Structural and functional characterization of an essential RTX subdomain of Bordetella pertussis adenylate cyclase toxin

The adenylate cyclase toxin (CyaA) is one of the major virulence factors of Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic cells by a unique mechanism that consists in a calcium-dependent, direct translocation of the CyaA catalytic domain across the plasma membrane of the target cells. CyaA possesses a series of a glycine- and aspartate-rich nonapeptide repeats (residues 1006-1613) of the prototype GGXG(N/D)DX(L/I/F)X (where X represents any amino acid) that are characteristic of the RTX (repeat in toxin) family of bacterial cytolysins. These repeats are arranged in a tandem fashion and may fold into a characteristic parallel beta-helix or beta-roll motif that constitutes a novel type of calcium binding structure, as revealed by the three-dimensional structure of the Pseudomonas aeruginosa alkaline protease. Here we have characterized the structure-function relationships of various fragments from the CyaA RTX subdomain. Our results indicate that the RTX functional unit includes both the tandem repeated nonapeptide motifs and the adjacent polypeptide segments, which are essential for the folding and calcium responsiveness of the RTX module. Upon calcium binding to the RTX repeats, a conformational rearrangement of the adjacent non-RTX sequences may act as a critical molecular switch to trigger the CyaA entry into target cells.     

Acylation of lysine 860 allows tight binding and cytotoxicity of Bordetella adenylate cyclase on CD11b-expressing cells

The Bordetella adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) forms cation-selective membrane channels and delivers into the cytosol of target cells an adenylate cyclase domain (AC) that catalyzes uncontrolled conversion of cellular ATP to cAMP. Both toxin activities were previously shown to depend on post-translational activation of proCyaA to CyaA by covalent palmitoylation of the internal Lys983 residue (K983). CyaA, however, harbors a second RTX acylation site at residue Lys860 (K860), and the role of K860 acylation in toxin activity is unclear. We produced in E. coli the CyaA-K860R and CyaA-K983R toxin variants having the Lys860 and Lys983 acylation sites individually ablated by arginine substitutions. When examined for capacity to form membrane channels and to penetrate sheep erythrocytes, the CyaA-K860R acylated on Lys983 was about 1 order of magnitude more active than CyaA-K983R acylated on Lys860, although, in comparison to intact CyaA, both monoacylated constructs exhibited markedly reduced activities in erythrocytes. Channels formed in lipid bilayers by CyaA-K983R were importantly less selective for cations than channels formed by CyaA-K860R, intact CyaA, or proCyaA, showing that, independent of its acylation status, the Lys983 residue may play a role in toxin structures that determine the distribution of charged residues at the entry or inside of the CyaA channel. While necessary for activity on erythrocytes, acylation of Lys983 was also sufficient for the full activity of CyaA on CD11b+ J774A.1 monocytes. In turn, acylation of Lys860 alone did not permit toxin activity on erythrocytes, while it fully supported the high-affinity binding of CyaA-K983R to the toxin receptor CD11b/CD18 and conferred on CyaA-K983R a reduced but substantial capacity to penetrate and kill the CD11b+ cells. This is the first evidence that acylation of Lys860 may play a role in the biological activity of CyaA, even if redundant to the acylation of Lys983.     

Characterization of the C-terminal domain essential for toxic activity of adenylate cyclase toxin

Adenylate cyclase toxin (CyaA) of Bordetella pertussis belongs to the RTX family of toxins. These toxins are characterized by a series of glycine- and aspartate-rich nonapeptide repeats located at the C-terminal half of the toxin molecules. For activity, RTX toxins require Ca²⁺, which is bound through the repeat region. Here, we identified a stretch of 15 amino acids (block A) that is located C-terminally to the repeat region and is essential for the toxic activity of CyaA. Block A is required for the insertion of CyaA into the plasma membranes of host cells. Mixing of a short polypeptide composed of block A and eight Ca²⁺ binding repeats with a mutant of CyaA lacking block A restores toxic activity fully. This in vitro interpolypeptide complementation is achieved only when block A is present together with the Ca²⁺ binding repeats on the same polypeptide. Neither a short polypeptide composed of block A only nor a polypeptide consisting of eight Ca²⁺ binding repeats, or a mixture of these two polypeptides, complement toxic activity. It is suggested that functional complementation occurs because of binding between the Ca²⁺ binding repeats of the short C-terminal polypeptide and the Ca²⁺ binding repeats of the CyaA mutant lacking block A.     

Third activity of Bordetella adenylate cyclase (AC) toxin-hemolysin. Membrane translocation of AC domain polypeptide promotes calcium influx into CD11b+ monocytes independently of the catalytic and hemolytic activities

The Bordetella adenylate cyclase toxin-hemolysin (CyaA) targets phagocytes expressing the alpha(M)beta2 integrin (CD11b/CD18), permeabilizes their membranes by forming small cation-selective pores, and delivers into cells a calmodulin-activated adenylate cyclase (AC) enzyme that dissipates cytosolic ATP into cAMP. We describe here a third activity of CyaA that yields elevation of cytosolic calcium concentration ([Ca2+]i) in target cells. The CyaA-mediated [Ca2+]i increase in CD11b+ J774A.1 monocytes was inhibited by extracellular La3+ ions but not by nifedipine, SK&F 96365, flunarizine, 2-aminoethyl diphenylborinate, or thapsigargin, suggesting that influx of Ca2+ into cells was not because of receptor signaling or opening of conventional calcium channels by cAMP. Compared with intact CyaA, a CyaA-AC- toxoid unable to generate cAMP promoted a faster, albeit transient, elevation of [Ca2+]i. This was not because of cell permeabilization by the CyaA hemolysin pores, because a mutant exhibiting a strongly enhanced pore-forming activity (CyaA-E509K/E516K), but unable to deliver the AC domain into cells, was also unable to elicit a [Ca2+]i increase. Further mutations interfering with AC translocation into cells, such as proline substitutions of glutamate residues 509 or 570 or deletion of the AC domain as such, reduced or ablated the [Ca2+]i-elevating capacity of CyaA. Moreover, structural alterations within the AC domain, because of insertion of various oligopeptides, differently modulated the kinetics and extent of Ca2+ influx elicited by the respective AC- toxoids. Hence, the translocating AC polypeptide itself appears to participate in formation of a novel type of membrane path for calcium ions, contributing to action of CyaA in an unexpected manner.     

An amphipathic alpha-helix including glutamates 509 and 516 is crucial for membrane translocation of adenylate cyclase toxin and modulates formation and cation selectivity of its membrane channels

The Bordetella pertussis adenylate cyclase toxin-hemolysin (ACT or CyaA) is a multifunctional protein. It forms small cation-selective channels in target cell and lipid bilayer membranes and it delivers into cell cytosol the amino-terminal adenylate cyclase (AC) domain, which catalyzes uncontrolled conversion of ATP to cAMP and causes cell intoxication. Here, we demonstrate that membrane translocation of the AC domain into cells is selectively dissociated from ACT membrane insertion and channel formation when a helix-breaking proline residue is substituted for glutamate 509 (Glu-509) within a predicted transmembrane amphipathic alpha-helix. Neutral substitutions of Glu-509 had little effect on toxin activities. In contrast, charge reversal by lysine substitutions of the Glu-509 or of the adjacent Glu-516 residue reduced the capacity of the toxin to translocate the AC domain across membrane and enhanced significantly its specific hemolytic activity and channel forming capacity in lipid bilayer membranes. Combination of the E509K and E516K mutations in a single molecule further exacerbated hemolytic and channel forming activity and ablated translocation of the AC domain into cells. The lysine substitutions strongly decreased the cation selectivity of the channels, indicating that Glu-509 and Glu-516 are located within or close to the membrane channel. These results suggest that the structure including glutamate residues 509 and 516 is critical for AC membrane translocation and channel forming activity of ACT.     

Calcium-dependent inactivation of the ATP-sensitive K+ channel of rat ventricular myocytes

Single-channel currents were recorded from ATP-sensitive K+ channels in inside-out membrane patches excised from isolated rat ventricular myocytes. Perfusion of the internal surface of excised membrane patches with solutions which contained between 5 and 100 microM free calcium caused the loss of K+ATP channel activity which was not reversed when the membranes were washed with Ca-free solution. K+ATP channel activity could be recovered by bathing the patches in Mg.ATP. The loss of K+ATP channel activity provoked by internal calcium was a process which occurred over a time scale of seconds. Channel closure evoked by internal ATP was essentially instantaneous. The speed of K+ATP channel inactivation increased with the concentration of calcium. Neither a phosphatase inhibitor (fluoride ions) nor a proteinase inhibitor (leupeptin) had any effect upon the loss of K+ channel activity stimulated by internal calcium.     

Involvement of calmodulin in the regulation of adenylate cyclase activity in guinea-pig enterocytes

The involvement of calmodulin as an activator of adenylate cyclase activity was examined in isolated guinea-pig enterocytes and in a membrane preparation. In enterocytes, which responded to prostaglandin E1, vasoactive intestinal peptide and cholera toxin with a significant increase in the rate of cAMP formation trifluoperazine, a calmodulin antagonist, completely inhibited cAMP formation. In a membrane preparation adenylate cyclase activity was stimulated 10-20-fold by the GTP analog, guanosine 5'-[beta-imido]5'-triphosphate (Gpp[NH]p). Prostaglandin E1 and vasoactive intestinal peptide enhanced cAMP formation in this system by 2-3- and 1.2-1.6-fold. respectively. Addition of 200 nM calmodulin to membranes, in which endogenous calmodulin was decreased from 1.4 microgram/mg protein to 0.5 microgram/mg protein by washing with buffer containing EGTA and EDTA, resulted in a 3-4-fold increase of adenylate cyclase activity. The absolute increment in adenylate cyclase activity caused by calmodulin (10-15 pmol cAMP/min per mg protein) was approximately the same in the absence or presence of Gpp[NH]p. The apparent Ka for Gpp[NH]p (6 . 10-7 M) was not significantly changed by the addition of calmodulin. Although endogenous calcium (approx. 10 microM) in the enzyme assay was adequate to affect stimulation by calmodulin, a maximal effect was observed at a calcium concentration of 100 microM. These findings indicate that a calmodulin-sensitive form of adenylate cyclase is present in guinea-pig enterocytes, and that stimulation of cAMP formation in the intestinal mucosa may involve a calmodulin-mediated mechanism.     

Effect of trifluoperazine on renal epithelioid Madin-Darby canine kidney cells

Following exposure to a number of hormones, the cell membrane in Madin-Darby Canine Kidney (MDCK) cells is hyperpolarized by increase of intracellular calcium activity. The present study has been performed to elucidate the possible role of calmodulin in the regulation of intracellular calcium activity and cell membrane potential. To this end trifluoperazine has been added during continuous recording of cell membrane potential or intracellular calcium. Trifluoperazine leads to a transient increase of intracellular calcium as well as a sustained hyperpolarization of the cell membrane by activation of calcium sensitive K+ channels. Half-maximal effects are observed between 1 and 10 mumol/L trifluoperazine. A further calmodulin antagonist, chlorpromazine, (50 mumol/L), similarly hyperpolarizes the cell membrane. The effects of trifluoperazine are virtually abolished in the absence of extracellular calcium. Pretreatment of the cells with either pertussis toxin or phorbol-ester TPA does not interfere with the hyperpolarizing effect of trifluoperazine. In conclusion, calmodulin is apparently involved in the regulation of calcium transfer across the cell membrane but not in the stimulation of K+ channels by intracellular calcium.     

Effect of thallium ions on gramicidin-induced conductance of muscle fiber membranes

Conductance of single fibres from m. ileofibularis of Rana esculenta was studied in isotonic K2SO4 solution under constant current conditions using the double sucrose gap method. It was found that Tl+ (at concentrations 5, 10, and 20 mM) blocked K+ currents in the gramicidin channel. The decrease of K+ conductance caused by Tl+ was associated with the changes of the membrane potential. Both the decrease of K+ conductance and value of permeability ratio (PTl/PK) found from the membrane potential changes depended on Tl+ concentration in the bathing solution. No effect of Tl+ on the potassium channels was registered in the absence of gramicidin channels. The Tl+ block described here proves the existence of Tl+ ion binding within gramicidin channels of the muscle membrane and interactions among ions in the channels.     

Imperatoxin A (IpTx(a)) from Pandinus imperator stimulates [(3)H]ryanodine binding to RyR3 channels.

The effect of imperatoxin A (IpTx(a)) on the ryanodine receptor type 3 (RyR3) was studied. IpTx(a) stimulates [(3)H]ryanodine binding to RyR3-containing microsomes, but this effect requires toxin concentrations higher than those required to stimulate RyR1 channels. The effect of IpTx(a) on RyR3 channels was observed at calcium concentrations in the range 0.1 microM to 10 mM. By contrast, RyR2 channels were not significantly affected by IpTx(a) in the same calcium ranges. Single channel current measurements indicated that IpTx(a) induced subconductance state in RyR3 channels that was similar to those observed with RyR1 and RyR2 channels. These results indicate that IpTx(a) is capable of inducing similar subconductance states in all three RyR isoforms, while stimulation of [(3)H]ryanodine binding by this toxin results in isoform-specific responses, with RyR1 being the most sensitive channel, RyR3 displaying an intermediate response and RyR2 the least responsive ones.     

Structural basis for the interaction of Bordetella pertussis adenylyl cyclase toxin with calmodulin

CyaA is crucial for colonization by Bordetella pertussis, the etiologic agent of whooping cough. Here we report crystal structures of the adenylyl cyclase domain (ACD) of CyaA with the C-terminal domain of calmodulin. Four discrete regions of CyaA bind calcium-loaded calmodulin with a large buried contact surface. Of those, a tryptophan residue (W242) at an alpha-helix of CyaA makes extensive contacts with the calcium-induced, hydrophobic pocket of calmodulin. Mutagenic analyses show that all four regions of CyaA contribute to calmodulin binding and the calmodulin-induced conformational change of CyaA is crucial for catalytic activation. A crystal structure of CyaA-calmodulin with adefovir diphosphate, the metabolite of an approved antiviral drug, reveals the location of catalytic site of CyaA and how adefovir diphosphate tightly binds CyaA. The ACD of CyaA shares a similar structure and mechanism of activation with anthrax edema factor (EF). However, the interactions of CyaA with calmodulin completely diverge from those of EF. This provides molecular details of how two structurally homologous bacterial toxins evolved divergently to bind calmodulin, an evolutionarily conserved calcium sensor.     

Mechanism of iberiotoxin block of the large-conductance calcium-activated potassium channel from bovine aortic smooth muscle.

The interaction of iberiotoxin (IbTX) with the large-conductance calcium-activated potassium (maxi-K) channel was examined by measuring single-channel currents from maxi-K channels incorporated into planar lipid bilayers. Addition of nanomolar concentrations of IbTX to the external side of the channel produced long nonconducting silent periods, which were interrupted by periods of normal channel activity. The distributions of durations of blocked and unblocked periods were both described by single exponentials. The mean duration of the unblocked periods decreased in proportion with the external concentration of IbTX, while the mean duration of the blocked periods was not affected. These results suggest that IbTX blocks the maxi-K channel through a simple bimolecular binding reaction where the silent periods represent times when a single toxin molecule is bound to the channel. In symmetric solutions of 150 mM KCl, with a membrane potential of 40 mV, the mean duration of the blocked periods produced by IbTX was 840 s, and the association rate was 1.3 x 10(6) M-1 s-1, yielding an equilibrium dissociation constant of about 1 nM. Raising the internal potassium concentration increased the dissociation rate constant of IbTX in a manner which was well described by a saturable binding function for potassium. External tetraethylammonium ion increased the average duration of the unblocked periods without affecting the blocked periods, suggesting that tetraethylammonium and IbTX compete for the same site near the conductance pathway of the channel. Increasing the external concentration of monovalent cations from 25 to 300 mM with either potassium or sodium decreased the rate of binding of IbTX to the channel by approximately 24-fold, with little effect on the rate of toxin dissociation.(ABSTRACT TRUNCATED AT 250 WORDS)     

A calcium-activated cation-selective channel in rat cultured Schwann cells

Calcium-activated channels, in the plasma membrane of rat cultured Schwann cells were studied in isolated 'inside-out' membrane patches. With identical (150 mM NaCl) solutions on either side of the membrane, a single channel conductance of 32 pS was calculated for inward current; the conductance was somewhat less for outward current. The channel is about equally permeable to sodium and potassium ions, but is not detectably permeable to either chloride or calcium. Under our experimental conditions the channel is activated by high (more than 10(-4) M) concentrations of calcium and is sensitive to voltage, channel activity increasing with membrane depolarization.     

Interaction of calmodulin with the red cell and its membrane skeleton and with spectrin

The binding of calmodulin to red cell membrane cytoskeletons and to purified spectrin from red cells and bovine brain spectrin (fodrin) has been examined. Under physiological solvent conditions binding can be measured by ultracentrifugal pelleting assays. The membrane cytoskeletons contained a single class of binding sites, with a concentration similar to that of spectrin dimers and an association constant of 1.5 X 10(5) M-1. Binding is calcium dependent and is suppressed by the calmodulin inhibitor trifluoperazine. The binding showed a marked dependence on ionic strength, with a maximum at 0.05 M, and a steep dependence on pH, with a maximum at pH 6.5. It was unaffected by 5 mM magnesium. An azidocalmodulin derivative, under the conditions of our experiments, did not label the spectrin-containing complex, although it could be used to demonstrate binding to fodrin. Binding of calmodulin to spectrin tetramers and fodrin in solution could be demonstrated by a pelleting assay after addition of F-actin. Calculations (which are necessarily rough) suggest that at the free calcium concentration prevailing in a normal red cell about 1 in 20 of the calmodulin binding sites in spectrin will be occupied; this proportion will rise rapidly with increasing intracellular calcium. To determine whether inhibition of calmodulin binding to red cell proteins disturbs the control of cell shape, as has been suggested, calcium ions were removed from the cell by addition of an ionophore and of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to the external medium. This did not affect the discoid shape. Trifluoperazine still induced stomatocytosis, exactly as in untreated cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

A calmodulin dependent Ca2+-activated K+ channel in the adipocyte plasma membrane

Increased membrane permeability (conductance) that is specific for K+ and directly activated by Ca2+ ions, has been identified in isolated adipocyte plasma membranes using the K+ analogue, 86Rb+. Activation of these K+ conductance pathways (channels) by free Ca2+ was concentration dependent with a half-maximal effect occurring at 32 +/- 4 nM free Ca2+ (n = 7). Addition of calmodulin further enhanced the Ca2+ activating effect on 86Rb+ uptake (K+ channel activity). Ca2+-dependent 86Rb+ uptake was inhibited by tetraethylammonium ion and low pH. It is concluded that the adipocyte plasma membrane possesses K+ channels that are activated by Ca2+ and amplified by calmodulin.     

Cyclic nucleotide-gated ion channels in sensory transduction

Cyclic nucleotide-gated (CNG) channels, directly activated by the binding of cyclic nucleotides, were first discovered in retinal rods, cones and olfactory sensory neurons. In the visual and olfactory systems, CNG channels mediate sensory transduction by conducting cationic currents carried primarily by sodium and calcium ions. In olfactory transduction, calcium in combination with calmodulin exerts a negative feedback on CNG channels that is the main molecular mechanism responsible for fast adaptation in olfactory sensory neurons. Six mammalian CNG channel genes are known and some human visual disorders are caused by mutations in retinal rod or cone CNG genes.     

Early response of cultured lepidopteran cells to exposure to delta-endotoxin from Bacillus thuringiensis: involvement of calcium and anionic channels.

The role of ion channels in the initial steps following exposure of SF-9 lepidopteran insect cells in culture to the delta-endotoxin CryIC from the insecticidal bacterium Bacillus thuringiensis was investigated using single ionic channel measurements and microspectrofluorescence of the calcium-sensitive probe fura-2. It was found that: (1) the toxin triggers an immediate rise in intracellular calcium; (2) the surge is due to calcium entering the cells via calcium channels; (3) the toxin recruits or introduces anionic channels in the cell's plasma membrane in a time-dependent manner. These channels, not seen in the absence of the toxin, are induced by toxin exposure to either side of the cell membrane. They have a conductance of 26 picosiemens (pS) and are mainly permeable to chloride. This study provides the first evidence of the primary role of calcium and chloride ions in the action of delta-endotoxin on cultured insect cells.     

Identification of a region that assists membrane insertion and translocation of the catalytic domain of Bordetella pertussis CyaA toxin

The adenylate cyclase (CyaA) toxin, one of the virulence factors secreted by Bordetella pertussis, the pathogenic bacteria responsible for whooping cough, plays a critical role in the early stages of respiratory tract colonization by this bacterium. The CyaA toxin is able to invade eukaryotic cells by translocating its N-terminal catalytic domain directly across the plasma membrane of the target cells, where, activated by endogenous calmodulin, it produces supraphysiological levels of cAMP. How the catalytic domain is transferred from the hydrophilic extracellular medium into the hydrophobic environment of the membrane and then to the cell cytoplasm remains an unsolved question. In this report, we have characterized the membrane-interacting properties of the CyaA catalytic domain. We showed that a protein covering the catalytic domain (AC384, encompassing residues 1-384 of CyaA) displayed no membrane association propensity. However, a longer polypeptide (AC489), encompassing residues 1-489 of CyaA, exhibited the intrinsic property to bind to membranes and to induce lipid bilayer destabilization. We further showed that deletion of residues 375-485 within CyaA totally abrogated the toxin's ability to increase intracellular cAMP in target cells. These results indicate that, whereas the calmodulin dependent enzymatic domain is restricted to the amino-terminal residues 1-384 of CyaA, the membrane-interacting, translocation-competent domain extends up to residue 489. This thus suggests an important role of the region adjacent to the catalytic domain of CyaA in promoting its interaction with and its translocation across the plasma membrane of target cells.     

Maitotoxin-Induced Intracellular Calcium Rise in PC 12 Cells: Involvement of Dihydropyridine-Sensitive and ω-Conotoxin-Sensitive Calcium Channels and Phosphoinositide Breakdown

The biological activities of maitotoxin are strictly dependent on the extracellular calcium concentration and are always associated with an increase of the free cytosolic calcium level. We tested the effects of voltage-sensitive calcium channel blockers (nicardipine and omega-conotoxin) on maitotoxin-induced intracellular calcium increase, membrane depolarization, and inositol phosphate production in PC12 cells. Maitotoxin dose dependently increased the cytosolic calcium level, as measured by the fluorescent probe fura 2. This effect disappeared in a calcium-free medium; it was still observed in the absence of extracellular sodium and was enhanced by the dihydropyridine calcium agonist Bay K 8644. Nicardipine inhibited the effect of maitotoxin on intracellular calcium concentration in a dose-dependent manner. The maitotoxin-induced calcium rise was also reduced by pretreating cells with omega-conotoxin. Pretreatment of cells with maitotoxin did not modify 125I-omega-conotoxin and [3H]PN 200-110 binding to PC12 membranes. Nicardipine and omega-conotoxin inhibition of maitotoxin-evoked calcium increase was reduced by pertussis toxin pretreatment. Maitotoxin caused a substantial membrane depolarization of PC12 cells as assessed by the fluorescent dye bisoxonol. This effect was reduced by pretreating the cells with either nicardipine or omega-conotoxin and was almost completely abolished by the simultaneous pretreatment with both calcium antagonists. Maitotoxin stimulated inositol phosphate production in a dose-dependent manner. This effect was reduced by pretreating the cells with 1 microM nicardipine and was completely abolished in a calcium-free EGTA-containing medium. The findings on maitotoxin-induced cytosolic calcium rise and membrane depolarization suggest that maitotoxin exerts its action primarily through the activation of voltage-sensitive calcium channels, the increase of inositol phosphate production likely being an effect dependent on calcium influx. The ability of nicardipine and omega-conotoxin to inhibit the effect of maitotoxin on both calcium homeostasis and membrane potential suggests that L- and N-type calcium channel activation is responsible for the influx of calcium following exposure to maitotoxin, and not that a depolarization of unknown nature causes the opening of calcium channels.     

Inhibition of cyclic nucleotide phosphodiesterase and calcineurin by spermine, a calcium-independent calmodulin antagonist

Spermine binding to calmodulin and its effects on two calmodulin-dependent enzymes were studied. Spermine bound to dansylated calmodulin with an apparent Ki of 0.7 mM, and to native calmodulin with a Kd of 1.1 mM in equilibrium dialysis experiments. Its binding was found to be independent of calcium. Spermine inhibited calmodulin-activated cyclic nucleotide phosphodiesterase noncompetitively with respect to calcium (Ki = 1.1 mM). Calmodulin activation of calcineurin was inhibited at similar concentrations (Ki = 1.2 mM). Spermine had little effect on basal phosphodiesterase activity or nickel-activated calcineurin activity. Inhibition of both enzymes correlated well with spermine binding to dansylcalmodulin. These findings suggest that spermine might modulate calcium-dependent events in the cell by inactivation of calmodulin via a novel calcium-independent mechanism.