Molecular analysis of the interaction of Bordetella pertussis adenylyl cyclase with fluorescent nucleotides

The calmodulin (CaM)-dependent adenylyl cyclase (AC) toxin from Bordetella pertussis (CyaA) substantially contributes to the pathogenesis of whooping cough. Thus, potent and selective CyaA inhibitors may be valuable drugs for prophylaxis of this disease. We examined the interactions of fluorescent 2',3'-N-methylanthraniloyl (MANT)-, anthraniloyl- and trinitrophenyl (TNP)-substituted nucleotides with CyaA. Compared with mammalian AC isoforms and Bacillus anthracis AC toxin edema factor, nucleotides inhibited catalysis by CyaA less potently. Introduction of the MANT substituent resulted in 5- to 170-fold increased potency of nucleotides. K(i) values of 3'MANT-2'd-ATP and 2'MANT-3'd-ATP in the AC activity assay using Mn(2+) were 220 and 340 nM, respectively. Natural nucleoside 5'-triphosphates, guanine-, hypoxanthine- and pyrimidine-MANT- and TNP nucleotides and di-MANT nucleotides inhibited CyaA, too. MANT nucleotide binding to CyaA generated fluorescence resonance energy transfer (FRET) from tryptophans Trp69 and Trp242 and multiple tyrosine residues, yielding K(d) values of 300 nM for 3'MANT-2'd-ATP and 400 nM for 2'MANT-3'd-ATP. Fluorescence experiments and docking approaches indicate that the MANT- and TNP groups interact with Phe306. Increases of FRET and direct fluorescence with MANT nucleotides were strictly CaM-dependent, whereas TNP nucleotide fluorescence upon binding to CyaA increased in the absence of CaM and was actually reduced by CaM. In contrast to low-affinity MANT nucleotides, even low-affinity TNP nucleotides generated strong fluorescence increases upon binding to CyaA. We conclude that the catalytic site of CyaA possesses substantial conformational freedom to accommodate structurally diverse ligands and that certain ligands bind to CyaA even in the absence of CaM, facilitating future inhibitor design.     

Differential interactions of the catalytic subunits of adenylyl cyclase with forskolin analogs

The diterpene forskolin (FS) binds to, and activates, mammalian membranous adenylyl cyclase (AC) isoforms I-VIII. Diterpenes without C₁-OH group do not activate ACs. The C₁-OH group forms a hydrogen bond with the backbone oxygen of Val506 of the C1 catalytic subunit of AC (isoform V numbering). To better understand the mechanism of AC activation we examined the interactions of FS and eight FS analogs with purified catalytic AC subunits C1 (AC V) and C2 (AC II) by fluorescence spectroscopy, using 2′,3′-O-(N-methylanthraniloyl)-guanosine 5′-triphosphate (MANT-GTP) as fluorescent reporter probe, and by enzymatic activity. FS analogs induced C1/C2 assembly as assessed by fluorescence resonance energy transfer from Trp1020 of C2 to MANT-GTP and by increased direct MANT-GTP fluorescence in the order of efficacy FS ∼ 7-deacetyl-FS ∼ 6-acetyl-7-deacetyl-FS ∼ 9-deoxy-FS > 7-deacetyl-7-(N-methylpiperazino-γ-butyryloxy)-FS > 1-deoxy-FS ∼ 1,9-dideoxy-FS ∼ 7-deacetyl-1-deoxy-FS ∼ 7-deacetyl-1,9-dideoxy-FS. In contrast, FS analogs activated catalysis in the order of efficacy FS > 7-deacety-FS ∼ 6-acetyl-7-deacetyl-FS ∼ 9-deoxy-FS > 7-deacetyl-7-(N-methylpiperazino-γ-butyryloxy)-FS ? 1-deoxy-FS, 1,9-dideoxy-FS, 7-deacetyl-1-deoxy-FS and 7-deacetyl-1,9-dideoxy-FS (all ineffective). 1-Deoxy-FS analogs inhibited FS-stimulated catalysis by an apparently non-competitive mechanism. Our data suggest a two-step mechanism of AC activation by diterpenes. In the first step, diterpenes, regardless of their substitution pattern, promote C1/C2 assembly. In the second and yet poorly understood step, diterpenes that form a hydrogen bond between C₁-OH and Val506 promote a conformational switch that results in activation of catalysis. The apparent non-competitive interaction of FS with 1-deoxy-FS analogs is explained by impaired ligand exchange due to strong hydrophobic interactions with C1/C2.     

Inhibition of nitric oxide-activated guanylyl cyclase by calmodulin antagonists

Background and purpose:

Nitric oxide (NO) controls numerous physiological processes by activation of its receptor, guanylyl cyclase (sGC), leading to the accumulation of 3′-5′ cyclic guanosine monophosphate (cGMP). Ca²⁺-calmodulin (CaM) regulates both NO synthesis by NO synthase and cGMP hydrolysis by phosphodiesterase-1. We report that, unexpectedly, the CaM antagonists, calmidazolium, phenoxybenzamine and trifluoperazine, also inhibited cGMP accumulation in cerebellar cells evoked by an exogenous NO donor, with IC₅₀ values of 11, 80 and 180 µM respectively. Here we sought to elucidate the underlying mechanism(s).

Experimental approach:

We used cerebellar cell suspensions to determine the influence of CaM antagonists on all steps of the NO-cGMP pathway. Homogenized tissue and purified enzyme were used to test effects of calmidazolium on sGC activity.

Key results:

Inhibition of cGMP accumulation in the cells did not depend on changes in intracellular Ca²⁺ concentration. Degradation of cGMP and inactivation of NO were both inhibited by the CaM antagonists, ruling out increased loss of cGMP or NO as explanations. Instead, calmidazolium directly inhibited purified sGC (IC₅₀= 10 µM). The inhibition was not in competition with NO, nor did it arise from displacement of the haem moiety from sGC. Calmidazolium decreased enzyme V_max and K_m, indicating that it acts in an uncompetitive manner.

Conclusions and implications:

The disruption of every stage of NO signal transduction by common CaM antagonists, unrelated to CaM antagonism, cautions against their utility as pharmacological tools. More positively, the compounds exemplify a novel class of sGC inhibitors that, with improved selectivity, may be therapeutically valuable.     

A 1H-NMR comparison of calmodulin activation by calcium and by cadmium

Our previous reports based on pharmacological and histochemical evidence suggest that calcium and cadmium can both activate calmodulin (CaM)-dependent functions. The study reported here was carried out to explain these observations in molecular terms, using 400 MHz 1H-NMR. Changes in the spectrum of bovine brain CaM induced by 0 to 4 molar equivalents of calcium and cadmium were practically the same. In particular, the chemical shifts and line shape of signals due to Tyr-138, Phe-65, Phe-89 and Tml-115 were similarly affected by either ion. In addition, the effects of N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7, a CaM antagonist) on the phenylalanine aromatic regions, methionine methyl regions and high-field methyl regions of the spectra of both calcium- and cadmium-saturated proteins were practically identical. The effect of W-7 on calcium- and cadmium-saturated CaM was reflected in changes in the signals of Ile-27, Phe-68, Phe-92, Ile-100 and Val-142, as well as Met-71, Met-72, Met-76, Phe-89 and Phe-141. The results show that cadmium binds to all calcium-binding sites of CaM, and induces conformational changes that are as extensive as those brought about by calcium. W-7 also inhibits CaM activation by calcium and cadmium. Combined with our previous toxicological evidence, these results suggest that cadmium binds indiscriminately to CaM and that subsequent activation or modulation of CaM-dependent functions is confused as a result. This may be a mechanism contributing to cadmium poisoning.     

Calmodulin antagonists' binding sites on calmodulin

Troponin I inhibited, concentration-dependently, [3H]-N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and [3H]-trifluoperazine (TFP) binding to purified bovine brain calmodulin (CaM). Selective oxidation of methionine residues of CaM by N-chlorosuccinimide resulted in a rapid decrease in [3H]-W-7, [3H]-TFP and [14C]-chlorpromazine binding concomitant with the loss of CaM activity. Carbethoxylation of histidine residues, nitration of tyrosine residues and chemical modification of arginine residues with 1,2-cyclohexanedione produced no significant changes either in [3H]-W-7 binding to CaM or in the ability of CaM to stimulate phosphodiesterase. Our results suggest that the binding sites of these CaM antagonists on CaM may be located between the second and third Ca2+-binding loops.     

Isoform-specific regulation of adenylyl cyclase: a potential target in future pharmacotherapy

Adenylyl cyclase (AC) is a target enzyme of multiple G-protein-coupled receptors (GPCRs). In the past decade, the cloning, structure and biochemical properties of nine AC isoforms were reported, and each isoform of AC shows distinct patterns of tissue distribution and biochemical/pharmacological properties. In addition to the conventional regulators of this enzyme, such as calmodulin (CaM) or PKC, novel regulators, for example, caveolin, have been identified. Most importantly, these regulators work on AC in an isoform dependent manner. Recent studies have demonstrated that certain classic AC inhibitors, i.e., P-site inhibitors, show an isoform-dependent inhibition of AC. The side chain modifications of forskolin, a diterpene extract from Coleus forskolii, markedly enhance its isoform selectivity. When taken together, these findings suggest that it is feasible to develop new pharmacotherapeutic agents that target AC isoforms to regulate various neurohormonal signals in a highly tissue-/organ-specific manner.     

Isoform-specific regulation of adenylyl cyclase: a potential target in future pharmacotherapy

Adenylyl cyclase (AC) is a target enzyme of multiple G-protein-coupled receptors (GPCRs). In the past decade, the cloning, structure and biochemical properties of nine AC isoforms were reported, and each isoform of AC shows distinct patterns of tissue distribution and biochemical/pharmacological properties. In addition to the conventional regulators of this enzyme, such as calmodulin (CaM) or PKC, novel regulators, for example, caveolin, have been identified. Most importantly, these regulators work on AC in an isoformdependent manner. Recent studies have demonstrated that certain classic AC inhibitors, i.e., P-site inhibitors, show an isoform-dependent inhibition of AC. The side chain modifications of forskolin, a diterpene extract from Coleus forskolii, markedly enhance its isoform selectivity. When taken together, these findings suggest that it is feasible to develop new pharmacotherapeutic agents that target AC isoforms to regulate various neurohormonal signals in a highly tissue-/organ-specific manner.     

Modulation of vascular and cardiac contractile protein regulatory mechanisms by calmodulin inhibitors and related compounds

The abilities of several calmodulin antagonists and other compounds belonging to different pharmacological classes to modulate Ca2+ X calmodulin mediated arterial myosin light chain phosphorylation and Ca2+-troponin C regulated cardiac myofibrillar ATPase activity have been quantitated in Triton X-100 purified preparations of bovine aortic actomyosin and canine ventricular myofibrils. At submaximal free Ca2+ concentrations, all calmodulin antagonists inhibited myosin phosphorylation; however, some (calmidazolium, trifluoperazine, chlorpromazine, pimozide) stimulated myofibrillar ATPase activity, some (compound 48/80, W-5) had no effect on activity, while others (W-7, haloperidol, mastoparan) inhibited ATPase activity. The relative order of potency for several agents in both preparations was the same, as IC50 values for inhibition of arterial myosin phosphorylation were: calmidazolium, 0.5 microM; trifluoperazine, 22 microM; perhexiline, 35 microM; and concentrations which stimulated cardiac myofibrillar ATPase activity by 50% were: calmidazolium, 9 microM; trifluoperazine, 45 microM; perhexiline, 90 microM. A common feature of stimulation of cardiac ATPase activity by these agents was a leftward shift in the pCa relationship, although different shape changes in the pCa curves were also apparent. Maximum ATPase activity was either not affected or inhibited (trifluoperazine). Several other agents belonging to diverse pharmacological classes also had differential effects on myosin phosphorylation and ATPase activity. These results show that structurally-distinct calmodulin antagonists and other compounds differentially affect cardiac myofibrillar ATPase activity. Moreover, several agents have been identified which inhibit arterial, and stimulate cardiac, contractile protein regulatory mechanisms. Thus, it may be possible to develop mechanistically novel cardiotonic/vasodilator agents, Ca2+ binding protein modulators, which function primarily by altering the Ca2+ sensitivity of contractile protein interactions.     

Bordetella pertussis adenylate cyclase toxin: a versatile screening tool.

The calmodulin-activated adenylate cyclase (AC) toxin is an essential virulence factor of Bordetella pertussis, the causative agent of whooping cough. This toxin has been exploited to devise screening techniques for investigating diverse biological processes. This mini-review describes several such applications. First, AC has been utilized as a selective reporter for protein translocation from bacteria to eukaryotic cells, in particular to study protein targeting by type III secretion machinery. More recently, AC has been used as a signal transducer in Escherichia coli to elaborate genetic screens for protein-protein interactions ("bacterial two-hybrid system") or site-specific proteolytic activities.     

The effect of calmodulin antagonists on the activation of RAW-264 macrophage-like cells for tumor cell killing

The effects of several calmodulin antagonists on the activation of RAW-264 macrophage-like cells for tumor cell killing were investigated. At concentrations ranging from 5 X 10(-8) to 5 X 10(-7) M, calmidazolium, trifluoperazine, chlorprothixene, chlorpromazine and W-13 inhibited the development of cytolytic activity, evoked in RAW-264 by treatment with lymphokine and lipopolysaccharide, in a dose-dependent manner. Since the order of the potency of these drugs against the activation of RAW-264 cells was much the same as their ability to inhibit calmodulin-dependent phosphodiestherase activity: calmidazolium greater than trifluoperazine greater than chlorprothixene greater than chlorpromazine greater than W-13, and because W-12, a nonactive analog of W-13, failed to inhibit the process of activation, we believe that the development of cytolytic activity in RAW-264 cells may be dependent on calmodulin. At micromolar concentrations, calmodulin antagonists (except calmidazolium) enhanced the process of activation. The enhancement of cytolytic activity was neither the result of the toxicity of these drugs nor related to their effects on intracellular calcium. It was entirely dependent on the presence of stimulants but occurred independently from the stage of macrophage activation, and most likely was due to the nonspecific interference of these agents with calmodulin-independent processes.     

Bioengineering of Bordetella pertussis Adenylate Cyclase Toxin for Vaccine Development and Other Biotechnological Purposes

The adenylate cyclase toxin, CyaA, is one of the key virulent factors produced by Bordetella pertussis, the causative agent of whooping cough. This toxin primarily targets innate immunity to facilitate bacterial colonization of the respiratory tract. CyaA exhibits several remarkable characteristics that have been exploited for various applications in vaccinology and other biotechnological purposes. CyaA has been engineered as a potent vaccine vehicle to deliver antigens into antigen-presenting cells, while the adenylate cyclase catalytic domain has been used to design a robust genetic assay for monitoring protein–protein interactions in bacteria. These two biotechnological applications are briefly summarized in this chapter.     

Comparative effects of calmodulin inhibitors on calmodulin's hydrophobic sites and on the activation of cyclic nucleotide phosphodiesterase by calmodulin

Experiments were designed to investigate the effect of inhibitors on calmodulin's hydrophobic sites and their consequences on the activation of a target enzyme, cyclic nucleotide phosphodiesterase. Two fluorescent probes, 2-(p-toluidinyl)-naphthalene-6-sulfonate (TNS) and 9-anthroylcholine (9AC) were used to study the interactions with calmodulin of inhibitors devoid of direct effect on the probes. Contrary to W-7, nicergoline, nicardipine and quercetin, which decreased the fluorescence of the two probes bound to calmodulin, bepridil only decreased 9AC fluorescence but increased the fluorescence intensity at the wavelength of the emission maximum of TNS. In spite of this difference, bepridil as well as W-7 and nicergoline competitively inhibited calmodulin activation of phosphodiesterase. In addition, nicergoline also inhibited phosphodiesterase activity competitively to cyclic GMP. These results show differences in the interactions of inhibitors with calmodulin; these differences are not detected in functional studies of the effect of inhibitors on phosphodiesterase activation.     

Symmetric covalent linkage of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) results in novel derivatives with increased inhibitory activities against calcium/calmodulin complex

A useful calmodulin (CaM) antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), was invented by Hidaka et al. in 1978 (J. Pharmacol. Exp. Ther. 207, 8-15). Here, we have designed new CaM antagonists on the basis of the three-dimensional structure of Ca2+/CaM complexed with W-7. Eleven new compounds all share a similar architecture, in which two W-7 molecules are linked between their aminohexyl termini by a linker with different functionalities. A wide range of inhibitory activities against Ca2+/CaM-dependent protein kinase I (CaM kinase I) has been observed with these self-crosslinked W-7 analogs, (W-7)2. In vitro competitive CaM kinase I assays using CaM kinase I and nuclear magnetic resonance studies indicated that one (W-7)2 molecule binds to one CaM molecule as expected, with the two chloronaphthalene rings of (W-7)2 being anchored separately to the N- and C-terminal hydrophobic pockets of Ca2+/CaM. The most potent compound, N,N'-bis[6-(5-chloro-1-naphthalenesulfonyl)-amino-1-hexyl]-p-xylen e-diamine ((W-7)2 - 10), inhibits CaM kinase I activity at an IC50 value of 0.23 microM; about 75 times more effectively than W-7. The length and basicity of the linker sequence in (W-7)2 significantly contribute to inhibitory activity. The present study opens an avenue for developing powerful CaM antagonists that could be used at low doses in vivo.     

Calmodulin mediates sulfur mustard toxicity in human keratinocytes

Sulfur mustard (SM) causes blisters in the skin through a series of cellular changes that we are beginning to identify. We earlier demonstrated that SM toxicity is the result of induction of both death receptor and mitochondrial pathways of apoptosis in human keratinocytes (KC). Because of its importance in apoptosis in the skin, we tested whether calmodulin (CaM) mediates the mitochondrial apoptotic pathway induced by SM. Of the three human CaM genes, the predominant form expressed in KC was CaM1. RT-PCR and immunoblot analysis revealed upregulation of CaM expression following SM treatment. To delineate the potential role of CaM1 in the regulation of SM-induced apoptosis, retroviral vectors expressing CaM1 RNA in the antisense (AS) orientation were used to transduce and derive stable CaM1 AS cells, which were then exposed to SM and subjected to immunoblot analysis for expression of apoptotic markers. Proteolytic activation of executioner caspases-3, -6, -7, and the upstream caspase-9, as well as caspase-mediated PARP cleavage were markedly inhibited by CaM1 AS expression. CaM1 AS depletion attenuated SM-induced, but not Fas-induced, proteolytic processing and activation of caspase-3. Whereas control KC exhibited a marked increase in apoptotic nuclear fragmentation after SM, CaM1 AS cells exhibited normal nuclear morphology up to 48h after SM, indicating that suppression of apoptosis in CaM1 AS cells increases survival and does not shift to a necrotic death. CaM has been shown to activate the phosphatase calcineurin, which can induce apoptosis by Bad dephosphorylation. Interestingly, whereas SM-treated CaM1-depleted KC expressed the phosphorylated non-apoptotic sequestered form of Bad, Bad was present in the hypophosphorylated apoptotic form in SM-exposed control KC. To determine if pharmacological CaM inhibitors could attenuate SM-induced apoptosis via Bad dephosphorylation, KC were pretreated with the CaM-specific antagonist W-13 or its less active structural analogue W-12. Following SM exposure, KC exhibited Bad dephosphorylation, which was inhibited in the presence of W-13, but not with W-12. Consequently, W-13 but not W-12 markedly suppressed SM-induced proteolytic processing and activation of caspase-3, as well as apoptotic nuclear fragmentation. Finally, while the CaM antagonist W-13 and the calcineurin inhibitor cyclosporin A attenuated SM-induced caspase-3 activation, inhibitors for CaM-dependent protein kinase II (KN62 and KN93) did not. These results indicate that CaM, calcineurin, and Bad also play a role in SM-induced apoptosis, and may therefore be targets for therapeutic intervention to reduce SM injury.     

Chlordecone interaction of calmodulin binding with phosphodiesterase.

The effects of organochlorine (O.C.) compounds, such as aldrin, dieldrin, endrin, isodrin, chlordecone and mirex, on calmodulin (CaM) activity were investigated. Changes induced by O.C. compounds on biological and physical properties of CaM were monitored in terms of phosphodiesterase stimulation and tyrosine fluorescence, respectively. None of the O.C. compounds altered tyrosine fluorescence of CaM in the presence of Ca2+. Except for chlordecone, none of the O.C. compounds inhibited CaM-activated phosphodiesterase (PDE). Chlordecone significantly decreased (P less than 0.05) CaM-activated PDE in a concentration-dependent manner without affecting the basal enzyme. Combination of chlordecone with W-7 (CaM antagonist) increased the inhibitory effect of W-7 on CaM activity. These results suggest that O.C. compounds may not be changing the tyrosine fluorescence of CaM. Among the O.C. compounds tested, chlordecone is a specific inhibitor of CaM-activated PDE.     

Two types of calmodulin antagonists: a structurally related interaction

The ability of several calmodulin (CaM) antagonists, such as N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7) and trifluoperazine, to displace [3H]-W-7 from CaM correlated with the inhibition of Ca2+-CaM-dependent phosphodiesterase (PDE) by these agents. These antagonists also suppressed the increase in fluorescence of n-phenyl-1-naphthylamine (NPN) by complex formation with CaM in the presence of Ca2+. However, the ability of some CaM antagonists, such as prenylamine and butaclamol, to displace [3H]-W-7 from CaM did not correlate with the inhibition of Ca2+-PDE. These antagonists enhanced the increase in fluorescence of NPN by complex formation with CaM in the presence of Ca2+. In a study employing 1H-nuclear magnetic resonance, the spectrum changes of the aromatic region of CaM induced by prenylamine were significantly more marked than the changes induced by W-7. These findings suggest two types of CaM antagonists. The compounds in each of the groups appear to have common molecular structures.     

Anti-ischaemic activity of various calmodulin antagonists

The anti-ischaemic activity of the calmodulin antagonists trifluperazine, felodipine, W-7 and calmidazolium has been investigated in electrically paced guinea-pig hearts, perfused according to Langendorff, which were subjected to 60 min of global ischaemia followed by 30 min of reperfusion. At concentrations that induced a comparable reduction in cardiac contractile force, trifluperazine, felodipine and to a lesser extent W-7, were associated with improvement of post-ischaemic functional (LVP and coronary flow) and biochemical parameters (CrP and ATP). Furthermore, felodipine and trifluperazine delayed the onset and suppressed the maximum tension of the ischaemic contracture was observed. In contrast, calmidazolium had no anti-ischaemic effects. This lack of anti-ischaemic activity of the most potent calmodulin antagonist calmidazolium, as well as the significant calcium entry blocking activity of both trifluperazine and felodipine suggest that additional factors besides calmodulin antagonism may contribute to the anti-ischaemic activity of these compounds.     

Calmodulin-stimulated adenylyl cyclase gene deletion affects morphine responses

To define the roles of the calmodulin-stimulated adenylyl cyclases (AC1 and AC8) in morphine-induced analgesia, tolerance, physical dependence, and conditioned place preference, we used mice having targeted disruptions of either the AC1 or AC8 genes or both genes [double knockout mice (DKO)]. Mice lacking either AC1 or AC8 genes or DKO did not differ from wild-type mice in short-term antinociceptive responses to morphine measured in the tail-flick analgesia assay. Morphine tolerance that developed immediately within 3 h of morphine administration (10 mg/kg s.c.) was significantly attenuated in DKO mice and AC8 single knockout mice. Tolerance induced continually by daily injections of morphine (10 mg/kg s.c.) was also reduced in DKO mice. In DKO mice continually treated with morphine, there was a significant reduction in withdrawal behaviors, including reduced wet-dog shakes and forepaw tremor after naloxone injection (10 mg/kg i.p.). Morphine produced hyperlocomotion and conditioned place preference in wild-type mice, whereas DKO mice displayed significantly less hyperlocomotion and conditioned place preference. Furthermore, the significant increase in phosphorylated cAMP-response element binding protein (CREB) staining in ventral tegmental area induced by long-term morphine treatment was not evident in DKO mice, suggesting that CREB activation by morphine requires cAMP generated by AC1 and AC8. These results support the hypothesis that calmodulin-stimulated adenylyl cyclases are important mediators of the neuronal responses to morphine.     

Calmodulin antagonists enhance calcium binding to calmodulin

The effects of calmodulin (CaM) antagonists, N-(6-aminohexyl)(-5-chloro-1-naphthalenesulfonamide (W-7) and its derivatives or trifluoperazine (TFP) on the Ca2+ binding to CaM were investigated. In the presence of these CaM antagonists, the extent of the Ca2+ binding to CaM increased. Stoichiometrical Examination showed that these CaM antagonists dose-dependently increased the sensitivity of all four Ca2+ binding sites on CaM. An analysis of CaM by UV spectroscopy revealed that the conformation of the Ca2+ CaM complex in the presence and absence of these CaM antagonists differed. Therefore, binding of this CaM antagonist to CaM increase the binding of Ca2+ to this protein.