The control of glucose 1,6-bisphosphatase by Ca2+ and calmodulin.

1. The control of glucose 1,6-bisphosphatase activity, the enzyme that degrades glucose 1,6-bisphosphate, a metabolite that regulates hexose phosphate metabolism, has been examined in a rat muscle extract. 2. The enzyme has been found to be activated by physiological Ca2+ concentrations. Ca2+ can be replaced by Sr2+ and Mn2+, but the effects are not so pronounced. 3. The Ca2+ effect is inhibited by EGTA. Trifluoperazine also inhibits enzyme activity. This effect can be reversed by adding exogenous calmodulin.     

Biphasic Ca2+ response of adenylate cyclase. The role of calmodulin in its activation by Ca2+ ions

The Ca2+-dependent regulation of human platelet membrane adenylate cyclase has been studied. This enzyme exhibited a biphasic response to Ca2+ within a narrow range of Ca2+ concentrations (0.1-1.0 microM). At low Ca2+ (0.08-0.3 microM) adenylate cyclase was stimulated (Ka = 0.10 microM), whereas at higher Ca2+ (greater than 0.3 microM) the enzyme was inhibited to 70-80% control (Ki = 0.8 microM). Membrane fractions, prepared by washing in the presence of LaCl3 to remove endogenous calmodulin (approximately equal to 70-80% depletion), exhibited no stimulation of adenylate cyclase by Ca2+ but did show the inhibitory phase (Ki = 0.4 microM). The activation phase could be restored to La3+-washed membranes by addition of calmodulin (Ka = 3.0 nM). Under these conditions it was apparent that calmodulin reduced the sensitivity of adenylate cyclase to Ca2+ (Ki = 0.8 microM). Prostaglandin E1 (PGE1) did not alter Ki or Ka values for Ca2+. Calmodulin did not alter the EC50 for PGE1 stimulation of adenylate cyclase but increased the Vmax (1.5-fold). The calmodulin antagonist trifluoperazine potently inhibited adenylate cyclase in native membranes (80%) and to a much lesser extent in La3+-washed membranes (15%). This inhibition was due to interaction of trifluoperazine with endogenous calmodulin since trifluoperazine competitively antagonized the stimulatory effect of calmodulin on adenylate cyclase in La3+-washed membranes. We propose that biphasic Ca2+ regulation of platelet adenylate cyclase functions to both dampen (low Ca2+) and facilitate (high Ca2+) the haemostatic function of platelets.     

The bell-shaped Ca2+ dependence of the inositol 1,4, 5-trisphosphate-induced Ca2+ release is modulated by Ca2+/calmodulin.

Calmodulin inhibits inositol 1,4,5-trisphosphate (IP3) binding to the IP3 receptor in both a Ca2+-dependent and a Ca2+-independent way. Because there are no functional data on the modulation of the IP3-induced Ca2+ release by calmodulin at various Ca2+ concentrations, we have studied how cytosolic Ca2+ and Sr2+ interfere with the effects of calmodulin on the IP3-induced Ca2+ release in permeabilized A7r5 cells. We now report that calmodulin inhibited Ca2+ release through the IP3 receptor with an IC50 of 4.6 microM if the cytosolic Ca2+ concentration was 0.3 microM or higher. This inhibition was particularly pronounced at low IP3 concentrations. In contrast, calmodulin did not affect IP3-induced Ca2+ release if the cytosolic Ca2+ concentration was below 0.3 microM. Calmodulin also inhibited Ca2+ release through the IP3 receptor in the presence of at least 10 microM Sr2+. We conclude that cytosolic Ca2+ or Sr2+ are absolutely required for the calmodulin-induced inhibition of the IP3-induced Ca2+ release and that this dependence represents the formation of the Ca2+/calmodulin or Sr2+/calmodulin complex.     

Molecular basis of calmodulin tethering and Ca2+-dependent inactivation of L-type Ca2+ channels.

Ca(2+)-dependent inactivation (CDI) of L-type Ca(2+) channels plays a critical role in controlling Ca(2+) entry and downstream signal transduction in excitable cells. Ca(2+)-insensitive forms of calmodulin (CaM) act as dominant negatives to prevent CDI, suggesting that CaM acts as a resident Ca(2+) sensor. However, it is not known how the Ca(2+) sensor is constitutively tethered. We have found that the tethering of Ca(2+)-insensitive CaM was localized to the C-terminal tail of alpha(1C), close to the CDI effector motif, and that it depended on nanomolar Ca(2+) concentrations, likely attained in quiescent cells. Two stretches of amino acids were found to support the tethering and to contain putative CaM-binding sequences close to or overlapping residues previously shown to affect CDI and Ca(2+)-independent inactivation. Synthetic peptides containing these sequences displayed differences in CaM-binding properties, both in affinity and Ca(2+) dependence, leading us to propose a novel mechanism for CDI. In contrast to a traditional disinhibitory scenario, we suggest that apoCaM is tethered at two sites and signals actively to slow inactivation. When the C-terminal lobe of CaM binds to the nearby CaM effector sequence (IQ motif), the braking effect is relieved, and CDI is accelerated.     

Mechanism of local and global Ca2+ sensing by calmodulin in complex with a Ca2+ channel

Calmodulin (CaM) in complex with Ca(2+) channels constitutes a prototype for Ca(2+) sensors that are intimately colocalized with Ca(2+) sources. The C-lobe of CaM senses local, large Ca(2+) oscillations due to Ca(2+) influx from the host channel, and the N-lobe senses global, albeit diminutive Ca(2+) changes arising from distant sources. Though biologically essential, the mechanism underlying global Ca(2+) sensing has remained unknown. Here, we advance a theory of how global selectivity arises, and we experimentally validate this proposal with methodologies enabling millisecond control of Ca(2+) oscillations seen by the CaM/channel complex. We find that global selectivity arises from rapid Ca(2+) release from CaM combined with greater affinity of the channel for Ca(2+)-free versus Ca(2+)-bound CaM. The emergence of complex decoding properties from the juxtaposition of common elements, and the techniques developed herein, promise generalization to numerous molecules residing near Ca(2+) sources.     

Ca2+-independent stimulation of cyclic GMP-dependent protein kinase by calmodulin

Calmodulin purified from bovine brain markedly stimulated cyclic GMP-dependent protein kinase from pig lung in the presence of cyclic GMP. This stimulation by calmodulin did not require Ca²⁺ and was dose-dependent up to optimal amounts, but the extent of stimulation decreased at concentrations over the optimal condition. The concentrations of cyclic GMP and cyclic AMP producing half-maximal stimulation were 4.5 × 10^?8 M and 5.0 × 10^?6 M respectively, under optimal conditions. Calmodulin increased maximum velocity without altering the Km for ATP. These effects of calmodulin on cyclic GMP-dependent protein kinase were similar to those of the stimulatory modulator described by Kuo and Kuo (J. Biol. Chem. $\text{251}$, 4283–4286, 1976). Ouf findings indicate that calmodulin regulates enzyme activity both Ca²⁺-dependently and independently.     

Regulation of the Ca2+-pump by calmodulin in intact cells

ATP-enriched human red cells display high rates of Ca²⁺-dependent ATP hydrolysis (16 mmol·litre cells^?1·h^?1) with a high Ca²⁺ affinity ($\text{K}{}_{0.5}\text{～0.2 μ}\text{M}$). The finding suggests a mechanism for regulation of cell Ca²⁺ levels, involving highly-cooperative stimulation of active Ca²⁺ extrusion following binding of calmodulin to the $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$.     

Regulation of the RYR1 and RYR2 Ca2+ release channel isoforms by Ca2+-insensitive mutants of calmodulin

Calmodulin (CaM) may function as a regulatory subunit of ryanodine receptor (RYR) channels, modulating both channel activation and inhibition by Ca2+; however, mechanisms underlying differences in CaM regulation of the RYR isoforms expressed in skeletal muscle (RYR1) and cardiac muscle (RYR2) are poorly understood. Here we use a series of CaM mutants deficient in Ca2+ binding to compare determinants of CaM regulation of the RYR1 and RYR2 isoforms. In submicromolar Ca2+, activation of the RYR1 isoform by each of the single-point CaM mutants was similar to that by wild-type apoCaM, whereas in micromolar Ca2+, RYR1 inhibition by Ca2+CaM was abolished by mutations targeting CaM's C-terminal Ca2+ sites. In contrast to the RYR1, no activation of the cardiac RYR2 isoform by wild-type CaM was observed, but rather CaM inhibited the RYR2 at all Ca2+ concentrations (100 nM to 1 mM). Consequently, whereas the apparent Ca2+ sensitivity of the RYR1 isoform was enhanced in the presence of CaM, the RYR2 displayed the opposite response (RYR2 Ca2+ EC50 increased 7-10-fold in the presence of 5 microM wild-type CaM). CaM inhibition of the RYR2 was nonetheless abolished by each of four mutations targeting individual CaM Ca2+ sites. Furthermore, a mutant CaM deficient in Ca2+ binding at all four Ca2+ sites significantly activated the RYR2 and acted as a competitive inhibitor of RYR2 regulation by wild-type Ca2+CaM. We conclude that Ca2+ binding to CaM determines the effect of CaM on both RYR1 and RYR2 channels and that isoform differences in CaM regulation reflect the differential tuning of Ca2+ binding sites on CaM when bound to the different RYRs. These results thus suggest a novel mechanism by which CaM may contribute to functional diversity among the RYR isoforms.     

Volume changes upon addition of Ca2+ to calmodulin: Ca2+-calmodulin conformational states

Measurement of the volume change by a rapid density method upon sequential addition of calcium ion to calmodulin showed relatively large, nonuniform increases for the first 4 moles Ca2+ per mole calmodulin. Substantially larger volume increases (approximately 15 ml/mol protein) were observed upon addition of the second and fourth moles Ca2+ relative to the first and third moles added per mole calmodulin. A total volume increase of approximately 170 ml/mol protein attended the addition of 4 moles Ca2+, as expected for multidentate carboxylate coordination to metal ion. Marginal changes in volume were observed upon further additions, the data showing a remarkably sharp transition after [Ca2+]/[calmodulin] = 4. The results are consistent with an ordered binding of Ca2+ in which pair-wise additions produce similar volume changes; the volume change behavior, however, does not indicate an absence of distinct conformational states for a Ca2+(1)-calmodulin and a Ca2+(3)-calmodulin complex as has been proposed on the basis of 1H-NMR evidences.     

Saccharomyces cerevisiae protein kinase dependent on Ca2+ and calmodulin.

A Ca2+- and calmodulin (CaM)-dependent protein kinase of Saccharomyces cerevisiae was partially purified by CaM affinity chromatography of the soluble fraction, and the properties of the enzyme were investigated. The protein kinase activity of the affinity-purified preparation was stimulated at least eightfold by the simultaneous presence of Ca2+ and CaM. The enzyme stimulation was strongly inhibited by trifluoperazine (TFP), a CaM antagonist. When the kinase was incubated in the presence of ATP, Ca2+, and CaM before the assay, the enzyme showed activity even in the presence of the Ca2+ chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and TFP. The conversion to this Ca2+- and CaM-independent form occurred very rapidly under the incubation conditions required for protein phosphorylation by the kinase. At the highest level of conversion, Ca2+- and CaM-independent kinase activity, which was measured in the presence of EGTA and TFP, was nearly equal to the total kinase activity, which was measured in the presence of Ca2+ and CaM. A protein with a molecular weight of 58,000 was the major species that was phosphorylated in a Ca2+- and CaM-dependent manner by incubation of the CaM affinity-purified proteins with [gamma-32P]ATP. The protein kinase activity of the protein with the same molecular weight was demonstrated by in situ protein phosphorylation in sodium dodecyl sulfate-polyacrylamide gels by using casein as the substrate, after removal of the detergent from electrophoresed CaM-binding proteins. These data indicate that phosphorylation of the kinase is responsible for the conversion of enzyme activity. Enzyme regulation by this mode may play an important role in integrating cellular functions during the cell cycle. A possible role for the Ca2+-and CaM-dependent protein kinase in the signal transduction of the mating pheromone alpha factor is also discussed.     

Molecular mechanism for divergent regulation of Cav1.2 Ca2+ channels by calmodulin and Ca2+-binding protein-1

Ca(2+)-binding protein-1 (CaBP1) and calmodulin (CaM) are highly related Ca(2+)-binding proteins that directly interact with, and yet differentially regulate, voltage-gated Ca(2+) channels. Whereas CaM enhances inactivation of Ca(2+) currents through Ca(v)1.2 (L-type) Ca(2+) channels, CaBP1 completely prevents this process. How CaBP1 and CaM mediate such opposing effects on Ca(v)1.2 inactivation is unknown. Here, we identified molecular determinants in the alpha(1)-subunit of Ca(v)1.2 (alpha(1)1.2) that distinguish the effects of CaBP1 and CaM on inactivation. Although both proteins bind to a well characterized IQ-domain in the cytoplasmic C-terminal domain of alpha(1)1.2, mutations of the IQ-domain that significantly weakened CaM and CaBP1 binding abolished the functional effects of CaM, but not CaBP1. Pulldown binding assays revealed Ca(2+)-independent binding of CaBP1 to the N-terminal domain (NT) of alpha(1)1.2, which was in contrast to Ca(2+)-dependent binding of CaM to this region. Deletion of the NT abolished the effects of CaBP1 in prolonging Ca(v)1.2 Ca(2+) currents, but spared Ca(2+)-dependent inactivation due to CaM. We conclude that the NT and IQ-domains of alpha(1)1.2 mediate functionally distinct interactions with CaBP1 and CaM that promote conformational alterations that either stabilize or inhibit inactivation of Ca(v)1.2.     

Stage-dependent inhibition of Plasmodium falciparum by potent Ca2+ and calmodulin modulators

The effects of Ca2+ channel blockers, verapamil, nicardipine and diltiazem, and of potent calmodulin (CaM) inhibitors, trifluoperazine (TFP), calmidazolium, W-7 and W-5, on Plasmodium falciparum in culture were examined. Among Ca2+ blockers, nicardipine was the most potent with the 50% inhibitory concentration (IC50) of 4.3 microM at 72 h after culture. Parasites were more sensitive to calmidazolium and W-7 with IC50 of 3.4 and 4.5 microM, respectively, than to TFP and W-5. All Ca2+ blockers and CaM inhibitors suppressed parasite development at later stages. Nicardipine, diltiazem, calmidazolium and W-5 also retarded parasite development at earlier stages and/or subsequent growth following pretreatment. Verapamil, nicardipine, TFP and calmidazolium reduced erythrocyte invasion by merozoites. Fluorescence microscopy with the cationic fluorescent dye rhodamine 123 revealed that nicardipine, TFP and calmidazolium depolarized both the plasma membrane and mitochondrial membrane potentials of the parasite. It is therefore considered that although all Ca2+ and CaM antagonists tested here influence parasite development at later stages, they are multifunctional, having effects not directly associated with Ca2+ channels or CaM.     

The stoichiometry of the Ca2+ pump in human erythrocyte vesicles: modulation by Ca2+, Mg2+ and calmodulin

Active Ca2+ uptake and the associated (Ca2+ + Mg2+)-ATPase activity were studied under the same conditions in an inside-out vesicle preparation of human red blood cells made essentially by the procedure of Quist and Roufogalis (Journal of Supramolecular Structure 6, 375-381, 1977). Some preparations were treated with 1 mM EDTA at 30 degrees to further deplete them of endogenous levels of calmodulin. As the Ca2+ taken up by the EDTA-treated inside-out vesicles, as well as the non-EDTA treated vesicles, was maintained after addition of 4.1 mM EGTA, the vesicles were shown to be impermeable to the passive leak of Ca2+ over the time course of the experiments. In the absence of added calmodulin, both active Ca2+ uptake and (Ca2+ + Mg2+)-ATPase were sensitive to free Ca2+ over a four log unit concentration range (0.7 microM to 300 microM Ca2+) at 6.4 mM MgCl2. Below 24 microM Ca2+ the stoichiometry of calcium transported per phosphate liberated was close to 2:1, both in EDTA and non-EDTA treated vesicles. Above 50 microM Ca2+ the stoichiometry approached 1:1. When MgCl2 was reduced from 6.4 mM to 1.0 mM, the stoichiometry remained close to 2:1 over the whole range of Ca2+ concentrations examined. In contrast to the results at 6.4 mM MgCl2, the Ca2+ pump was maximally activated at about 2 microM free Ca2+ and significantly inhibited above this concentration at 1 mM MgCl2. Calmodulin (0.5-2.0 microgram/ml) had little effect on the stoichiometry in any of the conditions examined. The possible significance of a variable stoichiometry of the Ca2+ pump in the red blood cell is discussed.     

Thermodynamics of Ca2+ binding to calmodulin and its tryptic fragments.

The binding of Ca2+ to calmodulin and its two tryptic fragments has been studied using microcalorimetry. The binding process is accompanied by the uptake or release of protons, depending on the ionic strength. With no added salt, the total enthalpy change for the binding of four calcium ions to calmodulin is -41 kJ mol-1 but in the presence of 0.15 mM KCl delta Htot is +17 kJ mol-1. The mode of binding of Ca2+ is also completely different with and without added salt. It is also shown that for the C-terminal fragment of calmodulin, TR2C, the drastic reduction in delta Gtot for the binding process on increasing the ionic strength is largely an enthalpic effect. Domain interactions in calmodulin are indicated by the fact that the sum of the enthalpies of calcium binding to the two tryptic fragments is not the same as the total binding enthalpy to calmodulin itself. The binding of Ca2+ to calmodulin has also been studied calorimetrically at different temperatures in the range 21-37 degrees C. delta Cp is large and negative in this interval.     

Ca2+-dependent interaction of the trpl cation channel and calmodulin.

The transient receptor potential-like ion channel from Drosophila melanogaster was originally identified as a calmodulin binding protein (Philips et al., 1992) involved in the dipterian phototransduction process. We used a series of fusion proteins and an epitope expression library of transient receptor potential-like fusion proteins to characterize calmodulin binding regions in the transient receptor potential-like channel through the use of [125I]calmodulin and biotinylated calmodulin and identified two distinct sites at the C-terminus of the transient receptor potential-like ion channel. Calmodulin binding site 1, predicted from searching of the primary structure for amphiphilic helices (Philips et al., 1992), covers a 16 amino acid sequence (S710-I725) and could only be detected through biotinylated calmodulin. Calmodulin binding site 2 comprises at least 13 amino acids (K859ETAKERFQRVAR871) and binds both [125I]calmodulin and biotinylated calmodulin. Both sites (i) bind calmodulin at least in a one to one stoichiometry, (ii) differ in their affinity for calmodulin revealing apparent Ki values of 12.3 nM (calmodulin binding site 1) and 1.7 nM (calmodulin binding site 2), respectively, (iii) bind calmodulin only in the presence of Ca2+ with 50% of site 1 and site 2, respectively, occupied by calmodulin in the presence of 0.1 microM (calmodulin binding site 1) and 3.3 microM Ca2+ (calmodulin binding site 2) and give evidence that (iv) a Ca2+-calmodulin-dependent mechanism contributes to transient receptor potential-like cation channel modulation when expressed in CHO cells.     

Direct detection of calmodulin tuning by ryanodine receptor channel targets using a Ca2+-sensitive acrylodan-labeled calmodulin

Calmodulin (CaM) activates the skeletal muscle ryanodine receptor (RyR1) at nanomolar Ca(2+) concentrations but inhibits it at micromolar Ca(2+) concentrations, indicating that binding of Ca(2+) to CaM may provide a molecular switch for modulating RyR1 channel activity. To directly examine the Ca(2+) sensitivity of RyR1-complexed CaM, we used an environment-sensitive acrylodan adduct of CaM. The resulting (ACR)CaM probe displayed high-affinity binding to, and Ca(2+)-dependent regulation of, RyR1 similar to that of unlabeled wild-type (WT) CaM. Upon addition of Ca(2+), (ACR)CaM exhibited a substantial (>50%) decrease in fluorescence (K(Ca) = 2.7 +/- 0.8 microM). A peptide derived from the RyR1 CaM binding domain (RyR1(3614)(-)(43)) caused an even more pronounced Ca(2+)-dependent fluorescence decrease, and a >or=10-fold leftward shift in its K(Ca) (0.2 +/- 0.1 microM). In the presence of intact RyR1 channels in SR vesicles, (ACR)CaM fluorescence spectra were distinct from those in the presence of RyR1(3614)(-)(43), although a Ca(2+)-dependent decrease in fluorescence was still observed. The K(Ca) for (ACR)CaM fluorescence in the presence of SR (0.8 +/- 0.4 microM) was greater than in the presence of RyR1(3614)(-)(43) but was consistent with functional determinations showing the conversion of (ACR)CaM from channel activator (apoCaM) to inhibitor (Ca(2+)CaM) at Ca(2+) concentrations between 0.3 and 1 microM. These results indicate that binding to RyR1 targets evokes significant changes in the CaM structure and Ca(2+) sensitivity (i.e., CaM tuning). However, changes resulting from binding of CaM to the full-length, tetrameric channels are clearly distinct from changes caused by the RyR1-derived peptide. We suggest that the Ca(2+) sensitivity of CaM when in complex with full-length channels may be tuned to respond to physiologically relevant changes in Ca(2+).     

Effects of calmodulin antagonists on the active Ca2+ uptake by rat liver mitochondria.

The mechanism of Ca2+ transport by rat liver mitochondria was investigated with respect to the possible involvement of calmodulin in this process. We studied the action of exogenous calmodulin isolated from brain tissue on the Ca2+-transport system, as well as the effect of two types of calmodulin antagonists; the phenothiazine drugs trifluoperazine and chlorpromazine and the more specific substance compound 48/80. Our results show that Ca2+ transport by mitochondria and mitochondrial ATPase activity are insensitive to exogenous calmodulin, although they can be inhibited by the phenothiazines. Since no effect of compound 48/80 was observed, we believe that the phenothiazines act through a mechanism that does not involve calmodulin. This is in accord with our inability to locate significant quantities of calmodulin in mitochondria by radioimmunoassay analysis. Our results further show that trifluoperazine and chlorpromazine also inhibit the electron-carrier system of the respiratory chain, and this effect may mediate their inhibitory action on Ca2+ transport when it is energized by respiration instead of ATP hydrolysis.     

Apocalmodulin and Ca2+ calmodulin bind to the same region on the skeletal muscle Ca2+ release channel.

The skeletal muscle Ca2+ release channel (RYR1) is regulated by calmodulin in both its Ca2+-free (apocalmodulin) and Ca2+-bound (Ca2+ calmodulin) states. Apocalmodulin is an activator of the channel, and Ca2+ calmodulin is an inhibitor of the channel. Both apocalmodulin and Ca2+ calmodulin binding sites on RYR1 are destroyed by a mild tryptic digestion of the sarcoplasmic reticulum membranes, but calmodulin (either form), bound to RYR1 prior to tryptic digestion, protects both the apocalmodulin and Ca2+ calmodulin sites from tryptic destruction. The protected sites are after arginines 3630 and 3637 on RYR1. These studies suggest that both Ca2+ calmodulin and apocalmodulin bind to the same or overlapping regions on RYR1 and block access of trypsin to sites at amino acids 3630 and 3637. This sequence is part of a predicted Ca2+ CaM binding site of amino acids 3614-3642 [Takeshima, H., et al. (1989) Nature 339, 439-445].     

Ca2+-dependent metarhodopsin inactivation mediated by calmodulin and NINAC myosin III

Phototransduction in flies is the fastest known G protein-coupled signaling cascade, but how this performance is achieved remains unclear. Here, we investigate the mechanism and role of rhodopsin inactivation. We determined the lifetime of activated rhodopsin (metarhodopsin = M( *)) in whole-cell recordings from Drosophila photoreceptors by measuring the time window within which inactivating M( *) by photoreisomerization to rhodopsin could suppress responses to prior illumination. M( *) was inactivated rapidly (tau approximately 20 ms) under control conditions, but approximately 10-fold more slowly in Ca2+-free solutions. This pronounced Ca2+ dependence of M( *) inactivation was unaffected by mutations affecting phosphorylation of rhodopsin or arrestin but was abolished in mutants of calmodulin (CaM) or the CaM-binding myosin III, NINAC. This suggests a mechanism whereby Ca2+ influx acting via CaM and NINAC accelerates the binding of arrestin to M( *). Our results indicate that this strategy promotes quantum efficiency, temporal resolution, and fidelity of visual signaling.     

Alcohols increase calmodulin affinity for Ca2+ and decrease target affinity for calmodulin

It has been proposed that alcohols and anesthetics selectively inhibit proteins containing easily disrupted motifs, e.g., alpha-helices. In this study, the calcineurin/calmodulin/Ca(2+) enzyme system was used to examine the effects of alcohols on calmodulin, a protein with a predominantly alpha-helical structure. Calcineurin phosphatase activity and Ca(2+) binding were monitored as indicators of calmodulin function. Alcohols inhibited enzyme activity in a concentration-dependent manner, with two-, four- and five-carbon n-alcohols exhibiting similar leftward shifts in the inhibition curves for calmodulin-dependent and -independent activities; the former was slightly more sensitive than the latter. Ca(2+) binding was measured by flow dialysis as a direct measure of calmodulin function, whereas, with the addition of a binding domain peptide, measured calmodulin-target interactions. Ethanol increased the affinity of calmodulin for Ca(2+) in the presence and absence of the peptide, indicating that ethanol stabilizes the Ca(2+) bound form of calmodulin. An increase in Ca(2+) affinity was detected in a calmodulin binding assay, but the affinity of calmodulin for calcineurin decreased at saturating Ca(2+). These data demonstrate that although specific regions within proteins may be more sensitive to alcohols and anesthetics, the presence of alpha-helices is unlikely to be a reliable indicator of alcohol or anesthetic potency.