Properties of calcium-dependent regulatory proteins from fungi and yeast

Calmodulins were isolated from vegetative mycelia of Basidiomycetes fungi, Agaricus campestris and Coprinus lagopus. These calmodulins showed similar mobilities to those of animal calmodulins on nondenaturing polyacrylamide gel electrophoresis in the presence or absence of Ca2+. The molecular weights of both calmodulins were determined to be 16,000. Agaricus calmodulin consisted of 148 amino acids including epsilon-N-trimethyllysine and cysteine. The UV-absorption spectrum showed the relatively high content of phenylalanine in Basidiomycetes calmodulins. The difference UV-absorption spectrum due to the blue shift by Ca2+ was observed. Both calmodulins activated muscle myosin light chain kinase and pea NAD+ kinase in a Ca2+-dependent manner, and the activities were inhibited by trifluoperazine or chlorpromazine. A calmodulin-like protein was partially purified from baker's yeast, Saccharomyces cerevisiae. However, detection of a calmodulin-like protein in prokaryotes was not successful.     

Response of three enzymes to oleic acid, trypsin, and calmodulin chemically modified with a reactive phenothiazine

Calmodulin was covalently modified with 10-(1-propionyloxysuccinimide)-2-trifluoromethylphenothiazine++ + to stoichiometries between 0 and 2 mol/mol in the presence of Ca2+. The modified calmodulins, oleic acid, and trypsin were assayed for their ability to activate pea plant NAD kinase, bovine brain 3',5'-cAMP phosphodiesterase, and human erythrocyte Ca2+-ATPase. All modified calmodulins activated both phosphodiesterase and Ca2+-ATPase; at the highest concentration assayed, calmodulin modified with 2 mol of reagent/mol activated phosphodiesterase and Ca2+-ATPase to 53% and 100%, respectively, of the activation obtained with unmodified calmodulin. However, higher concentrations of the modified calmodulins were required to observe the same activation; at least 900-fold and 100-fold higher concentrations were required for the two enzymes, respectively. NAD kinase was not activated by any calmodulin labeled to a stoichiometry greater than 1 mol/mol even when a concentration equal to 17,000 times the apparent dissociation constant of calmodulin for NAD kinase was assayed. Therefore, the modified protein (and not some fraction resistant to labeling) is active toward the mammalian enzymes but inactive toward plant NAD kinase. The different response of the three enzymes to the chemical modification suggests that the enzymes may utilize different binding domains on calmodulin. NAD kinase also was not activated by other known activators of the two mammalian enzymes, namely lipids and limited proteolysis. In parallel experiments using the same agents on each enzyme, NAD kinase was the only enzyme of the three that was not activated by oleic acid and several other lipids or by limited trypsin digestion. These results show that NAD kinase possesses several attributes which would not be predicted by current models of the mechanism of activation of enzymes by calmodulin.     

Further Characterization of Calmodulin from the Monocotyledon Barley (Hordeum vulgare)

We report here that calmodulin isolated from the monocotyledon barley is indistinguishable by a variety of criteria from calmodulin isolated from the dicotyledon spinach. In contrast to previous reports, we find that barley (Hordeum vulgare) calmodulin has an amino acid composition similar to that of vertebrate and spinach calmodulins, including the presence of a single trimethyllysinyl residue, and that barley calmodulin quantitatively activates cyclic nucleotide phosphodiesterase. Furthermore, spinach and barley calmodulins are similar in terms of tryptic peptide maps and immunoreactivity with various antisera that differ in their molecular specificities for calmodulins. Limited amino acid sequence analysis demonstrates that the region around the single histidinyl and trimethyllysinyl residues is identical among barley, spinach, and vertebrate calmodulins and that barley calmodulin, like spinach calmodulin, has a novel glutamine residue at position 96. We conclude that calmodulin is highly conserved among higher plants and that detailed sequence analysis is required before significant differences, if any, can be assigned to barley or other higher plant calmodulins. These studies suggest that calmodulin's fundamental importance to the eukaryotic cell may have been established prior to the evolutionary emergence of higher plants.     

Purification and biochemical properties of calmodulin from Saccharomyces cerevisiae

Calmodulin from the yeast Saccharomyces cerevisiae was purified to complete homogeneity by hydrophobic interaction chromatography and HPLC gel filtration. The biochemical properties of the purified protein as calmodulin were examined under various criteria and its similarity and dissimilarity to other calmodulins have been described. Like other calmodulins, yeast calmodulin activated bovine phosphodiesterase and pea NAD kinase in a Ca2+-dependent manner, but its concentration for half-maximal activation was 8-10 times that of bovine calmodulin. The amino acid composition of yeast calmodulin was different from those of calmodulins from other lower eukaryotes in that it contained no tyrosine, but more leucine and had a high ratio of serine to threonine. Yeast calmodulin did not contain tryptophanyl or tyrosyl residues, so its ultraviolet spectrum reflected the absorbance of phenylalanyl residues, and had a molar absorption coefficient at 259 nm of 1900 M-1 cm-1. Ca2+ ions changed the secondary structure of yeast calmodulin, causing a 3% decrease in the alpha-helical content, unlike its effect on other calmodulins. Antibody against yeast calmodulin did not cross-react with bovine calmodulin, and antibody against bovine calmodulin did not cross-react with yeast calmodulin, presumably due to differences in the amino acid sequences of the antigenic sites. It is concluded that the molecular structure of yeast calmodulin differs from those of calmodulins from other sources, but that its Ca2+-dependent regulatory functions are highly conserved and essentially similar to those of calmodulins of higher eukaryotes.     

Truncation of a mammalian myosin I results in loss of Ca2+-sensitive motility

MYR-1, a mammalian class I myosin, consisting of a heavy chain and 4-6 associated calmodulins, is represented by the 130-kDa myosin I (or MI(130)) from rat liver. MI(130) translocates actin filaments in vitro in a Ca(2+)-regulated manner. A decrease in motility observed at higher Ca(2+) concentrations has been attributed to calmodulin dissociation. To investigate mammalian myosin I regulation, we have coexpressed in baculovirus calmodulin and an epitope-tagged 85-kDa fragment representing the amino-terminal catalytic "motor" domain and the first calmodulin-binding IQ domain of rat myr-1; we refer to this truncated molecule here as MI(1IQ). Association of calmodulin to MI(1IQ) is Ca(2+)-insensitive. MI(1IQ) translocates actin filaments in vitro at a rate resembling MI(130), but unlike MI(130), does not exhibit sensitivity to 0.1-100 micrometer Ca(2+). In addition to demonstrating successful expression of a functional truncated mammalian myosin I in vitro, these results indicate that: 1) Ca(2+)-induced calmodulin dissociation from MI(130) in the presence of actin is not from the first IQ domain, 2) velocity is not affected by the length of the IQ region, and 3) the Ca(2+) sensitivity of actin translocation exhibited by MI(130) involves 1 or more of the other 5 IQ domains and/or the carboxyl tail.     

Can calmodulin function without binding calcium?

Calmodulin is a small Ca(2+)-binding protein proposed to act as the intracellular Ca2+ receptor that translates Ca2+ signals into cellular responses. We have constructed mutant yeast calmodulins in which the Ca(2+)-binding loops have been altered by site-directed mutagenesis. Each of the mutant proteins has a dramatically reduced affinity for Ca2+; one does not bind detectable levels of 45Ca2+ either during gel filtration or when bound to a solid support. Furthermore, none of the mutant proteins change conformation even in the presence of high Ca2+ concentrations. Surprisingly, yeast strains relying on any of the mutant calmodulins not only survive but grow well. In contrast, yeast strains deleted for the calmodulin gene are not viable. Thus, calmodulin is required for growth, but it can perform its essential function without the apparent ability to bind Ca2+.     

Isolation of the yeast calmodulin gene: calmodulin is an essential protein

Calmodulin was purified from Saccharomyces cerevisiae based on its characteristic properties. Like other calmodulins, the yeast protein is small, heat-stable, acidic, retained by hydrophobic matrices in a Ca2+-dependent manner, exhibits a pronounced Ca2+-induced shift in electrophoretic mobility, and binds 45Ca2+. Using synthetic oligonucleotide probes designed from the sequences of two tryptic peptides derived from the purified protein, the gene encoding yeast calmodulin was isolated. The gene (designated CMD1) is a unique, single-copy locus, contains no introns, and resides on chromosome II. The amino acid sequence of yeast calmodulin shares 60% identity with other calmodulins. Disruption or deletion of the yeast calmodulin gene results in a recessive-lethal mutation; thus, calmodulin is essential for the growth of yeast cells.     

A site-directed mutagenesis study of yeast calmodulin

A site-directed mutagenesis study was carried out in order to understand the regulatory mechanism of calmodulin. We started from the yeast (Saccharomyces cerevisiae) calmodulin gene since it has many differences in amino acid sequence and inferior functional properties compared with the vertebrate calmodulin. Recombinant yeast calmodulins were generated in Escherichia coli transformed by constructed expression plasmids. Three recombinant calmodulins were obtained. The first two were YCM61G, in which the Ca2(+)-binding site 2 (the four Ca2(+)-binding EF-hand structures in calmodulin were numbered from the N-terminus) was converted to the same as that in vertebrate calmodulin, and YCM delta 132-148, in which the C-terminal half sequence of site 4 was deleted. These two recombinant calmodulins had the same maximum Ca2+ binding (3 mol/mol) as yeast calmodulin, which indicates that site 4 of yeast calmodulin was the one losing Ca2+ binding capacity. YCM delta 132-148 could not activate target enzymes, whereas its Ca2+ binding profile was similar to those of yeast calmodulin and YCM61G. Therefore, the structure in site 4 which cannot bind Ca2+ is indispensable for the regulatory function of yeast calmodulin. The complete regulatory function of vertebrate calmodulin can be attained by the combination of 4 Ca2+ binding structures. The negative charge cluster in the central alpha-helix region is suggested to stabilize the active conformation of calmodulin, since the third yeast calmodulin mutant, YCM83E, which had the negative charge cluster, increased the maximum activation of myosin light chain kinase.     

An enzymatic assay for calmodulins based on plant NAD kinase activity

NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca2+-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K0.5) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The K0.5's ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K0.5's for the activation of Ca2+-ATPase ranged from 36.3 ng/ml for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca2+. Palmitic acid had a slightly stimulatory effect in the presence of Ca2+ (10% of maximum), but no effect in the absence of Ca2+. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures.     

Calmodulin activation and inhibition of skeletal muscle Ca2+ release channel (ryanodine receptor).

The calmodulin-binding properties of the rabbit skeletal muscle Ca2+ release channel (ryanodine receptor) and the channel's regulation by calmodulin were determined at < or = 0.1 microM and micromolar to millimolar Ca2+ concentrations. [125I]Calmodulin and [3H]ryanodine binding to sarcoplasmic reticulum (SR) vesicles and purified Ca2+ release channel preparations indicated that the large (2200 kDa) Ca2+ release channel complex binds with high affinity (KD = 5-25 nM) 16 calmodulins at < or = 0.1 microM Ca2+ and 4 calmodulins at 100 microM Ca2+. Calmodulin-binding affinity to the channel showed a broad maximum at pH 6.8 and was highest at 0.15 M KCl at both < or = 0.1 MicroM and 100 microM Ca2+. Under condition closely related to those during muscle contraction and relaxation, the half-times of calmodulin dissociation and binding were 50 +/- 20 s and 30 +/- 10 min, respectively. SR vesicle-45Ca2+ flux, single-channel, and [3H]ryanodine bind measurements showed that, at < or = 0.2 microM Ca2+, calmodulin activated the Ca2+ release channel severalfold. Ar micromolar to millimolar Ca2+ concentrations, calmodulin inhibited the Ca(2+)-activated channel severalfold. Hill coefficients of approximately 1.3 suggested no or only weak cooperative activation and inhibition of Ca2+ release channel activity by calmodulin. These results suggest a role for calmodulin in modulating SR Ca2+ release in skeletal muscle at both resting and elevated Ca2+ concentrations.     

Regulation of the erythrocyte Ca2(+)-ATPase by mutant calmodulins with positively charged amino acid substitutions.

Four mutant calmodulins with site-specific charge alterations have been used to activate the human erythrocyte Ca2(+)-ATPase. These charge alterations were accomplished either by insertion of new Lys residues or by substitution of Lys residues for Glu in two of the seven calmodulin alpha-helices. Two enzyme preparations, purified monomeric Ca2(+)-ATPase and erythrocyte ghost membranes, were used with comparable results. At 100 nM Ca2+, the Ca2(+)-ATPase activity was lowered significantly by charge reversal from negative to positive in both the central alpha-helix and the carboxy-terminal domain. While all mutant calmodulins with charge reversal ultimately stimulated the Ca2(+)-ATPase activity to the same extent, the concentration of mutant calmodulin required for half-maximal activation was from 36-fold (central alpha-helix) to 126-fold higher (alpha-helix in the carboxy-terminal domain) than that of the control calmodulin. There was also a significant difference in the stimulation of Ca2(+)-ATPase activity by the different mutant calmodulins as a function of Ca2+ concentration, being most pronounced at submicromolar Ca2+ concentrations where enzyme activation by calmodulin appears to be a physiologically relevant mechanism. In contrast to the mutant calmodulins with charge reversal, mutant calmodulins in which two positive charges were added in the central alpha-helix activated the Ca2(+)-ATPase in a way undistinguishable from the control calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of the erythrocyte Ca2+-ATPase and of bovine brain cyclic nucleotide phosphodiesterase by chemically modified calmodulin

Chemically modified calmodulins have been used to investigate structural features which are important for the interaction of the activator with targets. Carbamoylation of lysine residues had no influence on the ability of calmodulin to stimulate the plasma membrane Ca2+-ATPase whereas the stimulation of the bovine brain cyclic-nucleotide phosphodiesterase was reduced up to 50%. Different species of carbamoylated calmodulin have been isolated but no differences were detected in their interaction with the cyclic-nucleotide phosphodiesterase. Modification of arginine residues by 1,2-cyclohexanedione had no effect of the stimulation of the phosphodiesterase but reduced by 40% the stimulation of the erythrocyte Ca2+ ATPase. Mild oxidation of methionines by N-chlorosuccinimide produced a number of differently modified calmodulins. The different species have been purified and the modified residues have been identified. They affected the two different test enzymes to different extents indicating that methionines in the central helix of calmodulin are of greater importance for the interaction with the phosphodiesterase, whereas methionines located in the C-terminal half of calmodulin are more important for the interaction with the Ca2+-ATPase.     

Interactions of spin-labeled calmodulin with trifluoperazine and phosphodiesterase in the presence of Ca(II), Cd(II), La(III), Tb(III), and Lu(III)

Bovine calmodulin analogues, spin-labeled at methionine and tyrosine residues, have been utilized in electron paramagnetic resonance (EPR) studies designed to investigate calmodulin interactions with the antipsychotic drug trifluoperazine and the calmodulin-binding protein 3',5'-cyclic nucleotide phosphodiesterase. Trifluoperazine titrations of spin-labeled calmodulin analogues were carried out in the presence of Ca(II), Cd(II), and Tb(III). Similar experiments were performed with the phosphodiesterase in the presence of Ca(II), Cd(II), La(III), Tb(III), and Lu(III). EPR signals from the methionine-directed probe proved to be more sensitive to the binding of target molecules than signals from the tyrosine-directed probe, perhaps indicating that the spin-labeled methionine is at a site close to the target molecule binding site. While the binding of TFP, as monitored by EPR spectral changes in the methionine spin-labeled calmodulin, was in evidence with Ca(II), Cd(II), and all the lanthanides examined, no binding of phosphodiesterase to calmodulin could be detected in the presence of the lanthanide ions, perhaps due to inactivation of the phosphodiesterase by lanthanide ion binding. The abilities of the spin-labeled calmodulins to activate phosphodiesterase were also investigated. The spin-labeled tyrosine calmodulin was able to activate phosphodiesterase as well as native calmodulin, while a lower degree of activation was found when the spin-labeled methionine analogue was used.     

Circular dichroism studies on calcium binding to two series of Ca2+ binding site mutants of Drosophila melanogaster calmodulin.

The Ca(2+)-induced structural changes in mutant calmodulins from Drosophila melanogaster have been studied by circular dichroism. The proteins comprise eight site-specific mutants, in which a bidentate glutamic acid (at position 12 in each Ca2+ binding loop) is replaced with either glutamine (BQ series) or lysine (BK series). Previous studies of these proteins indicate that Ca2+ binding at the mutated site is effectively eliminated by each of these substitutions, with additional effects at nonmutated sites. Circular dichroism has now been used to assess Ca(2+)-induced changes in secondary and tertiary structure in these proteins. In the absence of Ca2+, the helical content of these mutant calmodulins is close to that of the wild-type protein. In excess Ca2+, calmodulins with a mutation in the N-terminal sites show Ca(2+)-induced increases in helicity (CD at 222 nm) that are similar to those of the wild-type protein. In contrast, much less additional helix is induced by Ca2+ in calmodulins with mutations in the C-terminal sites, with the two mutations to site IV showing a particularly poor response. Ca(2+)-induced changes to the environment of the single tyrosine of Drosophila calmodulin (Tyr-138 in site IV of the C-terminal domain) have been monitored via CD at 280 nm. The signal from this residue is significantly altered in the Ca(2+)-free form of almost all these mutants, including those in the N-terminal domain. This indicates significant interaction between the N- and C-terminal domains of these mutants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural analysis of wild-type and mutant yeast calmodulins by limited proteolysis and electrospray ionization mass spectrometry.

Calmodulin from Saccharomyces cerevisiae was expressed in Escherichia coli and purified. The purified protein was structurally characterized using limited proteolysis followed by ESI mass spectrometry to identify the fragments. In the presence of Ca2+, yeast calmodulin is sequentially cleaved at arginine 126, then lysine 115, and finally at lysine 77. The rapid cleavage at Arg-126 suggests that the fourth Ca(2+)-binding loop does not bind Ca2+. In the presence of EGTA, yeast calmodulin is more susceptible to proteolysis and is preferentially cleaved at Lys-106. In addition, mutant proteins carrying I100N, E104V or both mutations, which together confer temperature sensitivity to yeast, were characterized. The mutant proteins are more susceptible than wild-type calmodulin to proteolysis, suggesting that each mutation disrupts the structure of calmodulin. Furthermore, whereas wild-type calmodulin is cut at Lys-106 only in the presence of EGTA, this cleavage site is accessible in the mutants in the presence of Ca2+ as well. In these ways, the structural consequence of each mutation mimics the loss of a calcium ion in the third loop. In addition, although wild-type calmodulin binds to four proteins in a yeast crude extract in the presence of Ca2+, the mutants bind only to a subset of these. Thus, the inability to adopt the stable Ca(2+)-bound conformation in the third Ca(2+)-binding loop alters the ability of calmodulin to interact with yeast proteins in a Ca(2+)-dependent manner.     

Calcium-binding properties of two high affinity calcium-binding proteins from squid optic lobe

We have studied the calcium-binding properties of two high affinity calcium-binding proteins from squid optic lobes: one, squid calmodulin (SCaM), similar to bovine brain calmodulin (BCaM), the other, squid calcium-binding protein (SCaBP), distinct (Head, J.F., Spielberg, S., and Kaminer, B. (1983) Biochem J. 209, 797-802). Equilibrium dialysis measurements on the squid proteins (and BCaM) were made at 100 mM KCl in the presence and absence of 3 mM Mg2+, and at 400 mM KCl in the presence of 3 mM Mg2+, which more closely resembles the conditions in the squid. SCaM, SCaBP, and BCaM each bind a maximum of 4 Ca2+ ions/molecule of protein under the ionic conditions tested. SCaBP has a higher affinity than SCaM or BCaM for Ca2+ at 100 mM KCl in the absence of Mg2+. However, in the presence of Mg2+, half-maximal binding to SCaBP occurs at a similar pCa value to that observed with calmodulin. Increasing the KCl concentration reduces the affinity of all three proteins for Ca2+. UV absorption measurements showed that the binding of 4 Ca2+ ions/molecule is necessary to complete spectral changes in SCaBP, compared to two for the calmodulins. While Ca2+ causes perturbations in aromatic chromophores in SCaM and SCaBP, Mg2+ causes a significant perturbation only in SCaBP. These Mg2+-induced changes differ qualitatively from those induced by Ca2+.     

Two calmodulins in Naegleria flagellates: characterization, intracellular segregation, and programmed regulation of mRNA abundance during differentiation

Flagellates of Naegleria gruberi contain two calmodulins that differ in apparent molecular weight and intracellular location. Calmodulin-1, localized in flagella, has an apparent molecular weight of approximately 16,000, approximately the size of other protozoan calmodulins, whereas calmodulin-2, localized in cell bodies, is 15,300. Both proteins, purified, are calmodulins by several criteria, including Ca2+-dependent stimulation of calmodulin-dependent cyclic nucleotide phosphodiesterase and affinity for antibodies to vertebrate calmodulin. The finding of two calmodulins is unusual. Since the only known difference is apparent molecular weight, one calmodulin could be derived from the other, except that both calmodulins are synthesized in a wheat germ, cell-free system directed by RNA from differentiating Naegleria. Translatable mRNAs encoding calmodulins 1 and 2, not detected in amebas, appear and subsequently disappear concurrently during the 100-min differentiation of Naegleria from amebas to flagellates. Furthermore, these mRNAs increase and then decrease in abundance concurrently with those for flagellar tubulins, which suggests the possibility that the expression of the unrelated genes for calmodulin and tubulin may be under coordinate control during differentiation.     

alpha-Synuclein exhibits competitive interaction between calmodulin and synthetic membranes

alpha-Synuclein, a pathological component of Parkinson's disease by constituting the Lewy bodies, has been suggested to be involved in membrane biogenesis via induction of amphipathic alpha-helices. Since the amphipathic alpha-helix is also known as a recognition signal of calmodulin for its target proteins, molecular interaction between alpha-synuclein and calmodulin has been investigated. By employing a chemical coupling reagent of N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline, alpha-synuclein has been shown to yield a heterodimeric 1 : 1 complex with calmodulin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and even absence of calcium, whereas beta-synuclein was more dependent upon calcium for its calmodulin interaction. The selective calmodulin interaction of alpha-synuclein in the absence of calcium was also demonstrated with the aggregation kinetics of the synucleins in which only the alpha-synuclein aggregation was affected by calmodulin. A reversible binding assay confirmed that alpha-synuclein interacted with the Ca2+-free as well as the Ca2+-bound calmodulins with almost identical Kds of 0.35 micro m and 0.31 micro m, respectively, while beta-synuclein preferentially recognized the Ca2+-bound form with a Kd of 0.68 micro m. By using a C-terminally truncated alpha-synuclein of alpha-syn97, the calmodulin binding site(s) on alpha-synuclein was(were) shown to be located on the N-terminal region where the amphipathic alpha-helices have been suggested to be induced upon membrane interaction. By employing liposome and calmodulin in a state of being either soluble or immobilized on agarose, actual competition of alpha-synuclein between membranes and calmodulin was demonstrated with the observation that alpha-synuclein previously bound to the liposome was released upon specific interaction with the calmodulins. Taken together, these data may suggest that alpha-synuclein could act not only as a negative regulator for calmodulin in the presence and even absence of calcium, but it could also exert its activity at the interface between calmodulin and membranes.