Calmidazolium, a calmodulin antagonist, stimulates calcium-troponin C and calcium-calmodulin-dependent activation of striated muscle myofilaments

Although regulatory Ca2+-binding domains of calmodulin (CaM) and troponin C (TnC) are similar, it is interesting that agents that act as CaM antagonists appear to be TnC "agonists" in that they sensitize cardiac myofilaments to activation by Ca2+ (El-Saleh, S., and Solaro, R. J. (1987) Biophys. J. 51, 325 (abstr.). This indicates that the effects of agents that react with Ca2+-binding proteins may depend on protein-protein interactions involved in a particular Ca2+-dependent process. In experiments described here, we have explored this idea by testing effects of calmidazolium (CDZ), a potent calmodulin antagonist on striated muscle myofilaments regulated by cardiac TnC, skeletal TnC, and CaM. CDZ was shown to increase submaximal calcium activation of myofilament force and ATPase activity in both cardiac and skeletal muscle, but the effect was greater in the case of the cardiac preparations. In the presence of 10 microM CDZ, the free Ca2+ giving half-maximal activation was reduced to about 60% of the control value in the case of cardiac myofilaments. Analogous differential effects of CDZ were also seen in studies in which we measured direct effects of CDZ on Ca2+-dependent fluorescence changes of cardiac TnC and skeletal TnC labeled with probes reporting Ca2+ binding to the regulatory sites. Measurements were also done with myofibrillar preparations of psoas muscle in which the native skeletal TnC was removed and exchanged with cardiac TnC and CaM, both of which could substitute for skeletal TnC as a regulatory protein. CDZ was more effective in sensitizing Ca2+-dependent MgATPase activity of skeletal myofibrils containing CaM than in preparations containing the native TnC. However, CDZ was most effective in its Ca2+-sensitizing effect in the case of the preparations containing cardiac TnC. Our results indicate that effects of agents that bind to Ca2+-binding proteins depend not only on the particular variant, but also on the specific environment in which the Ca2+-binding proteins operate.     

Effects of calcium and calmodulin antagonists on calpain II subunit conformations

Only the 80-kD catalytic subunit of smooth muscle calpain II shows a change in intrinsic fluorescence on binding calcium, but both the 80-kD and 30-kD subunits show fluorescence changes in bound toluidinyl-naphthalenesulphonate as a result of calcium binding. Both subunits also show changes in intrinsic fluorescence in the presence of calmidazolium and felodipine. These studies indicate that both subunits have binding sites for calcium and the calmodulin antagonists, which are probably located in the calmodulin-like domain of each subunit.     

Effects of ruthenium red on activation of Ca2(+)-dependent cyclic nucleotide phosphodiesterase.

Ruthenium red inhibited Ca2(+)-dependent phosphodiesterase (Ca2(+)-PDE) selectively with an IC50 value of 15 microM. Increasing calmodulin concentration in the presence of both 100 microM and 4000 microM Ca2+ completely reversed the inhibition of Ca2(+)-PDE activity by ruthenium red. Ruthenium red-induced inhibition of Ca2(+)-PDE activity was also overcome by increasing the concentration of Ca2+ in the presence of both 200 ng and 2000 ng calmodulin, in sharp contrast to fluphenazine-induced inhibition of Ca2(+)-PDE. These results indicate that ruthenium red has distinct inhibitory mechanism which differs from that of calmodulin antagonists previously reported.     

A fluorescent calmodulin that reports the binding of hydrophobic inhibitory ligands.

Ca2+ binding to calmodulin in the pCa range 5.5-7.0 exposes hydrophobic sites that bind hydrophobic inhibitory ligands, including calmodulin antagonists, some Ca2+-antagonists and calmodulin-binding proteins. The binding of these hydrophobic ligands to calmodulin can be followed by the approx. 80% fluorescence increase they produce in dansylated (5-dimethylaminonaphthalene-1-sulphonylated) calmodulin (CDRDANS). In the presence of Ca2+, calmodulin binds the calmodulin inhibitor, R24571, with an affinity of approx. 2-3 nM and hydrophobic ligands, including trifluoperazine (TFP), W-7 [N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide], fendiline, felodipine and prenylamine, with affinities in the micromolar range. This binding is strongly Ca2+-dependent and Mg2+-independent. Calmodulin shows a reasonably high degree of specificity in its binding of these ligands over other ligands tested. CDRDANS, therefore, provides a convenient and simple means of monitoring the interaction of a variety of hydrophobic ligands with the Ca2+-dependent regulatory protein, calmodulin. CDRDANS binds to phospholipid vesicles made of (dimyristoyl)phosphatidylcholine (DMPC) or (dipalmitoyl)phosphatidylcholine (DPPC) and produces fluorescence increases only in the presence of Ca2+ and at temperatures above their gel-to-liquid crystalline phase transition. Although the fluorescence changes in CDRDANS accurately report phase transitions in these liposomes, its binding to these vesicles is weak. Calmodulin probably requires a high-affinity lipid-bound receptor protein for its high-affinity binding to natural membranes.     

Localization of a felodipine (dihydropyridine) binding site on calmodulin

The fluorescent dihydropyridine calcium antagonist drug felodipine binds to calmodulin (CaM) in a Ca2+-dependent manner. Its binding can be regulated by the interaction of CaM antagonist drugs through allosteric mechanisms [Mills, J.S., & Johnson, J.D. (1985) Biochemistry 24, 4897]. Here, we have examined the binding of a nonspecific hydrophobic fluorescent probe molecule TNS (toluidinylnaphthalenesulfonate) and of felodipine to CAM and several of its proteolytic fragments. While TNS interacts with sites on both the amino-terminal half of the protein [proteolytic fragment TR1C (1-77)] and carboxy-terminal half [proteolytic fragment TR2C (78-148)], felodipine binding shows more selectivity. It binds in a Ca2+-dependent manner to the proteolytic fragments TM1 (1-106) and TR2E (1-90) but exhibits only weak affinity for TR1C (1-77) and TR2C (78-148). Furthermore, felodipine exhibits selectivity over TNS and trifluoperazine (TFP) in blocking the tryptic cleavage between residues 77 and 78. These studies indicate a selective binding of felodipine to a hydrophobic site existing in residues 1-90 and suggest that productive binding requires amino acids in the region 78-90. Although the felodipine binding site is preserved in fragment 1-106, the allosteric interactions between the prenylamine and the felodipine binding sites that are observed with intact CaM are not observed in this fragment. Rather, prenylamine simply displaces felodipine from its binding site on this fragment. Our results are consistent with calmodulin containing not less than two allosterically related hydrophobic drug binding sites. One of these sites (felodipine) appears to be localized in region 1-90 and the other one in region 78-148.     

Tb~(3+)作为荧光探针研究钙调蛋白与拮抗药物的相互作用

本文报导以Tb~(3+)作为荧光探针,研究钙调蛋白(CaM)与其拮抗药物分子间相互作用的机制.所用方法简便、快速、灵敏.CaM的内源荧光研究表明,Tb~(3+)类似于Ca~(2+),也能诱导CaM分子构象发生改化,由于CaM分子中Ca~(2+)的第Ⅲ、Ⅳ结合位点上各有一个Tyr线基,如(?)280nm激发,则发生从Tyr向Tb~(3+)的能量转移,从而导致Tb~(3+)在490和545nm处的特征荧光发射大大加强.本文检测了药物分子与Tb~(3+)-CaM结合对该荧光发射的影响.实验表明,TFP与CaM的高亲和位点处于CaM分子C-末端部位,即含第Ⅲ、Ⅳ结构域的半分子上:丙拮抗药物酸枣仁皂甙A则优先结合在含第Ⅰ、Ⅱ的结构域的另一半分子(?).     

Interaction of calcium agonists and antagonists with Ca-binding proteins and their effect on cyclic nucleotide phosphodiesterase

The effects of Ca2+ antagonists (nicardipine, felodipine, nitrenedipine, isradipine, niphedipine, darodipine and riodipine) and Ca2+ agonists (BAY K8644 and CGP 28392), 1.4-dihydropyridine derivatives (1.2-DHP), on the calmodulin (CM)-dependent activation of cyclic nuxleotide phosphodiesterase (PDE) were studied. Both the blockers and activators of slow potential-dependent Ca2+ channels induced a un-competitive inhibition of the CM-dependent PDE activity. 1.4-DHP was found to replace the fluorescent probe, diS-C3-(5), from the Ca2(+)-dependent calmodulin-dye complex (K0.5 = 4-60 microM) but at concentrations below 100 microM had no effect on the Ca2(+)-dependent troponin C-dye complex. Darodipine (100 microM) did not interact with the proteins. The 1.4-DHP interaction with CM did not interfere with PDE activation. It is concluded that 1.4-DHP may affect Ca2+ dependent processes not only at the levels of activation or blocking of Ca2+ channels, but also through regulation of Ca2(+)-CM dependent enzymes.     

Hydrophobic regions function in calmodulin-enzyme(s) interactions

Certain naturally occurring lipids (phosphatidylinositol, phosphatidylserine, arachidonic acid) and sodium dodecyl sulfate activate at least two calmodulin-dependent enzymes, bovine brain 3':5'-cyclic nucleotide phosphodiesterase and chicken gizzard myosin light chain kinase in the absence of Ca2+. 2-p-Toluidinyl-naphthalene-6-sulfonate (TNS), which is often used as a probe for hydrophobic groups of proteins, inhibits these two calmodulin-dependent enzymes. Kinetic analysis of inhibition of chicken gizzard myosin kinase by TNS revealed a competitive fashion against calmodulin-induced activation. The interaction between TNS and purified bovine brain calmodulin as demonstrated in the appearance of TNS fluorescence in the presence of 3 microM or more of calcium ion was not observed in the presence of 2 mM EGTA. This suggests that TNS is able to bind to calmodulin in the presence of Ca2+. Moreover, a calmodulin-interacting agent N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide suppressed the TNS fluorescence induced by complex formation with calmodulin in the presence of Ca2+. These results suggest that when Ca2+ binds to the high affinity sites of calmodulin, it induces a conformational change which exposes hydrophobic groups, and the calmodulin is then capable of activating calmodulin-dependent enzymes. We propose that hydrophobic properties of Ca2+-calmodulin are important for the activation of Ca2+-calmodulin-dependent enzymes.     

Cooperativity among calmodulin's drug binding sites

The binding of felodipine, a dihydropyridine Ca2+ antagonist, to calmodulin has been studied by equilibrium dialysis and fluorescence techniques. Analysis using the Hill equation gives a Hill coefficient of 2. A plot of bound [felodipine] vs. free [felodipine]2 gives a Bmax of 1.9 mol/mol and a K0.5 of 22 microM. Two calmodulin antagonists, prenylamine and R24571, which have previously been shown to potentiate the fluorescent enhancement observed when felodipine binds to calmodulin [Johnson, J. D. (1983) Biochem. Biophys. Res. Commun. 112, 787], produce a reduction in Hill coefficient to 0.7 and 1.0, respectively, and account for the observed potentiation of felodipine binding. Titrations of felodipine with calmodulin in the absence and presence of prenylamine and R24571 suggest that these drugs decrease the K0.5 of calmodulin for felodipine by 25-fold. Thus, potentiating drugs (prenylamine and R24571) bind to either of the two felodipine binding sites and, through an allosteric mechanism, result in felodipine binding to the remaining site with greatly enhanced affinity. Two types of potentiating drugs are observed. Prenylamine exhibits a Hill coefficient of 0.8 whereas felodipine, R24571, and diltiazem exhibit Hill coefficients of 2 in their potentiation of felodipine binding. Titrations of felodipine and calmodulin with Ca2+ exhibit cooperativity with a Hill coefficient of 4. Half-maximal binding occurs near pCa 6.0. In the presence of R24571, the calcium dependence of felodipine binding is biphasic, now exhibiting a much higher affinity (pCa 7.6) component. A model is presented to explain the relationship of these various allosterically regulated conformers of calmodulin and their interactions and activation with its target proteins.     

Activation of protein kinase C is not required for exocytosis from bovine adrenal chromaffin cells. The effects of protein kinase C(19-31), Ca/CaM kinase II(291-317), and staurosporine

We examined whether protein kinase C activation plays a modulatory or an obligatory role in exocytosis of catecholamines from chromaffin cells by using PKC(19-31) (a protein kinase C pseudosubstrate inhibitory peptide), Ca/CaM kinase II(291-317) (a calmodulin-binding peptide), and staurosporine. In permeabilized cells, PKC (19-31) inhibited the phorbol ester-mediated enhancement of Ca2(+)-dependent secretion as much as 90% but had no effect on Ca2(+)-dependent secretion in the absence of phorbol ester. The inhibition of the phorbol ester-induced enhancement of secretion by PKC (19-31) was correlated closely with the ability of the peptide to inhibit in situ phorbol ester-stimulated protein kinase C activity. PKC(19-31) also blocked 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation of numerous endogenous proteins in permeabilized cells but had no effect on Ca2(+)-stimulated phosphorylation of tyrosine hydroxylase. Ca/CaM kinase II(291-317), derived from the calmodulin binding region of Ca/calmodulin kinase II, had no effect on Ca2(+)-dependent secretion in the presence or absence of phorbol ester. The peptide completely blocked the Ca2(+)-dependent increase in tyrosine hydroxylase phosphorylation but had no effect on TPA-induced phosphorylation of endogenous proteins in permeabilized cells. To determine whether a long-lived protein kinase C substrate might be required for secretion, the lipophilic protein kinase inhibitor, staurosporine, was added to intact cells for 30 min before permeabilizing and measuring secretion. Staurosporine strongly inhibited the phorbol ester-mediated enhancement of Ca2(+)-dependent secretion. It caused a small inhibition of Ca2(+)-dependent secretion in the absence of phorbol ester which could not be readily attributed to inhibition of protein kinase C. Staurosporine also inhibited the phorbol ester-mediated enhancement of elevated K(+)-induced secretion from intact cells while it enhanced 45Ca2+ uptake. Staurosporine inhibited to a small extent secretion stimulated by elevated K+ in the absence of TPA. The data indicate that activation of protein kinase C is modulatory but not obligatory in the exocytotoxic pathway.     

Allosteric interactions among drug binding sites on calmodulin

Felodipine is a fluorescent dihydropyridine Ca²⁺-antagonist. It binds to calmodulin in a Ca²⁺-dependent manner, and undergoes a fluorescence increase which allows us to monitor its interaction with calmodulin. Hydrophobic ligands including the calmodulin antagonist, R24571 and Ca²⁺ antagonists, prenylamine and diltiazem, bind to calmodulin and potentiate felodipine binding by as much as 20 fold. These studies suggest that allosteric interactions occur among different drug binding sites on calmodulin. Our results are discussed in terms of the mechanism of action of calmodulin.     

Calcium release from intact calmodulin and calmodulin fragment 78-148 measured by stopped-flow fluorescence with 2-p-toluidinylnaphthalene sulfonate. Effect of calmodulin fragments on cardiac sarcoplasmic reticulum

Calcium release from high and low-affinity calcium-binding sites of intact bovine brain calmodulin (CaM) and from the tryptic fragment 78-148, purified by high-pressure liquid chromatography, containing only the high-affinity calcium-binding sites, was determined by fluorescence stopped-flow with 2-p-toluidinylnaphthalene sulfonate (TNS). The tryptic fragments 1-77 and 78-148 each contain a calcium-dependent TNS-binding site, as shown by the calcium-dependent increase in TNS fluorescence. The rate of the monophasic fluorescence decrease in endogenous tyrosine on calcium dissociation from intact calcium-saturated calmodulin (kobs 10.8 s-1 and 3.2 s-1 at 25 degrees C and 10 degrees C respectively) as well as the rate of equivalent slow phase of the biphasic decrease in TNS fluorescence (kobsslow 10.6 s-1 and 3.0 s-1 at 25 degrees C and 10 degrees C respectively) and the rate of the solely monophasic decrease in TNS fluorescence, obtained with fragment 78-148 (kobs 10.7 s-1 and 3.5 s-1 at 25 degrees C and 10 degrees C respectively), were identical, indicating that the rate of the conformational change associated with calcium release from the high-affinity calcium-binding sites on the C-terminal half of calmodulin is not influenced by the N-terminal half of the molecule. The fast phase of the biphasic decrease of TNS fluorescence, observed by the N-terminal half of the molecule. The fast phase of the biphasic decrease of TNS fluorescence, observed with intact calmodulin only (kobsfast 280 s-1 at 10 degrees C) but not with fragment 78-148, is most probably due to the conformational change associated with calcium release from low-affinity sites on the N-terminal half. The calmodulin fragments 1-77 and 78-148 neither activated calcium/calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum nor inhibited calmodulin-dependent activation at a concentration approximately 1000-fold greater (5 microM) than that of the calmodulin required for half-maximum activation (5.9 nM at 0.8 mM Ca2+ and 5 mM Mg2+) of calmodulin-dependent phosphoester formation.     

Time-resolved fluorescence of erythrocyte plasma membrane Ca2(+)-ATPase in different functional states.

The fluorescence decay of the plasma membrane calmodulin-activated Ca2(+)-ATPase from the erythrocyte was measured for the first time. The availability of a novel procedure for on-line blank subtraction in frequency-domain lifetime data acquisition (G.G. Reinhart, B. Feddersen, D. Jameson and E. Gratton, Biophys. J. 57 (1990) 189a) permitted the elimination of background interference from detergent-solubilized purified plasma membrane ATPase samples. The fluorescence decay of the erythrocyte Ca2(+)-ATPase was measured in the absence of Ca2+, or in the presence of Ca2+ or Ca2+ plus calmodulin. In the three different experimental conditions the fluorescence decay was very heterogeneous and could be best described by Lorentzian distributions of lifetime values. In the absence of Ca2+ the decay was described by a broad lifetime distribution centered at 4.4 ns with a width of 3.2 ns, indicating heterogeneity of tryptophan microenvironments in the ATPase. Calcium ion binding promoted an 11% increase in the center and a 27% decrease in the width of the distribution. By contrast, addition of calmodulin in the presence of Ca2+ caused a 15% decrease in the center of the distribution, revealing structural difference between calmodulin-activated and Ca2(+)-activated states of the ATPase. These results indicate the usefulness of on-line blank subtraction in frequency-domain lifetime measurements to investigate conformational changes in detergent-solubilized membrane protein samples.     

The evaluation of the role of endogenous Ca-dependent regulators and protein kinases in activating and inhibiting ion-transport ATPases]

The data on hormonal regulation of ATP-driving ion pumps are contradictory depending on the object used: whether native cells or isolated membranes. To eliminate this contrariety, we studied the ion transporting ATPases in saponin-permeabilized cells in the presence of all endogenous regulators. In permeabilized erythrocytes we obtained the presence of Ca(2+)-dependent activation of Ca(2+)-ATPase by factor(s) not affected by calmodulin antagonist R24571. We obtained also Ca(2+)-dependent activation and inhibition of Na+,K(+)-ATPase. At a concentration of Mg(2+)-ions corresponding to the intracellular level (370 microM), the 0.5-0.7 microM Ca(2+)-activated Na+,K(+)-ATPase (up to 3-fold), whereas the 1-5 microM Ca2+ inhibited it. The cyclic AMP (10(-5) M) inhibited or eliminated Ca(2+)-dependent activation. The decrease in Mg(2+)-ion concentration to 50 microM eliminated the activation and strengthened the inhibition, which reached 100% at the 1-2 microM Ca2+ concentration. The washing of membranes with EGTA eliminated Ca2+ effects on Na+,K(+)-ATPase. These data suggest that the ion-transporting ATPases are activated or inhibited by Ca(2+)-dependent regulators whose activities may be changed by protein kinase catalysed phosphorylation.     

Possible participation of calpain in myosin light chain phosphorylation of human platelets

We previously demonstrated that myosin light chain kinase (MLCK) of gizzard is proteolyzed by platelet calpain. It has been also reported that partially cleaved MLCK may phosphorylate myosin light chain (20K) in the absence of calmodulin. Therefore, a possible participation of calpain in 20K phosphorylation was studied in human platelets, utilizing various inhibitors. An epoxy succinate derivative (E-64) or N-ethylmaleimide (NEM), used as calpain antagonist, inhibited 20K phosphorylation of Ca2+-stimulated lysed platelets. A synergistic effect between these calpain antagonists and calmodulin antagonist W-7 was observed. Also, the similar results were obtained in 20K phosphorylation of intact platelets. From these observations, it was suggested that 20K phosphorylation in platelets is mediated by two separate pathways, namely calmodulin and calpain dependent pathways, provided that calpain activity is specifically inhibited by the antagonists used.     

Evidence for membrane-associated calpain I in human erythrocytes. Detection by an immunoelectrophoretic blotting method using monospecific antibody

Low and high Ca2+-requiring forms of Ca2+-dependent cysteine proteinase are known as calpain I and calpain II, respectively. We have obtained, for the first time, monospecific antibodies for calpain I and for calpain II. Using these antibodies and an electrophoretic blotting method, we have found that a small, but reproducible, amount of calpain I was associated with human erythrocyte membranes while the bulk of the protease was contained in the cytosol. Most of membrane-associated calpain I was extractable with 1% Triton X-100, but not with 0.1% detergent. In the presence of 0.1 mM Ca2+ and 5 mM cysteine, membrane-associated calpain I degraded the membrane protein band 4.1 preferentially and band 3 protein only slowly. The Ca2+-induced autodigestion of the membrane preparation was inhibited by leupeptin but not by a cytosolic calpain inhibitor, calpastatin, added to the incubation medium. No calpain II was detected in either erythrocyte cytosol or membranes when anti-calpain II antibody was used under the same conditions as those for the detection of calpain I.     

The role of calmodulin on Ca2+ -dependent K+ transport regulation in the human red cell

Several lipophilic calmodulin antagonists (phenotiazines, butyrophenones and diphenylbutylpiperidines) inhibited Ca2+-induced loss of KC1 from human red cells. However, the Ki values for this effect did not bear good correlation with the Ki values reported for well-known calmodulin-dependent systems. In addition, the inhibition was strongly dependent on the haematocrit and valinomycin-induced KC1 fluxes were also affected. Added calmodulin did not have any effect on Ca2+-dependent 86Rb uptake by inside-out vesicles derived from red cell membranes whereas stimulation of Ca2+-dependent ATPase was apparent. Lipophilic anticalmodulins at high doses had all kinds of effects on 86Rb uptake by inside-out vesicles: increase, decrease or no change of the fraction of activated vesicles reached at submaximal Ca2+ concentrations, with or without modification of the relative rate of 86Rb uptake. The hydrophylic compound 48/80 decreased the fraction of activated vesicles reached at submaximal Ca2+ concentrations without affecting the relative rate of 86Rb uptake, but this effect took place only at concentrations 10-fold higher than the reported Ki for calmodulin-dependent systems. These results suggest that Ca2+-dependent K+ channels of red cells are not regulated by calmodulin.     

Calcium-dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin.

We investigated whether calmodulin mediates the stimulating effect of Ca2+ on nitric oxide synthase in the cytosol of porcine aortic endothelial cells. Nitric oxide was quantified by activation of a purified soluble guanylate cyclase. The Ca2(+)-sensitivity of nitric oxide synthase was lost after anion exchange chromatography of the endothelial cytosol and could only be reconstituted by addition of calmodulin or heat-denatured endothelial cytosol. The Ca2(+)-dependent activation of nitric oxide synthase in the cytosol was inhibited by the calmodulin-binding peptides/proteins melittin, mastoparan, and calcineurin (IC50 450, 350 and 60 nM, respectively), but not by the calmodulin antagonist, calmidazolium. In contrast, Ca2(+)-calmodulin-reconstituted nitric oxide synthase was inhibited with similar potency by melittin and calmidazolium. The results suggest that the Ca2(+)-dependent activation of nitric oxide synthase in endothelial cells is mediated by calmodulin.     

Endocytic vesicles contain a calmodulin-activated Ca2+ pump that mediates the inhibition of acidification by calcium

Endocytic vesicles isolated from rabbit reticulocytes contained an intrinsic Ca2(+)-pump activity that was dependent on ATP, activated by calmodulin and inhibited by vanadate. 45Ca2+ uptake and acidification studies indicated that acidification of the endocytic vesicles inversely correlated with the Ca2(+)-pump activity. Acidification was inhibited by externally added Ca2+ and calmodulin and activated by vanadate and EGTA. It is suggested that intravesicular Ca2+ can act as a modulator of endocytic vesicle acidification.     

Ca2(+)-dependent regulation of the spectrin/actin interaction by calmodulin and protein 4.1

The Ca2(+)-dependent regulation of the erythroid membrane cytoskeleton was investigated. The low-salt extract of erythroid membranes, which is mainly composed of spectrin, protein 4.1, and actin, confers a Ca2+ sensitivity on its interaction with F-actin. This Ca2+ sensitivity is fortified by calmodulin and antagonized by trifluoperazine, a potent calmodulin inhibitor. Additionally, calmodulin is detected in the low-salt extract. These results suggest that calmodulin is the sole Ca2(+)-sensitive factor in the low-salt extract. The main target of calmodulin in the erythroid membrane cytoskeleton was further examined. Under native conditions, calmodulin forms a stable and equivalent complex with protein 4.1 as determined by calmodulin affinity chromatography, cross-linking experiments, and fluorescence binding assays with an apparent Kd of 5.5 x 10(-7) M irrespective of the free Ca2+ concentration. Domain mapping with chymotryptic digestion reveals that the calmodulin-binding site resides within the N-terminal 30-kDa fragment of protein 4.1. In contrast, the interaction of calmodulin with spectrin is unexpectedly weak (Kd = 1.2 x 10(-4) M). Given the content of calmodulin in erythrocytes (2-5 microM), these results imply that the major target for calmodulin in the erythroid membrane cytoskeleton is protein 4.1. Low- and high-shear viscometry and binding assays reveal that an equivalent complex of calmodulin with protein 4.1 regulates the spectrin/actin interaction in a Ca2(+)-dependent manner. At a low Ca2+ concentration, protein 4.1 potentiates the actin cross-linking and the actin binding activities of spectrin. At a high Ca2+ concentration, the protein 4.1-potentiated actin cross-linking activity but not the actin binding activity of spectrin is suppressed by Ca2+/calmodulin. The Ca2(+)-dependent regulation of the spectrin/protein 4.1/calmodulin/actin interaction is discussed.