©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Sensitization of Calcium-induced Calcium Release by Cyclic ADP-ribose and Calmodulin (*)

Hon Cheung Lee (§) , Robert Aarhus , Richard M. Graeff

From the (1) Department of Physiology, University of Minnesota, Minneapolis, Minnesota 55455

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Cyclic ADP-ribose (cADPR) is emerging as an endogenous regulator of Ca-induced Carelease (CICR), and we have recently demonstrated that its action is mediated by calmodulin (CaM) (Lee, H. C., Aarhus, R., Graeff, R., Gurnack, M. E., and Walseth, T. F. (1994) Nature 370, 307-309). In this study we show by immunoblot analyses that the protein factor in sea urchin eggs responsible for conferring cADPR sensitivity to egg microsomes was CaM. This was further supported by the fact that bovine CaM was equally effective as the egg factor. In contrast, plant CaM was only partially active even at 10-20-fold higher concentrations. This exquisite specificity was also shown by binding studies using I-labeled bovine CaM. The effectiveness of various CaMs (bovine > spinach > wheat germ) in competing for the binding sites was identical to their potency in conferring cADPR sensitivity to the microsomes. A comparison between bovine and wheat germ CaM in competing for the sites suggests only 10-14% of the total binding was crucial for the activity. Depending on the CaM concentration, the sensitivity of the microsomes to cADPR could be changed by several orders of magnitude. The requirement for CaM could be alleviated by raising the divalent cation concentration with Sr. Results showed that CaM, cADPR, and caffeine all act synergistically to increase the divalent cation sensitivity of the CICR mechanism. The combined action of any of the three agonists was sufficient to sensitize the mechanism so much that even the nanomolar concentration of ambient Cawas enough to activate the release. Unlike the CICR mechanism, the microsomal inositol 1,4,5-trisphosphate-sensitive Carelease showed no dependence on CaM. Using an antagonist of CaM, W7, it was demonstrated that the cADPR- but not the inositol 1,4,5-trisphosphate-dependent release mechanism could be blocked in live sea urchin eggs. These results indicate cADPR can function as a physiological modulator of CICR and, together with CaM, can alter the sensitivity of the release mechanism to divalent cation by several orders of magnitude.


INTRODUCTION

Cyclic ADP-ribose (cADPR)() was discovered as a metabolite of NADwhich has Careleasing activity in an invertebrate cell, the sea urchin egg (1, 2, 3) . The generality of its function is shown by the fact that amphibian neurons and a variety of mammalian cells have since been found to be also responsive to cADPR (reviewed in Ref. 4). That the Carelease mechanism activated by cADPR is independent of the inositol 1,4,5-trisphosphate (IP) system is indicated by its insensitivity to heparin, an antagonist of the IPreceptor (5) . Conversely, 8-amino-cADPR blocks specifically the action of cADPR without affecting the IP-sensitive Carelease (6) . Also, microsomes desensitized to high concentrations of either cADPR or IPcan still respond, respectively, to the heterologous activator (5) . Specific binding of cADPR to the microsomal receptor is, likewise, unaffected by IPand heparin (7) . It is increasingly clear that the cADPR-dependent Carelease mechanism resembles the Ca-induced Carelease (CICR) in its pharmacology. Thus, agonists of CICR, such as Caand caffeine, potentiate cADPR-dependent Carelease (8, 9) . Antagonists of CICR, such as ruthenium red and procaine, selectively block only the Carelease induced by cADPR but not that induced by IP(8, 9) . Finally, ryanodine, an effector of CICR, can release Cafrom the cADPR-sensitive stores and desensitize them selectively to cADPR but not to IP(8, 9) . Therefore, the cADPR-sensitive Carelease mechanism is pharmacologically indistinguishable from the CICR mechanism.

Another notable feature of the cADPR-sensitive Carelease that differentiates it from the IPmechanism is its requirement for a soluble protein factor (1) . Separation of sea urchin egg microsomes from soluble protein components results in loss of cADPR responsiveness (1, 10) . Purification and characterizations of the cADPR-conferring factor indicate it is calmodulin (CaM) (10) . The cADPR sensitivity-conferring activity of CaM does not appear to be mediated through enzymatic means, but is more likely through direct interaction with the Carelease mechanism (10) . In this study, we show that the requirement for CaM is highly specific, with plant CaM being 10-20-fold less effective than either sea urchin or mammalian CaM. Analyses indicate CaM acts synergistically with cADPR to increase the sensitivity of the release mechanism to divalent cations. In addition to being a second messenger for Camobilization, these results suggest a new role for cADPR as a modulator of the Casensitivity of CICR.


EXPERIMENTAL PROCEDURES

Fractionation of Egg Microsomes and Microinjection of Intact Eggs

Homogenates of sea urchin egg ( Strongylocentrotus purpuratus) were prepared as described previously (7) . Frozen egg homogenates (25%) were thawed and incubated at 17 °C for 20-40 min. Egg homogenates (2 ml for each gradient) were layered on 10 ml of 25% Percoll prepared as described previously (10) . After centrifugation for 30 min at 25,000 rpm in a Beckman Ti50 rotor at 10 °C, the microsomes were collected by inserting a syringe needle directly into the band of vesicles on the upper part of the gradient. Microsomes collected from each gradient (1 ml) were diluted to 5 ml with a medium containing 250 m M N-methylglucamine, 250 m M potassium gluconate, 20 m M Hepes, 1 m M MgCl, 2 units/ml creatine kinase, 8 m M phosphocreatine, 0.5 m M ATP, and 1.5-3 µ M Fluo 3, pH 7.2, adjusted with acetic acid (GluIM). The final microsomal protein concentrations were about 0.6 mg/ml. Carelease activity was tested after incubating the microsomes with or without CaM (1-60 µg/ml) at 17° C for about 2 h.

Lytechinus pictus eggs were used for the microinjection experiments. The procedures for microinjection by pressure and for measuring the Cachanges in the injected eggs using Fluo 3 were as described previously (11) . Samples were dissolved in the injection buffer containing 0.5 M KCl, 50 µ M EGTA, 10 m M Hepes, pH 6.7.

Purification of the cADPR Sensitivity-conferring Factor

The supernatants from the Percoll density gradient centrifugation described above were collected and used as the source for the cADPR sensitivity-conferring factor. The supernatant (20 ml) was dialyzed against 2 liters of 20 m M Hepes, pH 7.2, overnight and centrifuged at 50,000 rpm for 1 h at 4 °C using a Beckman Ti50 rotor. The supernatant was collected and concentrated to 11 ml using Centriprep filters with M3,000 cutoff (Amicon, Beverly, MA). The concentrate was chromatographed on a DEAE 5PW column (Waters, Milford, MA) using a linear gradient of 0 to 2 M potassium acetate, pH 7.2, at a flow rate of 0.7 ml/min. The cADPR sensitivity-conferring activity was assayed by incubating 50 µl of each fraction with 0.55 ml of purified microsomes and challenging with 200 n M cADPR. All of the activity was eluted at about 1.3 M potassium acetate. The active fractions were combined (2 ml), concentrated to 0.3 ml, and finally purified on a 300 SW gel-filtration column (Waters) by elution with 0.2 M potassium acetate, pH 7.2, at 0.7 ml/min.

Calmodulin Binding

Egg microsomes were purified by Percoll density gradient centrifugation as described above. Microsomes collected from each gradient were diluted to 2.5 ml with GluIM (composition listed above) and 0.2-ml aliquots (1.2 mg protein/ml) were incubated for 2 h at 17 °C with 23,000-30,000 cpm of I-CaM (bovine, 69-88 µCi/µg) in the presence (1-60 µg/ml) or absence of unlabeled CaM. After incubation, the microsomes were pelleted by centrifugation at 4 °C for 10 min at 100,000 rpm in a Beckman TLA 100.1 rotor. The supernatant was discarded, and the pellets were counted in a -counter.

Immunoblot

The procedure used was similar to that described by Hulen et al. (12) . CaM samples were chromatographed on SDS-polyacrylamide gel electrophoresis and transferred to Immobilon-P (Millipore, Bedford, MA). The membrane was fixed for 45 min at room temperature with 0.2% glutaraldehyde. After rinsing, the membrane was incubated for 1 h at 37 °C with a monoclonal antibody against CaM (clone 6D4; Sigma). The antibody was diluted 2,000 times with 1% milk powder. After rinsing, the membrane was incubated with a secondary antibody labeled with horseradish peroxidase (Amersham Corp.) and developed using the ECL reagents and procedure supplied by Amersham.

Materials and Miscellaneous Methods

cADPR was synthesized by incubation of NADwith ADP-ribosyl cyclase purified from Aplysia ovotestis as described previously (13) . Fluo 3 was from Molecular Probes (Eugene, OR). Bovine and spinach CaM, activator-deficient 3`,5`-cyclic nucleotide phosphodiesterase, caffeine, and ryanodine were from Sigma. Wheat germ CaM was a generous gift from Dr. Gale Strasbourg, Michigan State University, East Lansing, MI. I-Labeled bovine CaM was from DuPont NEN. Concentrations of CaM were determined using the Coomassie reagent from Bio-Rad with bovine serum albumin as the standard.


RESULTS

Specificity of Calmodulin in Conferring cADPR Sensitivity to Microsomes

We have previously shown that the loss of cADPR responsiveness of sea urchin egg microsomes purified by Percoll density gradient can be restored by a soluble protein factor (10) . That the purified factor is CaM is indicated by the immunoblot shown in the inset of Fig. 1. On SDS-polyacrylamide gel electrophoresis, the factor ( SU, inset of Fig. 1) appeared as a protein of about 17 kDa ( lane 1b), identical to that of authentic bovine CaM ( lane 2b). It was recognized by a monoclonal antibody against CaM ( lane 1a) to a similar extent as bovine CaM ( lane 2a). The specificity of the monoclonal antibody is shown by its lack of reactivity to bovine serum albumin included in the samples as a control. The authentic bovine CaM could substitute for the sea urchin egg factor (SU) in conferring cADPR to the egg microsomes (Fig. 1). In the absence of either the egg factor or bovine CaM, purified egg microsomes did not response to cADPR (250 n M). Both the egg factor and bovine CaM were equally effective in reconstituting the Carelease induced by cADPR (250 n M) and both had half-maximal concentrations of about 2 µg/ml when assayed with the same preparation of egg microsomes.


Figure 1: Identification of the cADPR sensitivity-conferring factor in egg extracts as CaM. S. purpuratus egg microsomes were purified by Percoll density gradient centrifugation and incubated with bovine CaM or the purified conferring factor ( SU) at the indicated concentrations. Carelease activity was induced by 250 n M cADPR. The inset shows immunoblots of SU ( lane 1a) and bovine CaM ( lane 2a). Lanes 1b and 2b are the respective blots stained for protein. Bovine serum albumin ( BSA) was included in the samples as an internal control to demonstrate the specificity of the monoclonal antibody used.



Although animal CaMs such as bovine can substitute for sea urchin CaM, plant CaMs are much less effective. Fig. 2shows that microsomes treated with up to 30 µg/ml of wheat germ CaM remained unresponsive to cADPR. For comparison, bovine CaM was used as a control in the same preparation of egg microsomes, which produced close to a maximal effect at 2 µg/ml. The inset of Fig. 2 summarizes the results from three different preparations of microsomes. Spinach CaM at 37-73 µg/ml could produce only about half of the effect of bovine CaM at 2-5 µg/ml. Wheat germ CaM at 30 µg/ml was essentially ineffective.


Figure 2: Difference in effectiveness of animal and plant CaM in conferring cADPR sensitivity. Egg microsomes were incubated with either bovine, wheat germ, or spinach CaM at the indicated concentrations. Carelease activity was induced by 250 n M or 200-300 n M ( inset) cADPR. The error bars shown in the inset represent the S.E. of the number of determinations ( N).



The concentration-response curves for bovine and spinach CaM are shown in Fig. 3A. For bovine CaM, maximal response was seen at about 3-5 µg/ml. Spinach CaM could support only about one-third of the Carelease even at the highest concentration of 73 µg/ml tested with the same preparation of microsomes. Similar and dramatic differences between bovine and spinach CaM were seen when the concentrations of the CaM were, respectively, held constant while the concentration of cADPR was varied as shown in Fig. 3B. Microsomes treated with 10 µg/ml bovine CaM produced close to maximal Carelease when challenged with about 150 n M cADPR. The same preparation of microsomes treated with 37 µg/ml spinach CaM produced no response at 150 n M cADPR and only about half-maximal release when challenged with up to 400 n M cADPR. These results clearly show that the Carelease mechanism activated by cADPR exhibits exquisite specificity toward animal CaM. As will be discussed later, the sequences of bovine and spinach CaM differ by only 15 amino acids. This specificity is rather unique as bovine cyclic nucleotide phosphodiesterase shows relatively little preference between animal and plant CaM. The half-maximal concentrations of CaM for activating the phosphodiesterase were measured to be 0.30 ± 0.06 µg/ml (±S.E., n = 37) for bovine, 0.50 ± 0.07 µg/ml ( n = 39) for spinach, and 0.42 ± 0.1 µg/ml ( n = 16) for wheat germ. Also, with sufficient concentrations of the plant CaM, 100% activation of the phosphodiesterase was achieved. This was not the case for the cADPR sensitivity-conferring activity as shown in Fig. 3, maximal Carelease was not achieved even at the highest plant CaM used.


Figure 3: Concentration dependence of CaM in conferring cADPR sensitivity. A, egg microsomes were incubated with either bovine or spinach CaM at the indicated concentrations. Carelease activity was induced by 200 n M cADPR. B, egg microsomes were incubated with either 10 µg/ml bovine or 37 µg/ml spinach CaM and challenged with the indicated concentrations of cADPR.



Previous results suggest the cADPR sensitivity-conferring activity of CaM is not mediated by enzymatic reactions but is likely to be through direct interaction between CaM and the Carelease mechanism (10) . This is supported by the measurements of CaM binding to the microsomes shown in Fig. 4. The measurement was done under identical experimental conditions as the Carelease assays. The ambient Caconcentrations were generally in the range of 20-40 n M (9) and thus, the binding observed did not require high concentrations of Ca. The binding is specific since increasing the concentration of unlabeled bovine CaM progressively decreased the total binding of the I-labeled bovine CaM. The residual radioactivity observed in the presence of 20 µg/ml of unlabeled CaM was mainly due to trapping of the label in the microsomal pellets. Scatchard analysis of the binding data is shown in the inset. Binding appears to follow single component kinetics with a dissociation constant ( K) of 128 n M and a density ( B) of 10.7 pmol/mg. It is well documented that the ryanodine receptor has especially high affinity for CaM and the binding occurs even in the absence of Ca(14) .


Figure 4: Binding of I-labeled bovine CaM to egg microsomes. The binding was measured in the presence of the indicated concentrations of unlabeled bovine CaM as described under ``Experimental Procedures.'' The inset shows the Scatchard analysis of the binding data.



The effectiveness of various CaMs in competing for the binding of I-labeled bovine CaM to egg microsomes is shown in Fig. 5. The bovine CaM was the most effective competitor and, at 20 and 60 µg/ml, reduced the total binding to 30% and 23.4%, respectively. The residual 23% binding was nonspecific and represented mainly the trapping of the label in the microsomal pellets. Plant CaMs were less effective. The residual binding was significantly higher in the presence of either spinach ( p < 0.009, t test) or wheat germ ( p < 2 10) than bovine CaM at the same concentrations. The effectiveness sequence of bovine > spinach > wheat germ was identical to the cADPR-sensitivity conferring activity as depicted in the inset of Fig. 2. Since wheat germ CaM was essentially inactive in conferring the activity, the difference between the residual binding in the presence of wheat germ and bovine CaM may represent the crucial CaM binding sites for conferring cADPR sensitivity. This amounted to about 10-14% of the total CaM binding or 1.1-1.5 pmol/mg using the Bvalue of Fig. 4.

Mechanism of the cADPR Sensitivity-conferring Activity of Calmodulin

Fig. 6 shows the remarkably strong dependence of the microsomal cADPR sensitivity on calmodulin. In the absence of calmodulin, the microsomes would not release Caeven when challenge with as high a concentration of cADPR as 200 µ M. In the presence of a low concentration of calmodulin, the cADPR-responsiveness of the microsomes was restored only at high concentrations of cADPR. At saturating concentrations of calmodulin, the microsomes responded maximally to about 0.1 µ M of cADPR. Therefore, depending on how much calmodulin is present, the microsomal responsiveness to cADPR can be varied by more than 3-4 orders of magnitude.


Figure 6: Sensitization of the Ca release mechanism to cADPR by CaM. Percoll density purified microsomes were incubated with the indicated concentrations of bovine CaM. The Carelease was induced by various concentrations of cADPR as indicated.



The biological effects of CaM generally are related to Ca. We, therefore, investigated the relationship between divalent cations and the microsomal cADPR sensitivity. The results are shown in Fig. 7. Srwas used instead of Casince it minimally interfered with Fluo 3, which was used for monitoring Carelease from microsomes. In the absence of CaM, cADPR as high as 1 µ M produced no Carelease (Fig. 7 a). Fig. 7 b shows that adding 40 µ M Srgreatly sensitized the release system such that cADPR as low as 13 n M produces significant Carelease, and 75 n M cADPR produced close to a maximal response. Another way of interpreting these results is that the Casensitivity of the release mechanism is increased by the presence of cADPR. In the absence of cADPR, addition of 40 µ M of Srdid not produce any Carelease (Fig. 7 b). In the presence of cADPR, the release mechanism is sensitized to divalent cations and the same concentration of Srcould now induce a large Carelease (Fig. 7 a).


Figure 7: Effects of strontium on the cADPR-dependent Ca release. Microsomes were purified by Percoll density centrifugation and incubated without CaM. Strontium ( Sr) and cADPR were added to the final concentrations indicated.



This is shown more clearly in Fig. 8 A. In the absence of CaM, adding less than 100 µ M Srto microsomes did not produce any Carelease. Above 100 µ M Sr, significant Carelease was eventually induced. In the presence of calmodulin, the dose-response curve is shifted about 2-5-fold to the left. A low concentration of cADPR alone was more effective in sensitizing the release mechanism and produces a larger shift of the dose-response curve to the left. At 25 n M, cADPR was too low to induce Carelease even in the presence of CaM ( cf. Fig. 3B). The sensitivity of the release mechanism to Srwas increased more than 2 orders of magnitude in the presence of both cADPR and CaM, and only 10-20 µ M Srwas sufficient to produce maximal Carelease. The dose-response curve could, in fact, be shifted further to the left if higher concentrations of cADPR were used (data not shown). Therefore, cADPR and CaM can act synergistically to increase the sensitivity of the Carelease system to divalent cations.


Figure 8: Sensitization of Ca-induced Ca release by CaM, cADPR, and caffeine. Percoll density purified microsomes were incubated without (- CaM) or with (+ CaM) 0.6 µ M (10 µg/ml) bovine CaM. CICR was activated using the indicated concentrations of strontium in the presence of 25 µ M cADPR (+ cADPR) or caffeine (1 m M).



It is generally accepted that caffeine also acts by sensitizing CICR to Ca. This is shown in Fig. 8B. A low concentration of caffeine (1 m M), which was not sufficient to release Caon its own ( cf. Fig. 9), could sensitize the release mechanism to Srto an extent similar to that of CaM. Together, CaM and caffeine could increase the sensitivity even further. With both caffeine and cADPR present, the Carelease mechanism was so much sensitized that maximal Carelease was induced without any added divalent cation. These results indicate that CaM, cADPR and caffeine all act synergistically to increase the Casensitivity of CICR. When two of these sensitizers are present in sufficient concentrations, even ambient concentrations of Ca, which generally are in the nanomolar range, are sufficient to activate maximal release.

Similar to its effects on cADPR, CaM can also potentiate the Carelease activity of caffeine and ryanodine, which are modulators of CICR. In the absence of CaM, at least 10-11 m M of caffeine was needed to induce minimal Carelease. In the presence of CaM, 8-9 m M of caffeine was enough to produce maximal Carelease. Similar sensitizing effects of CaM were also seen with ryanodine as shown in Fig. 9 B. Compared with the large sensitization of CaM on cADPR (Fig. 6), its stimulating effects on these two artificial modulators of CICR were rather minimal.


Figure 9: Sensitization of the Ca release mechanism to caffeine and ryanodine by CaM. Percoll density-purified microsomes were incubated without or with 7 µg/ml bovine CaM. The Carelease was induced by various concentrations of caffeine or ryanodine as indicated.



Although CaM can sensitize all four known modulators of CICR, Ca, cADPR, caffeine, and ryanodine, it has no effect on the IP-dependent Casystem as shown in Fig. 10. In the presence of CaM, about 200 n M of cADPR was sufficient to induce maximal Carelease. In the absence of CaM, 200 n M of cADPR produced no release. In contrast, the same preparation of microsomes responded equally well to IPwith or without CaM. The sensitizing effect of CaM is, therefore, specific for the CICR system.

CaM also appears to be responsible for regulating cADPR-sensitivity in intact sea urchin eggs. To monitor intracellular Cachanges, eggs were pre-loaded with the Caindicator Fluo 3, and the change in its fluorescence was expressed as a ratio between initial fluorescence ( F) and that at various times afterward ( F). Fig. 11shows that microinjection of cADPR into an unfertilized egg elicited Carelease resulting in about a 10-fold increase in the Fluo 3 fluorescence ratio ( F/ F). To inhibit the endogenous CaM, the antagonist, W7, was used; W7 has previously been shown to block CaM from sensitizing the cADPR-induced Carelease in egg homogenates (10) . In the presence of W7, injection of similar concentrations of cADPR did not produce much change in Fluo 3 fluorescence. The inset of Fig. 11 summarizes the results. Of the 12 eggs injected with 313 ± 50 n M (mean ± S.E.) of cADPR in the presence of 80-120 µ M W7, 10 showed very little Cachange, while two had a normal response. The average increase in Fluo 3 fluorescence ratio ( F/ F- 1) was 1.69 ± 0.82 for the 12 eggs. In the absence of W7, the increase in the ratio value ( F/ F- 1) of 5.70 ± 1.09 was measured for the nine eggs injected with 337 ± 64 n M cADPR, more than 3-fold higher than in the presence of W7. In contrast, IP-induced changes in the fluorescence ratio were independent of W7 (80-120 µ M). The increase of the ratio of 5.17 ± 0.78 measured after injection of 456 ± 62 n M IPto eight eggs in the presence of W7 was identical to the ratio of 5.18 ± 1.03 measured in six eggs injected with 318 ± 55 n M of IPin the absence of W7. These results indicate cADPR sensitivity in live cells can be regulated by CaM.


Figure 11: Inhibition of cADPR-induced Ca release in intact sea urchin eggs by a CaM antagonist. Methods for microinjection are described under ``Experimental Procedures.'' Intracellular Cachanges were monitored with Fluo 3. For normalization, fluorescence intensity at various times ( F) was divided by the initial fluorescence ( F). The CaM antagonist, W7, was added to a final concentration of 120 µ M (+ W7). When indicated, cADPR was microinjected into L. pictus eggs to a final concentration of 270 n M (+ W7) or 350 n M (- W7). The inset shows the average increase in the fluorescence ratio ( F/ F - 1) induced by microinjection of cADPR or IPin the absence (- W7) or presence (+ W7) of W7 (80-120 µ M). The numbers inside the data bars indicate the number of eggs injected, and the error bars indicate the S.E.




DISCUSSION

The simplest model for the Carelease action of cADPR is through activation of a ligand-gated Cachannel. This would be analogous to that described for the IPreceptor (reviewed in Ref. 15). However, unlike the IPreceptor, the cADPR-gated channel appears to have close to an absolute requirement for CaM. In its absence, cADPR as high as 200 µ M cannot activate the channel. The fact that caffeine, a known Casensitizer of CICR and Sr, can substitute for CaM suggests it may also act by sensitizing the release channel to Ca. This was directly demonstrated by showing that both CaM and cADPR can indeed potentiate the Sr-induced Carelease in a manner similar to caffeine. That all three agonists act synergistically to increase the Casensitivity indicates the central role of Cain the activation of the Carelease mechanism. These results are consistent with cADPR functioning as a modulator of CICR. There appears to be a certain limit on the maximal extent of sensitization that can be achieved by cADPR alone. When the ambient Caconcentration is below that level, Carelease cannot be activated by cADPR alone; the synergistic action by CaM or caffeine is also needed. Raising the ambient divalent cation concentration alleviates the additional requirement. This interpretation postulates Caas the final activator of the release channel, while cADPR and CaM play a modulator role by increasing the Casensitivity of the release system to such an extent that ambient Cacan activate. It is generally believed that CICR is important in propagation of Cawaves and in amplifying the Casignals due to influx (reviewed in Ref. 4). The mechanism of how the Casensitivity of this important signaling pathway can be regulated has not been described. Results described in this study provide just such a regulatory mechanism. As directly shown in Fig. 8, depending on the endogenous levels of cADPR and CaM, the Casensitivity of CICR can be shifted by several orders of magnitude.

The modulator mechanism described apparently is operative in live cells since W7, an antagonist of CaM, can selectively inhibit the cADPR-sensitive Carelease in sea urchin eggs without affecting that of IP. The modulator model depicted in Fig. 12 summarizes these results. Low concentrations of cADPR could be present in cells at all times, which together with CaM are sufficient to sensitize the Carelease system. An increase in intracellular Caconcentration, either by receptor activated influx or by Camobilization through the IPpathway, can then activate further Carelease. This is, in fact, what was observed in amphibian neurons, where cADPR was shown to potentiate the Cainflux elicited by membrane depolarization (16) . The modulator function of cADPR, of course, does not exclude the possibility that cADPR may also be a second messenger. Indeed, it has previously been shown that ADP-ribosyl cyclase in sea urchin eggs, the synthetic enzyme of cADPR, can be activated by a cGMP-dependent mechanism (17) . This has led to the proposal that nitric oxide, through its stimulation of cGMP production, may activate the cADPR-signaling mechanism (18) . Alternatively, as depicted in the messenger model in Fig. 12, binding of an external ligand to its receptor could result in increased synthesis of cADPR which, in the presence of endogenous CaM, can then activate Carelease from the internal stores. The main difference between the modulator and the messenger models is that the former emphasizes the major role of Caas the activation signal, with the endogenous levels of cADPR being relatively constant. These two models are not mutually exclusive. Depending on how much calmodulin, cADPR, and Caare present in cells, the signaling mechanism can operate anywhere between these two extremes.


Figure 12: Models depicting two modes of action for cADPR. The activation signal in the Messenger Model is the ligand-induced activation of the ADP-ribosyl cyclase ( C) leading to an increase in cytosolic cADPR, while in the Modulator Model, it is the agonist-gated influx or internal release of Ca(not shown). In the Modulator Model, the primary function of cADPR and CaM is to sensitize the Carelease channel ( R) to Ca.



The CaM requirement of the cADPR-dependent Carelease described in this study is remarkable in its exquisite specificity toward animal CaMs. Both sea urchin and bovine CaM are equally effective while plant CaMs were much less so. The sequences of bovine and spinach CaM show a difference of 15 out of a total of 148 amino acids, with 12 of them being conservative substitutions (19, 20) . The three nonconservative changes from bovine to spinach CaM are: Thr-26 Cys, Gly-96 Gln, and Thr-146 Met. The first two are within the two of the four Cabinding sites of CaM, while the third change is also very close to the fourth Casite. That these changes are concentrated in the Cabinding regions of CaM is consistent with the Casensitizing effect of CaM on the release system as described in this study. The three apparently crucial amino acids are identical between bovine and sea urchin CaM (19, 21) , accounting for their equal potency in conferring the cADPR sensitivity. Perhaps the most remarkable specificity is that observed between spinach and wheat germ CaM. There are only two differences in their sequences, a replacement of Asp-97 by Asn and an insertion of an additional Asn at position 9 (21, 22) . That point mutations on CaM can dramatically affect its regulation on the Ca-dependent Kchannel has been well documented in Paramecium (reviewed in Ref. 23). Comparisons of the competition between wheat germ and bovine CaM for the microsomal binding sites shown in this study suggest that only 10-14% of the total binding sites are crucial for cADPR-sensitive Carelease. This exquisite specificity of the cADPR-dependent mechanism for CaM could potentially be exploited for the identification of the relevant CaM binding sites.


FOOTNOTES

*
This study was supported in part by National Institutes of Health Grants HD17484 and HD32040 (to H. C. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: 6-182 Lyon Laboratory, Dept. of Physiology, University of Minnesota, Minneapolis, MN 55455. Tel.: 612-625-7120 (office), 612-625-4641 (laboratory); Fax: 612-625-0991 (office), 612-625-5941 (department).

The abbreviations used are: cADPR, cyclic ADP-ribose; CaM, calmodulin; CICR, Ca-induced Carelease; IP, inositol 1,4,5-trisphosphate.


ACKNOWLEDGEMENTS

We thank Gale Strasbourg for providing the wheat germ calmodulin.

Note Added in Proof-Bovine serum albumin (six different types from Sigma, Boeringer Mannheim and United States Biochemicals) and egg albumin (Sigma), do not potentiate the calcium releasing activity of cADPR even at 1 mg/ml, further demonstrating the specificity of CaM.


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