Membrane-bound calmodulin in Xenopus laevis oocytes as a novel binding site for melatonin

^a Department of Medical Biochemistry and Molecular Biology, The University of Seville School of Medicine and Virgen Macarena Hospital, 41009 Seville, Spain

	ABSTRACT

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Melatonin has been suggested as a physiological antagonist of calmodulin. In this work, we have characterized melatonin binding sites in Xenopus laevis oocyte membranes. Binding of [¹²⁵I]melatonin by X. laevis oocyte membranes fulfills all criteria for binding to a receptor site. Binding was dependent on time, temperature, and membrane concentration and was stable, reversible, saturable, and specific. The binding site was also pharmacologically characterized. Stoichiometric studies showed a high-affinity binding site with a K_d of 1.18 nM. These data are in close agreement with data obtained from kinetic studies (K_d=0.12 nM). In competition studies, we observed a low-affinity binding site (K_d=63.41 µM). Moreover, the binding site was characterized as calmodulin. Thus, binding was dependent on calcium and blocked by anti-CaM antibodies in a concentration-dependent manner. Calmodulin inhibitor chlorpromazine also inhibited binding of the tracer. From these results, it is suggested that membrane-bound calmodulin acts as a melatonin binding site in Xenopus laevis oocytes, where it might couple cellular activities to rhythmic circulating levels of melatonin. This hypothesis correlates with the previous findings describing melatonin as a physiological antagonist of calmodulin.—Romero, M. P., García-Pergañeda, A., Guerrero, J. M., Osuna, C. Membrane-bound calmodulin in Xenopus laevis oocytes as a novel binding site for melatonin. FASEB J. 12, 1401–1408 (1998)

Key Words: membranes pd; CaM • indoleamine • protein kinase

	INTRODUCTION

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MELATONIN (N-acetyl-5-methoxytryptamine),² the major endocrine product of the pineal gland, plays an important role in the regulation of circadian rhythms as well as many physiological functions in mammals (1). This indoleamine exhibits a circadian secretory rhythm that conveys environmental information to the organism. Four mechanisms of action have been described for melatonin: binding to membrane receptors (2, 3) and possibly to nuclear (4, 5) binding sites, as a scavenger of free oxygen radicals (6), or binding to Ca²⁺ binding proteins such as calmodulin (Ca²⁺/CaM) (7) or protein kinase C (8).

CaM is a highly conserved 17 kDa Ca²⁺ binding protein found in all eukaryotic cells. It is a multifunctional, ubiquitous molecule (9) that mediates many Ca²⁺-regulated enzyme systems and cellular processes when complexed with this divalent cation (10–13). CaM has been shown to be essential for cell cycle progression in many eukaryotic organisms. Thus, CaM is known to be important in controlling progression at three points in the cell cycle: the initiation of DNA synthesis (G1/S boundary), the initiation of mitosis (G2/M), and at the metaphase/anaphase transition of mitosis to permit chromosome segregation and the completion of mitosis (14). Available data suggest that CaM also modulates gene transcription through CaM-dependent protein kinases (15), as well as DNA replication through the control of the activities of DNA polymerases (16, 17).

Ca²⁺-induced conformational change allows CaM to interact with enzymes, peptides, and pharmacological agents such as the phenothiazines. Benítez-King et al. (18) and Antón-Tay et al. (19) have demonstrated that melatonin binds to CaM with high affinity, and this could be the mechanism through which melatonin modulates many intracellular Ca²⁺ functions such as cytoskeletal rearrangements (20), cellular CaM levels and CaM-dependent phosphodiesterase activity (21), Ca²⁺/Mg²⁺ ATPase (22), CaM-dependent protein kinase II (23) or, as previously reported by our group, nitric oxide synthase activity (24–26). Through CaM, melatonin may directly affect Ca²⁺ signaling by interacting with target enzymes such as adenylate cyclase and phosphodiesterase, as well as with structural proteins.

The development of 2-[¹²⁵I]iodomelatonin ([¹²⁵I]melatonin), a high-affinity melatonin receptor agonist, as a radioligand has allowed the distribution and pharmacological characteristics of melatonin binding sites to be examined in various tissues of a number of species (27–29). The existence of a melatonin receptor in Xenopus laevis dermal melanophores is well known. The ability of the hormone to cause melanin aggregation in these cells is one of the earliest described actions of melatonin (for review, see ref 30). Moreover, cultured Xenopus dermal melanophores were recently used to successfully clone a high-affinity melatonin receptor cDNA (31).

Here we demonstrate the presence of a high-affinity binding site for melatonin in Xenopus laevis oocyte membranes. The binding of melatonin to membranes was dependent on time, temperature, and membrane concentration and was stable, reversible, saturable, and specific. Moreover, the binding was dependent on calcium and the binding site was characterized as calmodulin. Melatonin binding to membranes was blocked by anti-CaM antibodies. These results support the evidence of melatonin-specific binding to CaM in biological systems and emphasize the importance of calmodulin as a modulator of hormone action (32).

	METHODS

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Materials
All reagents were of analytical grade and obtained from commercial sources. [¹²⁵I]Melatonin was purchased from the Radiochemical Centre (Amersham Int., Amersham, U.K.). The specific activity of the radioligand was 1900–2175 Ci/mmol and was used for 60 days. Purity of the radioligand was checked by silica gel column chromatography (SGCC) and was >95%. Melatonin, 6-hydroxymelatonin, 5-methoxyindole-3-acetic acid, 5-methoxytryptophol, 5-methoxy-DL-tryptophan, 5-methoxytryptamine, N-acetylserotonin, 5-hydroxyindole-3-acetic acid, 5-hydroxytryptophol, 5-hydroxytryptamine, 5-hydroxy-DL-tryptophan, tryptamine, and DL-tryptophan were purchased from Sigma (St. Louis, Mo.), as were the calmodulin inhibitors chlorpromazine and calmidazolium, a mixture of three monoclonal antibodies against calmodulin, and the purified human immunoglobulin G. Other drugs were obtained from commercial sources.

Preparation of Xenopus oocyte membranes
Female Xenopus laevis frogs (Centre d'Elevage de Xenopes du CRBM, Montpellier, France) were anesthetized by hypothermia and ovarian lobes were removed through an abdominal incision and placed into 50 mM Tris-HCl buffer, pH 7.4, containing 4 mM CaCl₂. Oocytes were dissected from the ovarian tissue and incubated for 20 min in 0.05 U/ml collagenase (type II, Sigma), then washed extensively in Tris-HCl buffer and mechanically disrupted in the presence of PMSF (50 µg/ml). Membranes were isolated by centrifugation at low speed (2500xg, 10 min), followed by centrifugation of the supernatant (30,000xg, 30 min), and resuspended in Tris-HCl buffer. Protein concentration was determined by the method described by Bradford (33).

Binding studies
Binding assay conditions were essentially as described previously (34). Briefly, membranes (200 µg/ml) were incubated with [¹²⁵I]melatonin (100 pM) in 50 mM Tris-HCl buffer (pH 7.4) in the absence or the presence of 4 mM CaCl₂ and, when necessary, native melatonin and other drugs, in a total reaction volume of 400 µl. Reaction was initiated by the addition of the membranes, and performed at the times and temperatures indicated. Membrane-bound [¹²⁵I]melatonin was separated by centrifugation, washed with incubation buffer containing 10% (w/v) sucrose, and the radioactivity was measured in a LKB gamma counter. Data are reported as specific binding, i.e., total tracer bound minus the amount of the tracer that was not displaced by 100 µM melatonin. No detectable degradation of [¹²⁵I]melatonin during the incubation occurred as SGCC of aliquots of the incubation medium, taken before and after incubation, revealed similar amounts of radioactivity (95%) in the position of [¹²⁵I]melatonin. The dissociation constant (K_d) and maximum binding capacity (B_max) were calculated by the method described by Scatchard (35).

Gel filtration
Membranes (1 mg/ml) were heated for 60 min at 85°C and centrifuged (30,000xg, 30 min, 4°C) in order to deplete them from CaM. The pellets obtained were saved and supernatant was chromatographed on a Sephadex G-50 column (Pharmacia Biotech, Uppsala, Sweden) to separate its components according to their sizes (36). Elution was performed with 50 mM Tris-HCl buffer, pH 7.4, containing 4 mM CaCl₂, and 800 µl fractions were collected and assayed for protein content (33). Previously saved pellets were then resuspended using either Tris-HCl buffer, the supernatant, 200 µg/ml calmodulin, or the chromatography fractions; [¹²⁵I]melatonin binding assays were performed in order to determine the binding capacity restoration. The column was calibrated with marker proteins of known molecular mass (Pharmacia Biotech) under the same conditions used to analyze the supernatant, and the logarithm of the molecular mass was plotted against the fraction number to estimate the calmodulin fraction.

	RESULTS

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Kinetic studies
The specific binding of [¹²⁵I]melatonin to Xenopus oocyte membranes was a time- and temperature-dependent process ( Fig.1, top) . At 37°C, extraction of radioligand after 60 min incubation showed that more than 95% of radioactivity comigrated on SGCC with authentic [¹²⁵I]melatonin; no other radiolabeled peaks were observed. Thus, [¹²⁵I]melatonin is stable during the course of a standard binding assay at 37°C. Binding of [¹²⁵I]melatonin to oocyte membranes was also proportional to membrane concentration ( Fig. 1, bottom). Therefore, additional experiments were performed at 37°C with 200 µg protein/ml for 60 min to provide better binding conditions, with no observable degradation of the tracer.

View larger version (20K): [in this window] [in a new window]   Figure 1. Effect of time, temperature, and cell concentration on binding of [125I]melatonin to Xenopus oocyte membranes. Top: association of [125I]melatonin to membranes at 0, 15, and 37°. Membranes (200 µg/ml) were incubated with [125I]melatonin (100 pM) at the times indicated. Bottom: effect of membrane concentration on [125I]melatonin binding. Membranes (up to 300 µg/ml) were incubated with [125I]melatonin (100 pM) for 60 min at 37°. In both experiments, each point is the mean of three experiments performed in triplicate. Standard errors were always lower than 15% of the mean.

Binding of [¹²⁵I]melatonin to membranes was a trypsin-sensitive process ( Fig. 2). Trypsin pretreatment (30 min at 37°C) was stopped by the addition of 300 µg/ml TLCK and centrifugation (30,000xg, 30 min), and membranes were resuspended in Tris-HCl buffer prior to binding assay performance. Binding decreased to 36.23% of control after 10 µg/ml trypsin pretreatment and to 18.84% after 50 µg/ml trypsin pretreatment.

View larger version (35K): [in this window] [in a new window]   Figure 2. Effect of trypsin pretreatment on binding of [125I]melatonin to Xenopus oocyte membranes. Membranes (200 µg/ml) were preincubated at 37°C for 30 min either in Tris-HCl buffer (control), 10 µg/ml (trypsin-10), or 50 µg/ml trypsin (trypsin-50). Binding assay was performed at 37°C for 60 min with 100 pM [125I]melatonin. Each point is the mean of three experiments performed in triplicate.

Under optimal conditions, binding of [¹²⁵I]melatonin to membranes increased during the first 60 min and equilibrated after 60–90 min ( Fig. 3, top), with K₊₁ = 5.79 x 10⁸ M^-1 · min^-1 ( Fig. 3, left bottom). After equilibrium, binding of [¹²⁵I]melatonin to membranes was reversible. Dissociation of the tracer–membrane complex was studied by the addition of 100 µM melatonin; this test indicated a half-time of dissociation of 10 min, with K_-1 = 0.0693 min^-1 ( Fig. 3, right bottom). The kinetically derived value of K_d was 0.12 nM, obtained from the ratio of the rate constants (K_-1/K₊₁).

View larger version (25K): [in this window] [in a new window]   Figure 3. Association and dissociation of [125I]melatonin to Xenopus oocyte membranes. Top: membranes (200 µg/ml) were incubated with [125I]melatonin (100 pM) at 37°C at the times indicated (). After 60 min incubation, 100 µM melatonin was added and the specifically bound radioactivity was determined at the appropriated times (), and expressed as the percentage of radioactivity at time zero. Left bottom: pseudo first-order plot of the association data. Bmax is the specific binding at equilibrium. B is the specific binding at the time point measured. Right bottom: first-order plot of the dissociation data. Bmax is the specific binding when dissociation is initiated.

Stoichiometric studies
In competition studies, the addition of increasing concentrations of unlabeled melatonin caused a competitive inhibition of [¹²⁵I]melatonin binding to Xenopus oocyte membranes ( Fig. 4). Half-maximal inhibition (IC₅₀) was observed at 25 µM native melatonin. The Scatchard analysis of the data gave a curvilinear plot ( Fig. 4, inset) that could be resolved into two straight lines, suggesting the presence of two different classes of melatonin binding sites: a class with high affinity (K_d=1.18 nM) and low binding capacity (B_max=345 fmol/mg protein), and a class with low affinity (K_d=63.41 µM) and high binding capacity (B_max=1.3x10⁹ fmol/mg protein).

View larger version (19K): [in this window] [in a new window]   Figure 4. Competitive inhibition of [125I]melatonin binding to Xenopus oocyte membranes by native melatonin. Membranes (200 µg/ml) were incubated with [125I]melatonin (100 pM) and increasing concentrations of native melatonin for 60 min at 37°C. Each point is the mean of three experiments performed in triplicate. Inset: Scatchard analysis of the data.

Specific binding of [¹²⁵I]melatonin to membranes increased with increasing concentrations of the radioligand up to 1 nM, but no saturation was reached.

Pharmacological studies
The pharmacological characterization of [¹²⁵I]melatonin binding to Xenopus laevis oocyte membranes was carried out with tracer concentrations of radioligand (100 pM) and with melatonin or different metabolites involved in the melatonin synthesis pathway. Results showed that the melatonin binding site on Xenopus laevis oocyte membrane is highly specific for the hormone. Binding of [¹²⁵I]melatonin was inhibited by increasing concentrations of native melatonin; 50% inhibition (IC₅₀) was observed at 25 µM melatonin. N-acetylserotonin, 5-hydroxytryptophol, and tryptamine also displaced the radioligand, but were 3-, 20-, and 31-fold less effective than melatonin, respectively. Other indoles—5-hydroxyindoles or 5-methoxyindoles—were shown to be ineffective ( Table 1).

Implication of calmodulin
A set of criteria was defined in order to ascertain whether melatonin-specific binding to Xenopus laevis oocyte membranes was a calmodulin-dependent process.

1) The depletion of endogenous CaM by appropriate means should alter the level of specific binding. One of the most prominent features of CaM is its thermal stability; this characteristic was used to deplete oocyte membranes from CaM. Membranes (1 mg/ml) were heated for 60 min at 85°C and centrifuged (30,000xg, 30 min); pellets were then resuspended in Tris-HCl buffer and a binding assay was performed. [¹²⁵I]Melatonin-specific binding decreased to 21% of maximum, whereas to only 97.61% when pellets were resuspended in the supernatant ( Fig. 5).

View larger version (46K): [in this window] [in a new window]   Figure 5. Effect of CaM depletion on [125I]melatonin binding to Xenopus oocyte membranes. Membranes (1 mg/ml) were heated at 85°C for 60 min prior to centrifugation (30,000xg, 30 min). Pellets were resuspended using either Tris-HCl buffer, the supernatant, or 200 µg/ml CaM, prior to [125I]melatonin binding assay. Native membranes were used as control. Each point is the mean of three experiments performed in triplicate.

2) The experimental system depleted of endogenous CaM should respond to exogenous CaM. Membranes were depleted of CaM, as described, and pellets were resuspended in 200 µg/ml CaM for binding assay. Exogenous CaM restored the membrane [¹²⁵I]melatonin binding capacity to 108.81% ( Fig. 5). Binding was also restored by endogenous CaM. Supernatant was loaded onto a Sephadex G-50 column and eluted with Tris-HCl buffer. Collected fractions were used to resuspend the pellets in order to determine the elution profile of binding capacity restoration. A single peak corresponding to an apparent molecular mass of 19 kDa was found ( Fig. 6).

View larger version (19K): [in this window] [in a new window]   Figure 6. Binding restoration by endogenous calmodulin. Membranes (1 mg/ml) were heated at 85°C for 60 min prior to centrifugation (30,000xg, 30 min). Supernatant was chromatographed on a Sephadex G-50 column, fractions were used to resuspend pellets, and binding assay was performed. Native membranes were used as control. Column was calibrated with proteins of known molecular mass; V0 indicates the void volume, Ch indicates chymotrypsinogen. Two independent experiments yielded similar results.

3) Since CaM requires Ca²⁺ for activity, sequestering Ca²⁺ in the reaction system by an appropriate chelator should alter the binding characteristics. Binding assay was performed in Tris-HCl buffer containing or not 4 mM CaCl₂, either in the presence or in the absence of 10 mM EGTA. [¹²⁵I]melatonin-specific binding dramatically decreased when Ca²⁺ was eliminated from the medium ( Fig. 7).

View larger version (36K): [in this window] [in a new window]   Figure 7. Calcium dependence of [125I]melatonin binding to Xenopus oocyte membranes. Membranes (200 µg/ml) were incubated at 37°C for 60 min with [125I]melatonin (100 pM) either in the presence of 4 mM CaCl2, 10 mM EGTA, both, or none of them. Each point is the mean of three experiments performed in triplicate.

4) Activity should also be altered by the use of CaM inhibitors, molecules that prevent calmodulin from activating target enzymes in the presence of Ca²⁺. Two of these inhibitors, chlorpromazine and calmidazolium, were tested. Binding of the tracer was inhibited by increasing concentrations of chlorpromazine, and IC₅₀ was observed at 0.5 mM. Calmidazolium showed no effect on binding inhibition (IC₅₀>1 mM).

5) The effect of CaM should be reversed by its antibody. A mixture of three monoclonal antibodies against CaM was used to block melatonin-specific binding to oocyte membranes. Purified human immunoglobulin G was used as a control. Binding assays were performed after 30 min preincubation at 37°C with different antibody dilutions. Binding of [¹²⁵I]melatonin to membranes was blocked by the specific antibody in a dilution-dependent manner, but was not altered when membranes were incubated with nonspecific antibodies ( Fig. 8). Being positive, this criterion also provides evidence than CaM is accessible to its antibody in this system.

View larger version (16K): [in this window] [in a new window]   Figure 8. Effect of anti-calmodulin antibodies on [125I]melatonin binding to Xenopus oocyte membranes. Membranes (200 µg/ml) were preincubated at 37°C for 30 min with a mixture of three monoclonal antibodies against CaM () or purified human IgG (). Binding assay was performed at 37°C for 60 min with [125I]melatonin (100 pM). Each point is the mean of two experiments performed in triplicate.

	DISCUSSION

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We demonstrate here, for the first time, the behavior of calmodulin as a melatonin binding site in a biological system. Binding of [¹²⁵I]melatonin by Xenopus laevis oocyte membranes fulfills all criteria for binding to a receptor binding site. Thus, the binding of melatonin to membranes was dependent on time, temperature, and membrane concentration and was stable, reversible, saturable, and specific. Binding was calcium dependent and blocked by anti-CaM antibodies, suggesting that CaM is involved in this binding.

Competition studies were performed using increasing concentrations of native melatonin. Interpretation of the data by the Scatchard analysis (35) disclosed two binding sites with different affinities (1.18 nM and 63.41 µM). Kinetic studies yielded only one class of binding site (K_d=0.12 nM); affinity of this receptor is quite similar to that found for the high-affinity binding site described by competition studies. The use of lower concentrations of native melatonin in kinetic studies (100 pM) than in competition studies (up to 100 µM) may explain the apparent disagreement in the number of binding sites. The affinity of the melatonin binding site in Xenopus laevis oocyte membranes is in the same range as that described for binding of melatonin to calmodulin (18).

The pharmacological characterization of the melatonin binding site indicated that only N-acetylserotonin, 5-hydroxytryptophol, and tryptamine approached the effectiveness of melatonin in displacing [¹²⁵I]melatonin bound to oocyte membranes ( Table 1), but were 3-, 20-, and 31-fold less effective than melatonin, respectively. Other indoles including 5-hydroxy- and 5-methoxyindoles were ineffective.

The pioneering work of Cohen et al. (37) provided the first evidence for a cytoplasmic melatonin receptor. Fifteen years later, Benítez-King et al. (18) described the specific binding of melatonin to calmodulin in vitro, which suggested an explanation to many previously reported melatonin effects involving microtubules (38) and the cytoskeleton (8). These results were confirmed by Pozo et al. (26). Following this line of evidence and because the characteristics of the binding site are different from those described for the typical membrane receptor, i.e., its resistance to heating, we tested whether melatonin-specific binding to Xenopus laevis oocyte membranes was a calmodulin-mediated process. Binding of [¹²⁵I]melatonin to membranes was undoubtedly dependent on calcium; the absence of CaCl₂ and presence of EGTA, a calcium-chelating agent, dramatically altered the binding capacity of the membranes ( Fig. 7). Moreover, CaM-depleted membranes lost their binding capacity, which could be restored by both exogenous ( Fig. 5) and endogenous ( Fig. 6) calmodulin. CaM-depleted membranes showed a remanent melatonin binding ( Fig. 5), possibly due to remaining calmodulin not extractable even on the presence of EGTA or heat. The role of CaM as a melatonin receptor was also demonstrated by using anti-CaM antibodies; [¹²⁵I]melatonin binding to membranes was blocked in a dilution-dependent manner by a mixture of three monoclonal antibodies against calmodulin, whereas nonspecific immunoglobulins did not alter the binding ( Fig. 8).

We also tested the effect of two different calmodulin antagonists. The first calmodulin inhibitors to be discovered were the neuroleptic phenothiazines (39). Using radiolabeled inhibitors, Levin and Weiss (40) demonstrated two or three high-affinity binding sites (K_d=1-10 µM) and a great number of low-affinity binding sites (K_d>100 µM) for these compounds. Chlorpromazine (2-chloro-10-[3-dimethylaminopropyl]phenothiazine) is known to inhibit cholera-toxin-stimulated intestinal adenylate cyclase and fluid secretion (41). This effect could be due to the role of chlorpromazine as a calmodulin inhibitor by blocking the Ca²⁺-CaM inhibition of NaCl transport, a fundamental mechanism for fluid absorption in the intestine (42). Chlorpromazine inhibited melatonin-specific binding, and IC₅₀ was observed at 0.5 mM. Calmidazolium (1-[bis(p-chlorophenol)methyl]-3-[2,4-dichloro-ß-(2,4-dichlorobenzyloxi) phenethyl]imidazolium chloride), formerly known as R24571, is a lipophilic cation derived from the antimycotic miconazole that has been shown to be a very potent inhibitor of CaM-dependent enzymes (43, 44). Thus, calmidazolium inhibits the endothelium-derived relaxing factor/nitric oxide synthesis in endothelial cells (45) as well as platelet and sarcoplasmic reticulum Ca²⁺-ATPase activities (46, 47), both processes regulated by free Ca²⁺ and calmodulin. According to Anderson et al. (46), CaM might not be involved in these inhibitory effects of calmidazolium, which supports previous observations with a variety of anti-CaM drugs (44, 48, 49). On the contrary, the calmodulin inhibitory role is proposed to be exerted through an effect on membrane phospholipids, probably by disrupting lipid-protein interactions in the membrane. These data are consistent with the lack of effect of the drug on the binding of melatonin by Xenopus laevis membrane oocytes.

CaM is essential for life. This Ca²⁺ binding protein is present in all eukaryotic cells and serves as the primary intracellular receptor for Ca²⁺. It is well documented that numerous cellular functions, including cytoskeletal rearrangements (20), cell cycle progression (14), and many enzymatic activities (50), are CaM-mediated processes. In this context, our results suggest that melatonin might play an important role in the regulation of many cellular activities. Through binding to calmodulin, the hormone may directly affect calcium signaling. Thus, melatonin-calmodulin interaction appears to represent an essential mechanism for cell activity regulation and synchronization.

In conclusion, the present work describes calmodulin as a melatonin receptor in Xenopus laevis oocyte membranes. This evidence suggests that melatonin, besides other known functions, may directly participate in many CaM-regulated processes and cellular functions, coupling cellular activities to rhythmic circulating levels of melatonin.

	ACKNOWLEDGMENTS

 
This work was supported by grants from the DGICYT (PB94–425, PM95–0159). M.P.R. was supported by a fellowship from the Spanish government (F.P.I. AP-94). A.G.-P. was the recipient of a predoctoral fellowship from the Junta de Andalucía government (F.P.D.I. 94).

	FOOTNOTES

 
¹ Correspondence: Department of Medical Biochemistry and Molecular Biology, The University of Seville School of Medicine and Virgen Macarena Hospital, Avda. Sánchez Pizjuán 4, 41009 Seville, Spain. E-mail: carmen18{at}arrakis.es

² Abbreviations: melatonin, N-acetyl-5-methoxytryptamine; CaM, calmodulin; [¹²⁵I]melatonin, 2-[¹²⁵I]iodomelatonin; SGCC, silica gel column chromatography; K_d, dissociation constant; B_max, maximum binding capacity; IC₅₀, half-maximal inhibition; chlorpromazine, 2-chloro-10-[3-dimethylaminopropyl]phenothiazine; calmidazolium, 1-[bis(p-chlorophenol)methyl]-3-[2,4-dichloro-ß-(2,4-dichlorobenzyloxi) phenethyl]imidazolium chloride.

Received for publication February 25, 1998. Accepted for publication May 27, 1998.

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