single channel activities of the type 1 InsP₃R (InsP₃R-1), the major brain isoform (38, 39), in its native ER membrane environment under rigorously defined conditions on both the cytoplasmic and luminal sides of the channel. Our results demonstrate that cytoplasmic ATP free acid, but not cytoplasmic MgATP complex, activates the gating of the InsP₃R primarily by allosteric regulation of the [Ca²⁺]_i sensitivity of the Ca²⁺ activation sites of the channel. By regulating the Ca²⁺-induced Ca²⁺ release properties of the InsP₃R, ATP may play an important role in shaping the extent and duration of [Ca²⁺]_i signals, possibly linking cell metabolic state to important Ca²⁺-dependent process including synaptic plasticity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Patch Clamping the Oocyte Nucleus-- Patch clamp experiments were performed as described in Refs. 8, 35, 36, and 40. Briefly, stage V or VI oocytes were opened mechanically just prior to use. The nucleus was separated from the cytoplasm and transferred to a dish on the stage of a microscope for patch-clamping. The oocyte expresses only a single InsP₃R isoform (type 1) and lacks other (e.g. ryanodine receptor) Ca²⁺ release channels (41). Experiments were done in "on-nucleus" configuration, with the solution in the perinuclear lumen between the outer and inner nuclear membranes in apparent equilibrium with the bath solution (35) and with the cytoplasmic aspect of the InsP₃R channel facing into the patch pipette. Following standard conventions, the applied potential is that of the pipette electrode minus the reference bath electrode (positive current flows from pipette outward). Experiments were performed at room temperature with the pipette electrode at +20 mV relative to the reference bath electrode.

Data Acquisition and Analysis-- Single channel currents were amplified with an Axopatch-1D amplifier (Axon Instruments, Foster City, CA) with antialiasing filtering at 1 kHz, transferred to a Power Macintosh 8100 via an ITC-16 interface (Instrutech Corp., Great Lake, NY), digitized at 5 kHz, and written directly onto the hard disc by Pulse+PulseFit software (HEKA Elektronik, Lambrecht, Germany). Data were analyzed to identify channel opening and closing events and evaluate channel open probabilities using MacTac 3 (Bruxton, Seattle, WA). 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 S.E. Theoretical curves were fitted to experimental data using Igor Pro 3 (WaveMetrics, Lake Oswego, OR).

Solutions for Patch Clamp Experiments-- All patch clamp experiments were performed with solutions containing 140 mM KCl and 10 mM HEPES with pH adjusted to 7.1 using KOH. Since the luminal [Ca²⁺] or [ATP] have no systematic effects on the open probability response of the InsP₃R (8), a bath solution containing no ATP and 250 nM free Ca²⁺ was used in all experiments. Pipette solutions contained various concentrations of nucleotides (sodium salts of ATP, ADP, AMP, GTP, and UTP and adenosine, from Sigma) as specified. Because of chelation of Mg²⁺ by ATP, the actual free [Mg²⁺] and free [ATP] in the solutions containing Mg²⁺ and ATP were calculated by the Maxchelator software (C. Patton, Stanford University, Stanford, CA). By using K⁺ as the current carrier and appropriate quantities of the high affinity Ca²⁺ chelator, BAPTA (100-500 µM; Molecular Probes, Inc., Eugene, OR), the low affinity Ca²⁺ chelator, 5,5'-dibromo-BAPTA (100-350 µM; Molecular Probes), or just ATP (0 or 0.5 mM) to buffer [Ca²⁺] in the experimental solutions, [Ca²⁺] was tightly controlled in our experiments. Total Ca²⁺ content (60-370 µM) in the solutions was determined by induction-coupled plasma mass spectrometry (Mayo Medical Laboratory, Rochester, MN). Free [Ca²⁺] was calculated using the Maxchelator software. Pipette solutions contained 10 µM InsP₃ (Molecular Probes).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Activation of Channel Gating by ATP-- To examine the effects of ATP on the permeation and gating properties of the InsP₃R, we included 10 µM InsP₃, 250 nM free Ca²⁺, and 0.5 mM free ATP in the pipette solution. Under these conditions, the endogenous Xenopus type 1 InsP₃R channel exhibited channel conductance properties and kinetics similar to those observed previously under similar conditions (8, 40). The channels gated with a moderately high open probability (P_o) of ~0.5 (Fig. 1A). In similar experiments employing pipette solutions that lacked ATP, the InsP₃R channel P_o was significantly lower (~0.2) in either the absence or presence of 3 mM Mg²⁺ (Fig. 1, B and C). To determine whether the ATP activation of the channel P_o was mediated by MgATP, which could suggest a role for ATP hydrolyisis or phosphorylation, similar experiments were undertaken with 3 mM total Mg²⁺ and 0.5 mM total ATP in the pipette. Under these conditions, [MgATP] is approximately 0.5 mM, and the free Mg²⁺ concentration ([Mg²⁺]_i) and the cytoplasmic free ATP concentration ([ATP]_i) were calculated to be 2.5 and 0.012 mM, respectively. Nevertheless, P_o remained low (Fig. 1D). The low P_o in the presence of MgATP (Fig. 1) was solely caused by ATP complexation by Mg²⁺, since it was fully reversed by adding more ATP to the pipette solution to restore [ATP]_i (Fig. 1E). Thus, MgATP has no effect, stimulatory or inhibitory, on InsP₃R activity. We previously demonstrated that the P_o of the Xenopus type 1 InsP₃R is independent of [Mg²⁺]_i up to 9.5 mM (40). Taken together, these results suggest that ATP free acid (ATP³ or ATP⁴) was the relevant ionic species and that ATP hydrolysis was not involved in the stimulation of InsP₃R channel gating.

View larger version (50K): [in this window] [in a new window]   Fig. 1.   Typical current traces of InsP3R channels in outer nuclear membrane at [Ca2+]i = 250 ± 20 nM under various [ATP]i and [Mg2+]i. The arrows indicate closed channel current levels. A, [ATP]i = 0.5 mM; [Mg2+]i = 0 mM. B, [ATP]i = 0 mM; [Mg2+]i = 0 mM. C, [ATP]i = 0 mM; [Mg2+]i = 3.0 mM. D, [ATP]i = 12 µM; [Mg2+]i = 2.5 mM (0.5 mM total ATP; 3 mM total Mg2+). Reduction of InsP3R channel conductance in the presence of Mg2+ (C and D) is caused by permeant ion block of the channel by the divalent cation (40). E, [ATP]i = 1.9 mM; [Mg2+]i = 113 µM (4.8 mM total ATP; 3 mM total Mg2+).

Effects of ATP on the [Ca²⁺]_i Dependence of InsP₃R Gating-- InsP₃ activates the InsP₃R by modulating the sensitivity of the channel to [Ca²⁺]_i (8). To determine the mechanism of ATP activation of the InsP₃R channel gating, we investigated in detail the effects of ATP on the channel kinetics of the InsP₃R over a wide range of [Ca²⁺]_i. A systematic series of patch clamp experiments were performed using pipette solutions containing various [Ca²⁺]_i with 0.5 mM ATP alone, 3 mM Mg²⁺ alone, 0.5 mM ATP, and 3 mM Mg²⁺ (calculated [ATP]_i = 0.012 mM; calculated [Mg²⁺]_i = 2.5 mM) or no ATP or Mg²⁺. To avoid possible effects of Ca²⁺ on InsP₃ binding, a functionally saturating InsP₃ concentration of 10 µM was used (8). The [Ca²⁺]_i sensitivity of the InsP₃R in the absence of cytoplasmic free ATP was biphasic (Fig. 2) and could be well fitted with a biphasic Hill equation similar to the following one previously derived for the InsP₃R in the presence of 0.5 mM cytoplasmic free ATP (8).

(Eq. 1)

Similar results were obtained independent of the presence or absence of either Mg²⁺ or MgATP in the pipette solutions. This result indicates that the InsP₃R can achieve a maximum open probability P_max of 0.79 in the absence of cytoplasmic free ATP, a level of activity very similar to the P_max of 0.81 found in the presence of 0.5 mM cytoplasmic free ATP (8). Thus, ATP does not activate the channel by increasing P_max. The Hill coefficient for Ca²⁺ activation H_act was 2.4 ± 0.6 in the absence of free ATP, similar to H_act = 1.9 ± 0.3 in the presence of 0.5 mM free ATP. This result suggests that Ca²⁺ probably activates the InsP₃R via the same cooperative process in either the presence or absence of cytoplasmic free ATP. Thus, ATP does not activate the channel by modulating H_act. The observed activation of the InsP₃R by cytoplasmic free ATP (Fig. 1) was associated with a reduction of the half-maximal activating [Ca²⁺]_i (K_act) from 500 ± 50 nM in the absence of free ATP to 190 ± 20 nM in the presence of 0.5 mM free ATP. Thus, ATP activates the channel by sensitizing it to Ca²⁺. ATP therefore enhances Ca²⁺-induced Ca²⁺ release (CICR) by the InsP₃R.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   [Ca2+]i dependence of the InsP3R channel Po in the presence of low (~0 mM) [ATP]i. Open triangles represent data with [ATP]i = 0 mM and [Mg2+]i = 0 mM. Open squares represent data with [ATP]i = 0 mM and [Mg2+]i = 3.0 mM. Open circles represent data with [ATP]i = 12 µM and [Mg2+]i = 2.5 mM (0.5 mM total ATP; 3 mM total Mg2+). The solid curve is the biphasic Hill equation fit using Equation 1. For comparison, Po data of the InsP3R in the presence of 0.5 mM [ATP]i (small filled circles) and the corresponding biphasic Hill equation fit (dotted curve; published in Ref. 8) are also shown.

Because InsP₃ activates channel gating by modifying the [Ca²⁺]_i inhibition phase of the channel [Ca²⁺]_i dependence (8), we were also interested in examining the effects of ATP on this aspect of the response. However, investigations of InsP₃R channel activity in the absence of free ATP at [Ca²⁺]_i which inhibit channel gating (>20 µM; Ref. 8) were not possible because of the unavailability of a Ca²⁺ chelator with the appropriate Ca²⁺ affinity. In our previous experiments that examined the effects of high [Ca²⁺]_i on InsP₃R channel gating (8), ATP was used as the Ca²⁺ chelator for buffering [Ca²⁺]_i at high [Ca²⁺]_i. The data we were able to obtain in the absence of ATP in the present study indicated that P_o began to decrease as [Ca²⁺]_i was increased beyond 10 µM, but the inhibitory half-maximal [Ca²⁺]_i, K_inh, or Hill coefficient, H_inh, could not be determined accurately (Fig. 2).

Effects of ATP on InsP₃R Channel Kinetics-- Analysis of the mean open and closed durations of the InsP₃R revealed that the mean open duration (_o) in the absence of free ATP lay within a narrow range between 5 and 15 ms over a wide range of [Ca²⁺]_i (1-10 µM). At both very low (<400 nM) or very high (>10 µM) [Ca²⁺]_i, _o was shorter (~3 ms) (Fig. 3). In contrast, the mean closed duration (_c) in the absence of free ATP decreased about 2 orders of magnitude, from 200 to 3 ms, as [Ca²⁺]_i was increased from 200 nM to 1 µM. _c remained low between 1 and 10 µM [Ca²⁺]_i (Fig. 4). These same basic kinetics were observed in all experiments conducted in the absence of cytoplasmic free ATP, regardless of the presence or absence of free Mg²⁺ or MgATP complex. Similar [Ca²⁺]_i dependences of _o and _c (Figs. 3 and 4) were also observed in the presence of 0.5 mM free ATP (8). An examination of the differences in the [Ca²⁺]_i dependences of _o and _c in either the presence and absence of cytoplasmic free ATP reveals that the mechanism whereby free ATP enhances channel activity is by stabilization of the open channel state(s) and destabilization of the closed channel state(s) in the low [Ca²⁺]_i regime (30-500 nM). Our more limited data indicate that free ATP may also stabilize the open channel state(s) at very high [Ca²⁺]_i (>15 µM).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   [Ca2+]i dependence of the mean open durations of the InsP3R channels in the absence of free cytoplasmic ATP (A) and in the presence of 0.5 mM free ATP (B). The symbols used are the same as those in Fig. 2.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   [Ca2+]i dependence of the mean closed durations of the InsP3R channels in the absence of free cytoplasmic ATP (A) and in the presence of 0.5 mM free ATP (B). The symbols used are the same as those in Fig. 2.

ATP Concentration Dependence of Ca²⁺ Activation of InsP₃R Gating-- We undertook a systematic study of the activation of the InsP₃R by [Ca²⁺]_i over a wide range of free [ATP]_i. In the presence of 10 µM InsP₃, the activating Hill equation (Equation 2) agreed well the experimental data for [ATP]_i of 4.8 and 9.5 mM, with no significant effects of [ATP]_i on H_act or P_max (Fig. 5).

(Eq. 2)

[ATP]_i decreased K_act of the InsP₃R by over an order of magnitude, from 500 ± 50 nM in 0 mM [ATP]_i to 29 ± 4 nM in 9.5 mM [ATP]_i. The effects of [ATP]_i on K_act of the InsP₃R were analyzed by fitting the data with a modified Michaelis-Menten equation (Fig. 6).

(Eq. 3)

A modification to the standard Michaelis-Menten equation was necessary because ATP alone is insufficient to activate the channel in absence of InsP₃, and therefore the activating half-maximal [Ca²⁺]_i (K_act) should not approach 0 even in presence of saturating concentrations of ATP. The results indicate that the range over which K_act of the InsP₃R varies in response to [ATP]_i, K_r = 480 ± 20 nM; the minimum K_act under saturating [ATP]_i, K_min = 17 ± 3 nM; and the functional dissociation coefficient for cytoplasmic free ATP activation of the InsP₃R, K_ATP = 0.27 ± 0.04 mM. The good fit of this equation to the data suggests that ATP stimulation of channel activity is not cooperative, requiring binding of only one ATP molecule to the InsP₃R tetramer to stimulate it.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Effect of [ATP]i on activation of the InsP3R by [Ca2+]i. The different symbols plotted correspond to various [ATP]i used, as tabulated in the graph (in mM). The curves are Hill equation fits for the corresponding experimental data: for 0 mM [ATP]i, Hact = 2.4, Kact = 500 nM, and Pmax = 0.79; for 0.5 mM [ATP]i, Hact = 1.9, Kact = 190 nM, and Pmax = 0.81; for 4.8 mM [ATP]i, Hact = 2.3, Kact = 45 nM, and Pmax = 0.71; for 9.5 mM [ATP]i, Hact = 2.4, Kact = 29 nM, and Pmax = 0.82. For 0.3 mM [ATP]i, assuming that Hact = 2 and Pmax = 0.8, Hill equation fitting gives Kact = 230 nM.

View larger version (9K): [in this window] [in a new window]   Fig. 6.   Effect of [ATP]i on the activating half-maximal [Ca2+]i (Kact) of the InsP3R. The curve is the theoretical fit based on the modified Michaelis-Menten equation (Equation 3).

Nucleotide Specificity-- To determine the nucleotide specificity of the stimulatory effects we observed for free ATP, we also investigated the effects of adenosine, AMP, ADP, GTP, and UTP. Each nucleotide was present as 0.5 mM free nucleotide in the absence of Mg²⁺. The [Ca²⁺]_i was fixed at 220 ± 15 nM, because the P_o is very sensitive to activation by free ATP at this [Ca²⁺]_i (Fig. 2). Using the channel P_o (0.14) in the absence of any nucleotide as the reference, the relative P_o of the channel was determined in the presence of the various nucleotide species (Fig. 7). Similar to the MgATP complex, free UTP, ADP, and adenosine had no effects on the P_o of the InsP₃R (p > 0.05). In contrast, free ATP, AMP, and GTP each activated the InsP₃R (p < 0.05). Free ATP had the greatest effect, more than tripling the P_o, whereas both free AMP and GTP doubled the channel P_o.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Relative open probabilities of InsP3R in the presence of 0.5 mM free cytoplasmic nucleotides. [Ca2+]i = 220 ± 15 nM. Po = 0.14 in the absence of any nucleotide. Total [Mg2+] is 3 mM for data labeled MgATP and 0 mM for the rest.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We previously described the detailed permeation and gating properties of the Xenopus type 1 InsP₃R channel by patch clamp studies of isolated oocyte nuclei (8, 35, 36, 40). Under optimal conditions, gating of the channel is robust, with maximum open probability of ~80% over a wide range of [Ca²⁺]_i (8). Importantly, the gating of the channel is regulated by both InsP₃ as well as by [Ca²⁺]_i. The regulation of the InsP₃-liganded channel activity by [Ca²⁺]_i is biphasic, with half-maximal activation at 210 nM and half-maximal inhibition at 45 µM in 10 µM InsP₃. InsP₃ binds noncooperatively to a high affinity site (K_D ~ 50 nM) on each monomer of the channel tetramer. Binding of InsP₃ to the channel has the sole effect of decreasing the Ca²⁺ affinity of the Ca²⁺ inhibition site on each monomer, in a process which has high cooperativity (Hill coefficient of 4) (8). When cytoplasmic InsP₃ concentration ([InsP₃]_i) is low, under conditions of no or weak stimulation, the channel is inhibited by relatively low [Ca²⁺]_i, whereas it becomes much less sensitive to Ca²⁺ inhibition at higher [InsP₃]_i, enabling it to become activated. Thus, InsP₃ activates the channel by tuning the inhibition efficacy of the Ca²⁺ ligand. Of particular significance, [Ca²⁺]_i activation of the channel was not modified by InsP₃. However, it is unknown whether other modulators of InsP₃R activity similarly impinge on the Ca²⁺ inhibition properties of the channel or whether the Ca²⁺ activation properties of the channel are exploited as an alternate method of channel regulation. The results from the present study suggest that ATP stimulates gating of the InsP₃R by modulating the Ca²⁺ sensitivity of the Ca²⁺ activation sites.

Regulation of InsP₃-mediated Ca²⁺ Release by ATP-- We performed a systematic investigation of the effects of nucleotides on gating of the Xenopus type 1 InsP₃R Ca²⁺ release channel. Our study focused on the effects of ATP on single channel activity, and included additional examination of effects of other nucleotides for comparison. Stimulation of InsP₃R by nucleotides in the presence of InsP₃ has been previously reported (20-23, 42, 43). The majority of published studies measured the effects of nucleotides on InsP₃-induced Ca²⁺ fluxes either from intracellular stores in permeabilized cells or into lipid vesicles in vitro. ATP stimulation of InsP₃-induced Ca²⁺ fluxes was reported in all these studies, but the considerable qualitative as well as quantitative variability among them makes comparison with our results difficult. Maximum stimulation by ATP of Ca²⁺ release by the InsP₃R has been reported to range from 1.5- to 2-fold (20-22) the activity with InsP₃ alone. Considerable discrepancies exist regarding the ATP concentration required for maximal stimulation, ranging from 10 µM (20) to 1 mM (21, 22). In terms of the nucleotide specificity, ADP was reported to be as potent as ATP (21, 22) or to be only 40% as effective (20) or ineffective (24); AMP has been reported to stimulate to ~70% (21, 22) or 10% (20) of the ATP stimulation level; GTP was ineffective (21) or stimulated to 30% of the ATP stimulation level (22). Regarding the specific ATP species, MgATP complex was as effective as free ATP (20) or only 50% as effective (21).

Some reports indicate that the effects of ATP on InsP₃R activity are biphasic, being stimulatory at low [ATP]_i and inhibitory at high [ATP]_i (20-23). The concentrations at which increased [ATP]_i starts to reduce InsP₃R activity have varied from 0.1 (20) to 1 mM (21, 22) in Ca²⁺ flux experiments. In bilayer experiments, ATP concentrations of >5 mM inhibited channel activity with a half-maximal concentration of 11 mM (23). In contrast, recent Ca²⁺ flux studies found no inhibitory effects of ATP at 5 (24) or 10 mM (42) on InsP₃-induced Ca²⁺ release from the ER. The results from our nuclear patch clamp experiments are in agreement with these latter studies. We detected no inhibitory effects of cytoplasmic ATP up to 10 mM. It has been suggested that the inhibitory effects of high [ATP]_i are caused by competitive inhibition of InsP₃ binding to the InsP₃R (23). Therefore, the different observations may be caused by different [InsP₃]_i used in these studies, <5 µM in studies reporting ATP inhibition (20-23) and >5 µM in studies reporting no ATP inhibition (Refs. 24 and 42 and this study).

Stimulation of Single Channel Gating of the InsP₃R by ATP-- Many of the discrepancies in the results of Ca²⁺ flux studies may have been caused by species differences, different concentrations of InsP₃, or other important parameters, including Ca²⁺ and other divalent cations (i.e. Mg²⁺) whose concentrations in the vicinity of the InsP₃R might not have been adequately controlled. Furthermore, the measurements of Ca²⁺ fluxes involved populations of unknown numbers and multiple types of InsP₃R, which may also have contributed to the discrepant results (42). Importantly, the effects of nucleotides on the activity of the InsP₃R on the single molecule level can only be inferred from these studies. There has been only one previous detailed investigation of the effects of ATP on single channel activity of the InsP₃R (23). In the presence of 0.2 µM [Ca²⁺]_i and 2 µM InsP₃, ATP enhanced the P_o of canine cerebellum type 1 InsP₃R reconstituted from microsomes into artificial planar lipid bilayers with a binding coefficient of 40 µM and Hill coefficient of 1. The MgATP complex was as effective as free ATP, whereas GTP was only 20% as effective and AMP was ineffective.

The effects of nucleotides on the Xenopus type 1 channel observed by nuclear patch clamp in the present study have some similarities but also differ in several important respects. In agreement with the results from the bilayer study, the Hill coefficient for ATP activation was also 1 in our study, although the apparent binding coefficient K_ATP for free ATP was ~300 µM in our study, compared with 40 µM in the bilayer study. In both studies, ATP activation of channel gating was associated with stabilization of the open state and destabilization of the closed state. Both studies concluded that ATP hydrolysis was not involved, since nonhydrolyzable analogs were as effective as ATP in the bilayer study. In contrast to the lipid bilayer results, the MgATP complex had no stimulatory effects in our study; furthermore, free GTP and AMP had potent stimulatory effects (both about 60% of the ATP stimulation level) in the present study. The difference in nucleotide specificity observed in the two studies is difficult to reconcile. It may be caused by the different sources (canine versus Xenopus), lipid environment (artificial lipid bilayers versus native ER), and/or the isolation/reconstitution protocols. Of particular interest is our finding that MgATP was without effect on the channel activity. This distinction is important; whereas the MgATP concentration in the cytoplasm is in the range of 3-8 mM (44-48), the cytoplasmic free ATP concentration is in the range of 400-600 µM. The apparent affinity of 40 µM for ATP stimulation of the InsP₃R in bilayers and the apparent effectiveness of MgATP substituting for ATP in InsP₃R stimulation would suggest that ATP (free or complexed with Mg²⁺) plays no role in channel modulation under most conditions, since the total concentration of free and magnesium-complexed ATP always far exceeds its apparent affinity for the channel. In contrast, and as discussed in more detail below, our determination that only free ATP is effective, with an apparent affinity (apparent K_D ~ 270 µM) that is nearly coincident with levels in the cytoplasm, suggests that physiological modulation of ATP levels in cells will have profound effects on Ca²⁺ release activity of the InsP₃R by modifying its CICR properties. Of note, a recent study using permeabilized lymphocytes that were engineered to express only the type 1 InsP₃R found that free ATP enhanced InsP₃-induced Ca²⁺ release with an apparent K_D of 390 µM (42), in quite good agreement with our channel results.

A second important distinction between our results and those of the bilayer study concerns the mechanism by which ATP stimulates channel gating. Our results demonstrate that ATP binding to the InsP₃R increases the Ca²⁺ sensitivity of the Ca²⁺ activation site of the channel. The bilayer experiments were carried out at a single [Ca²⁺]_i (0.2 µM). The P_o observed in 2 µM InsP₃ increased from ~0.01 in the absence of ATP to ~0.07 in 1 mM ATP (the order of magnitude difference in absolute values of P_o observed in the two studies may be due to an InsP₃ insensitivity of the reconstituted channels in the bilayer study (8)). It was concluded that ATP binding to InsP₃R increases the intrinsic efficacy of InsP₃ to activate the InsP₃R (23). However, our results demonstrate that ATP does not affect P_max. The increased P_o observed in the bilayer study can be accounted for by the increase in affinity of the Ca²⁺ activation site observed in the present study (Fig. 5).

Regulation of the [Ca²⁺]_i Dependence of InsP₃R Gating by ATP-- The present study represents the first systematic investigation of the effects of ATP on single channel activity of the InsP₃R over a wide range of [Ca²⁺]_i (10 nM to 20 µM). Our results reveal the major mechanism by which elevations of cytoplasmic free ATP stimulate gating of the InsP₃R, by demonstrating that ATP increases the affinity of the Ca²⁺ activating site of the channel specifically. ATP decreased the half-maximal activating [Ca²⁺]_i (K_act), without affecting the maximum P_o. Although channel P_o decreased at low [ATP]_i, this could be fully reversed by increased [Ca²⁺]_i. Therefore, ATP is not a necessary agonist for activation of the InsP₃R, but rather it is an allosteric regulator, tuning the efficacy of [Ca²⁺]_i to stimulate the activity of the InsP₃-liganded InsP₃R over a limited range of [Ca²⁺]_i (10 nM to 1 µM as shown in Fig. 5). We previously showed that InsP₃ activates the InsP₃R solely by tuning the half-maximal inhibitory [Ca²⁺]_i (K_inh) of the channel, whereas activation of the InsP₃R by [Ca²⁺]_i is unaffected (8). InsP₃ is therefore a regulator of Ca²⁺-inhibition of Ca²⁺ release, whereas ATP is a regulator of CICR. Thus, the effect of free ATP on the activation of the InsP₃R by [Ca²⁺]_i complements the effect of InsP₃. Together, [ATP]_i and [InsP₃]_i each act as allosteric regulators to tune the activation and inhibition, respectively, of the InsP₃R by [Ca²⁺]_i.

It is not yet possible to completely describe the gating of the channel under all conditions of [Ca²⁺]_i, [InsP₃]_i, and [ATP]_i, because we have yet to fully characterize the effects of ATP on Ca²⁺ inhibition of the channel under submaximal [InsP₃]. The data we obtained under saturating [InsP₃] suggest the possibility that ATP also affects the Ca²⁺ inhibition phase of the [Ca²⁺]_i dependence curve by making the channel less sensitive to [Ca²⁺]_i inhibition. This mode of regulation is therefore analogous to that of InsP₃. Further studies are required to define the effects of free ATP on Ca²⁺ inhibition of the channel and the relationship of those effects to that of InsP₃. Nevertheless, it is interesting to consider the similarities involved in the regulation of channel gating by ATP and InsP₃. As mentioned earlier, InsP₃ is a channel activator because it decreases the affinity of the Ca²⁺ inhibition site of the channel. It is important to note, however, that it is not the absolute magnitude of the Ca²⁺ affinity of that site that is critical for InsP₃R activity, but rather its relationship to that of the Ca²⁺ activation site. The higher Ca²⁺ affinity of the inhibition site keeps the channel inactive in the absence of InsP₃. However, the Ca²⁺ affinity of the inhibition site becomes less than that of the activation site when the channel binds InsP₃. It follows, therefore, that an alternate mechanism to activate the channel would be to increase the Ca²⁺ affinity of the activation site. Because this is a major effect of ATP, we speculate that suitable conditions could be defined in which channel gating could be activated by an increase in [ATP]_i without any change in [InsP₃]_i. Because the relative affinities for Ca²⁺ of the InsP₃R activation and inhibition sites is the critical factor in determining the level of channel activity, it follows that allosteric regulation of antagonistic Ca²⁺-binding sites by ATP and InsP₃, by together tuning the Ca²⁺ dependence of channel gating, render the Ca²⁺ dependence of Ca²⁺ release by the InsP₃R a dynamic property, dependent upon stimulus intensity and cell metabolic state.

Physiological Implications-- The results of our study suggest that complex features of InsP₃-induced [Ca²⁺]_i signals will be dependent upon an elaborate regulation of Ca²⁺ release through the InsP₃R by [Ca²⁺]_i, [InsP₃]_i, and [ATP]_i. Importantly, modulation of channel activity by both ATP and InsP₃ is achieved by regulating the [Ca²⁺]_i dependence of channel gating. The interplay between [ATP]_i and [Ca²⁺]_i in the control of InsP₃R channel activities observed in the present study probably has important physiological significance, particularly if the InsP₃R actually experiences various [ATP]_i. Our results indicate that the apparent affinity of the ATP-binding site on the InsP₃R is physiologically relevant, since it approximates the free ATP concentration in the cytoplasm. Estimates of total Mg²⁺ (44, 45) and total ATP (46-48) concentrations in cells are each in the range of 5-10 mM. Assuming equal concentrations of each, the free ATP concentration is calculated to vary roughly from 420 µM (5 mM total Mg²⁺) to 540 µM (8 mM total Mg²⁺). For comparison, the apparent affinity of the ATP-binding site on the InsP₃R was determined in the present study to be 270 µM. Interestingly, not only is the apparent affinity of the ATP-binding site on the InsP₃R coincident with the normal cytoplasmic free ATP concentration, but changes in free ATP concentration are very sensitive to changes in total ATP concentration in the cytoplasm. For example, at 5 mM total Mg²⁺, an increase of total ATP concentration from 5 to 5.5 mM (10% change) will result in a 1.5-fold increase in the concentration of free ATP (from ~420 µM to ~600 µM). Thus, relatively small changes in total cytoplasmic ATP can have pronounced effects on free ATP concentration and, therefore, on the CICR properties of the InsP₃R. Thus, our results suggest that the channel is poised in vivo to respond to changes in the free ATP concentration, for example those that may occur during ischemia. Therefore, the nucleotide sensitivity may enable Ca²⁺ release properties of the InsP₃R to be tuned to the metabolic state of the cell.

It has become evident from several recent studies that mitochondria and the ER form a tightly coupled, complex signaling unit. Imaging studies have revealed that mitochondria are in close physical proximity to the ER (25), especially to sites of Ca²⁺ release (26). In cerebellar Purkinje cells, ER cisternae containing high densities of InsP₃R-1 are often wrapped around or closely apposed to mitochondria (49, 50). A physiological implication of this structural arrangement is that it enables the rise in [Ca²⁺]_i caused by agonist-stimulated InsP₃R activity to be effectively transmitted as a transient increase in mitochondrial matrix [Ca²⁺] that closely parallels the [Ca²⁺]_i rise, due to the locally high [Ca²⁺]_i in the microdomain of the release channels and rapid uptake of released Ca²⁺ by the mitochondria (28-30, 33, 51). The resulting changes in mitochondrial matrix [Ca²⁺] affect the mitochondrial membrane potential (32) and the activities of the mitochondrial dehydrogenases that are crucial in ATP synthesis and intracellular ATP levels (27, 31, 33, 34, 52). The cytosolic ATP concentration in turn affects processes that contribute to [Ca²⁺]_i regulation, including InsP₃-induced Ca²⁺ release (Refs. 20-24 and 43 and this study), passive leak from Ca²⁺ stores (53), plasma membrane store-operated Ca²⁺ entry (24, 54, 55), and Ca²⁺ extrusion and uptake into the ER by Ca²⁺-ATPases (24, 56, 57). Thus, in addition to buffering [Ca²⁺]_i directly by active Ca²⁺ sequestration and export (58-62), mitochondria indirectly participate in intracellular Ca²⁺ signaling, using cytosolic ATP as a global cytoplasmic messenger. We speculate that the close physical proximity of mitochondria and ER may enable local changes in ATP concentration, due to release from mitochondria into the microdomains of close ER-mitochondria apposition, to rapidly effect local InsP₃R-mediated Ca²⁺ release. Of significance, the ATP released by mitochondria is free ATP, the InsP₃R ligand, not MgATP (63). Thus, communication between these two organelles may be two-way, with local Ca²⁺ release as the currency of communication from ER to mitochondria and local ATP release providing the cross-talk from mitochondria to ER.

The phosphoinositide signaling system is highly expressed throughout the brain (64, 65). Recent observations suggest that Ca²⁺ release from type 1 InsP₃ receptors is involved in nerve growth (66) and synaptic plasticity, including long term potentiation (67-69) and depression (LTD) (67, 67, 70-72). Disruption of the mouse InsP₃R-1 gene eliminates LTD in the cerebellum (70), and the competitive InsP₃R inhibitor heparin blocks LTD in the neocortex (73). Metabotropic glutamate receptors (mGluR), which couple to the InsP₃ signaling pathway, have been implicated in synaptic plasticity (65, 69, 73), and mice with targeted disruption of mGluR1 show impaired LTD (74, 75). InsP₃-mediated LTD in Purkinje cell dendrites was recently shown to be spatially restricted to sites where both mGluR and InsP₃R are located (71). Ca²⁺ influx through N-methyl-D-aspartate receptors and voltage-gated channels is considered to be of major importance in synaptic plasticity (65). Synaptic plasticity in several different brain regions requires both Ca²⁺ entry and mGluR/InsP₃R (72, 76, 77), and it been suggested that Ca²⁺ influx might serve to trigger Ca²⁺ release by CICR (65, 69, 78), with InsP₃Rs therefore playing a critical role in amplifying the Ca²⁺ influx signal (65). Importantly, [Ca²⁺]_i signaling in nonexcitable cells is also associated with both Ca²⁺ release from stores and Ca²⁺ influx, and Ca²⁺ influx has been demonstrated to play a similar role in amplifying and modifying InsP₃-mediated [Ca²⁺]_i signals (79). By demonstrating that the Ca²⁺ sensitivity of CICR by the InsP₃R-1 can be regulated, our data raise the possibility that synaptic plasticity and other cellular processes involving InsP₃Rs may be modulated by physiological stimuli that impinge on the Ca²⁺ sensitivity of the release channel. The results of the present study suggest that cytoplasmic ATP, and therefore the metabolic status of the cell, may be relevant in this respect. Of note, phospholipase C activity in brain tissue is enhanced in response to ischemia (80), and exposure of hippocampal slices to anoxia (81) or 2-deoxyglucose (13), manipulations expected to alter cytoplasmic ATP concentrations, induce long term potentiation. Directed studies of the role of cell metabolic state and cytoplasmic ATP concentrations in controlling InsP₃-mediated Ca²⁺ release will be required to determine the relevance of ATP regulation of InsP₃R gating for synaptic plasticity as well as other cellular processes.

	FOOTNOTES

^* 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.

^¶ To whom correspondence and reprint requests should be addressed: Dept. of Physiology, University of Pennsylvania, B39 ANAT-CHEM, Philadelphia, PA 19104-6085. Tel.: 215-898-1354; Fax: 215-573-6808; E-mail: foskett@mail.med.upenn.edu.

	ABBREVIATIONS

The abbreviations used are: InsP₃, inositol 1,4,5-trisphosphate; InsP₃R, InsP₃ receptor; ER, endoplasmic reticulum; BAPTA, 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; LTD, long term depression; mGluR, metabotropic glutamate receptor(s).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				M. J. Betzenhauser, L. E. Wagner II, H. S. Park, and D. I. Yule ATP Regulation of Type-1 Inositol 1,4,5-Trisphosphate Receptor Activity Does Not Require Walker A-type ATP-binding Motifs J. Biol. Chem., June 12, 2009; 284(24): 16156 - 16163. [Abstract] [Full Text] [PDF]

				V. K. Nagaleekar, S. A. Diehl, I. Juncadella, C. Charland, N. Muthusamy, S. Eaton, L. Haynes, L. A. Garrett-Sinha, J. Anguita, and M. Rincon IP3 Receptor-Mediated Ca2+ Release in Naive CD4 T Cells Dictates Their Cytokine Program J. Immunol., December 15, 2008; 181(12): 8315 - 8322. [Abstract] [Full Text] [PDF]

				H. S. Park, M. J. Betzenhauser, J. H. Won, J. Chen, and D. I. Yule The Type 2 Inositol (1,4,5)-Trisphosphate (InsP3) Receptor Determines the Sensitivity of InsP3-induced Ca2+ Release to ATP in Pancreatic Acinar Cells J. Biol. Chem., September 19, 2008; 283(38): 26081 - 26088. [Abstract] [Full Text] [PDF]

				M. J. Betzenhauser, L. E. Wagner II, M. Iwai, T. Michikawa, K. Mikoshiba, and D. I. Yule ATP Modulation of Ca2+ Release by Type-2 and Type-3 Inositol (1, 4, 5)-Triphosphate Receptors: DIFFERING ATP SENSITIVITIES AND MOLECULAR DETERMINANTS OF ACTION J. Biol. Chem., August 1, 2008; 283(31): 21579 - 21587. [Abstract] [Full Text] [PDF]

				L. E. Wagner II, S. K. Joseph, and D. I. Yule Regulation of single inositol 1,4,5-trisphosphate receptor channel activity by protein kinase A phosphorylation J. Physiol., August 1, 2008; 586(15): 3577 - 3596. [Abstract] [Full Text] [PDF]

				J. M. Power and P. Sah Competition between Calcium-Activated K+ Channels Determines Cholinergic Action on Firing Properties of Basolateral Amygdala Projection Neurons J. Neurosci., March 19, 2008; 28(12): 3209 - 3220. [Abstract] [Full Text] [PDF]

				J. K. Foskett, C. White, K.-H. Cheung, and D.-O. D. Mak Inositol Trisphosphate Receptor Ca2+ Release Channels Physiol Rev, April 1, 2007; 87(2): 593 - 658. [Abstract] [Full Text] [PDF]

				C. White, J. Yang, M. J. Monteiro, and J. K. Foskett CIB1, a Ubiquitously Expressed Ca2+-binding Protein Ligand of the InsP3 Receptor Ca2+ Release Channel J. Biol. Chem., July 28, 2006; 281(30): 20825 - 20833. [Abstract] [Full Text] [PDF]

				L. E. Wagner II, M. J. Betzenhauser, and D. I. Yule ATP Binding to a Unique Site in the Type-1 S2- Inositol 1,4,5-Trisphosphate Receptor Defines Susceptibility to Phosphorylation by Protein Kinase A J. Biol. Chem., June 23, 2006; 281(25): 17410 - 17419. [Abstract] [Full Text] [PDF]

				F.-X. Yi and I. M. Bird Pregnancy-Specific Modulatory Role of Mitochondria on Adenosine 5'-Triphosphate-Induced Cytosolic [Ca2+] Signaling in Uterine Artery Endothelial Cells Endocrinology, November 1, 2005; 146(11): 4844 - 4850. [Abstract] [Full Text] [PDF]

				R. Dumollard, P. Marangos, G. Fitzharris, K. Swann, M. Duchen, and J. Carroll Sperm-triggered [Ca2+] oscillations and Ca2+ homeostasis in the mouse egg have an absolute requirement for mitochondrial ATP production Development, July 1, 2004; 131(13): 3057 - 3067. [Abstract] [Full Text] [PDF]

				D.-O. D. Mak, S. M.J. McBride, and J. K. Foskett Spontaneous Channel Activity of the Inositol 1,4,5-Trisphosphate (InsP3) Receptor (InsP3R). Application of Allosteric Modeling to Calcium and InsP3 Regulation of InsP3R Single-channel Gating J. Gen. Physiol., October 27, 2003; 122(5): 583 - 603. [Abstract] [Full Text] [PDF]

				R. Dumollard, K. Hammar, M. Porterfield, P. J. Smith, C. Cibert, C. Rouviere, and C. Sardet Mitochondrial respiration and Ca2+ waves are linked during fertilization and meiosis completion Development, February 15, 2003; 130(4): 683 - 692. [Abstract] [Full Text] [PDF]

				J. C. Lenz, H. P. Reusch, N. Albrecht, G. Schultz, and M. Schaefer Ca2+-controlled competitive diacylglycerol binding of protein kinase C isoenzymes in living cells J. Cell Biol., October 28, 2002; 159(2): 291 - 302. [Abstract] [Full Text] [PDF]

				R. Dumollard, J. Carroll, G. Dupont, and C. Sardet Calcium wave pacemakers in eggs J. Cell Sci., September 15, 2002; 115(18): 3557 - 3564. [Abstract] [Full Text] [PDF]

				S. H. Yoo, Y. S. Oh, M. K. Kang, Y. H. Huh, S. H. So, H. S. Park, and H. Y. Park Localization of Three Types of the Inositol 1,4,5-Trisphosphate Receptor/Ca2+ Channel in the Secretory Granules and Coupling with the Ca2+ Storage Proteins Chromogranins A and B J. Biol. Chem., November 30, 2001; 276(49): 45806 - 45812. [Abstract] [Full Text] [PDF]

				L. Missiaen, H. DeSmedt, G. Bultynck, S. Vanlingen, P. Desmet, G. Callewaert, and J. B. Parys Calmodulin Increases the Sensitivity of Type 3 Inositol-1,4,5-trisphosphate Receptors to Ca2+ Inhibition in Human Bronchial Mucosal Cells Mol. Pharmacol., March 1, 2000; 57(3): 564 - 567. [Abstract] [Full Text]

				D. Boehning and S. K. Joseph Functional Properties of Recombinant Type I and Type III Inositol 1,4,5-Trisphosphate Receptor Isoforms Expressed in COS-7 Cells J. Biol. Chem., July 7, 2000; 275(28): 21492 - 21499. [Abstract] [Full Text] [PDF]

				J. S. Marchant, V. Ramos, and I. Parker Structural and functional relationships between Ca2+ puffs and mitochondria in Xenopus oocytes Am J Physiol Cell Physiol, June 1, 2002; 282(6): C1374 - C1386. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mak, D.-O. D. Articles by Foskett, J. K. Search for Related Content PubMed PubMed Citation Articles by Mak, D.-O. D. Articles by Foskett, J. K. Social Bookmarking                      What's this?