Possible involvement of calmodulin in the regulation of ATPase activity in guard cells

Calcium ions play an important role in the regulation of stomatal movement and the mechanism underlying this action is yet to be determined. It is suggested that guard cell plasma membrane ATPase is a target for calcium action and that this effect is mediated by calmodulin. In this study, the effects of calcium and two calmodulin antagonists on ATPase activity in a crude homogenate of Commelina communis L. guard cell protoplasts were examined. The homogenate contained Mg²⁺-dependent, K⁺-simulated ATPase activity, which was inhibited by CaCl² while stimulated by the calmodulin antagonists, compound 48/80 and chlorpromazine. The calmodulin antagonists partially reversed the inhibitory effect of calcium ions. The results support the possibility of calmodulin involvement in the regulation of guard cell ATPase activity by calcium ions.     

Metal-ATP complexes as substrates and free metal ions as activators of the red cell calcium pump

The plasma membrane calcium pump in most mammalian cells is the basic mechanism for assuring a low cytoplasmic calcium concentration. In inside-out human red cell membrane vesicles /IOVs/ the substrate and metal specificity as well as the intracellular protein /calmodulin/ regulation of the ATP-dependent active calcium transport can be investigated $\text{in}$$\text{situ}$. In this paper we demonstrate that Me²⁺. ATP^4? /in the following MeATP/ complexes, including MgATP, MnATP, CoATP, FeATP, and NiATP, can serve as substrates for the calcium pump in IOVs. Calcium pumping is activated by the above metals, while Sr, Ba, Cu, Cd ions or the trivalent cations are ineffective in this respect. Calmodulin-stimulation of the calcium transport is present independent of the metal ions used for the activation of the pump. Based on kinetic studies we suggest that divalent metal ions interact with the red cell calcium pump at four different sites: 1./ MeATP complex is the true substrate of the pump; 2./ Ca or Sr ions activate the system by binding to the transport site/s/ and other metal ions competitively inhibit this binding; 3./ the presence of free divalent metal ions /Mg, Mn, Co, Fe, or Ni, but not Ca, Sr, Ba/ is required for activating calcium translocation; 4./ interaction with a Ca — calmodulin complex specifically stimulates calcium pumping.     

Plant Ca<Superscript>2+</Superscript>-ATPases

Plant calcium pumps, similarly to animal Ca²⁺ pumps, belong to the superfamily of P-type ATPase comprising also the plasma membrane H⁺-ATPase of fungi and plants, Na⁺/K⁺ ATPase of animals and H⁺/K⁺ ATPase of mammalian gastric mucosa. According to their sensitivity to calmodulin the plant Ca²⁺-ATPases have been divided into two subgroups: type IIA (homologues of animal SERCA) and type IIB (homologues of animal PMCA). Regardless of the similarities in a protein sequence, the plant Ca²⁺ pumps differ from those in animals in their cellular localization, structure and sensitivity to inhibitors. Genomic investigations revealed multiplicity of plant Ca²⁺-ATPases; they are present not only in the plasma membranes and ER but also in membranes of most of the cell compartments, such as vacuole, plastids, nucleus or Golgi apparatus. Studies using yeast mutants made possible the functional and biochemical characterization of individual plant Ca²⁺-ATMPases. Plant calcium pumps play an essential role in signal transduction pathways, they are responsible for the regulation of [Ca²⁺] in both cytoplasm and endomembrane compartments. These Ca²⁺-ATPases appear to be involved in plant adaptation to stress conditions, like salinity, chilling or anoxia.     

Molecular properties of the red cell calcium pump: I. Effects of calmodulin,proteolytic digestion and drugs on the kinetics of active calcium uptake in inside-out red cell membrane vesicles

In calmodulin-stripped inside-out human red cell membrane vesicles /IOV/ ATP + Mg²⁺-dependent active calcium uptake is stimulated by the addition of calmodulin. Calmodulin increases the maximum calcium transport rate /V_max/, decreases K_Ca, and does not affect K_ATP of calcium uptake. The action of both membrane bound and external calmodulin is competitively inhibited by phenothiazines. Drugs reacting with SH groups of proteins reversibly inhibit calcium pumping by decreasing V_max and not affecting K_Ca and K_ATP. The relative magnitude of calmodulin stimulation of calcium transport is unaltered by SH reagents.Mild proteolytic digestion of IOVs stimulates active calcium uptake and mimics the effects of calmodulin on the kinetic parameters — that is converts the system to a “high calcium-affinity” state. Proteolysis eliminates calcium-dependent calmodulin binding to IOV membranes and any further stimulation of calcium uptake by calmodulin. Based on these results the presence of a calmodulin-binding regulatory subunit of the red cell calcium pump at the internal membrane surface is postulated.     

Interaction of calmodulin with phospholamban and caldesmon: comparative studies by 1H-NMR spectroscopy.

In order to identify comparative aspects of the interaction of calmodulin with its target proteins, proton magnetic-resonance studies of complex formation between calmodulin and defined segments of phospholamban and caldesmon have been undertaken. Residues 3-15 in the cytoplasmic region of phospholamban, an integral membrane protein of cardiac sarcoplasmic reticulum believed to regulate the calcium pumping ATPase, are shown to contribute to interaction with calmodulin. Using wheat germ calmodulin specifically modified with a spin-label to provide the spectral means for spatial localisation, these residues of phospholamban were correlated with binding in the vicinity of the probe attached to Cys-27 in the N-terminal domain of calmodulin. This interaction, relevant to the mechanism of calmodulin-dependent phosphorylation of phospholamban that relieves its inhibitory influence on the calcium pump, provides a useful model system for comparative study of the properties of calmodulin-binding domains. We contrast here a calmodulin-binding segment in the C-terminal region of caldesmon localised by 1H-NMR study of the interface(s) between the two proteins. These observations are discussed in the context of other calmodulin-binding sequences.     

Calmodulin-stimulated calcium pumping ATPases located at higher plant intracellular membranes: a significant divergence from other eukaryotes?

The plasma membrane (PM) of all eukaryotes so far investigated contains a P-type Ca²⁺-pumping ATPase responsible for maintaining low cytosolic free calcium concentrations. In animal cells this has been shown to be a type of Ca²⁺-pump which is directly stimulated by binding the calcium-dependent regulator protein calmodulin. These PM Ca²⁺-pumps have been named 'PM-type' as they appear to be exclusively located at the PM and not in intracellular membrane (IM) fractions. Recent progress on higher plant cells reveals that they possess calmodulin-stimulated Ca²⁺-pumps of the 'PM-type'. However, these calmodulin-stimulated Ca²⁺-pumps appear to be located not only at the PM but also in intracellular membranes, probably the endoplasmic reticulum (ER). The evidence is also convincing that these IM-located Ca²⁺-pumps are directly stimulated by calmodulin (possess a calmodulin-binding region) and are true 'PM-type' Ca²⁺-pumps. This appears to represent a marked divergence between plant and animal cell Ca²⁺-pumps. Recently, molecular cloning has revealed that plant cells also contain a Ca²⁺-pump which is not directly stimulated by calmodulin and which strongly resembles the mammalian ER/SR type of Ca²⁺-pump. The significance of these findings for plant cell function is discussed.     

The liver plasma membrane Ca2+ pump: hormonal sensitivity

The liver plasma membrane Ca2+ pump is supposed to extrude cytosolic calcium out of the cell. This system has now been well defined on the basis of its plasma membrane origin, its high affinity Ca2+ -stimulated ATPase activity, its Ca2+ transport activity, its phosphorylated intermediate. The liver calcium pump appears to be a target of hormonal action since it has been shown that glucagon and calcium mobilizing hormones namely alpha 1-adrenergic agonists, vasopressin, angiotensin II inhibit this system. The present review details the mechanism of calcium pump inhibition by glucagon and points out its difference from the inhibition process induced by calcium mobilizing hormones. We conclude that the inhibitory action of the Ca2+ mobilizing hormones and glucagon on the liver plasma membrane Ca2+ pump might play a key role in the actions of these hormones by prolonging the elevation in cytosolic free Ca2+.     

Cell death suppressor Arabidopsis bax inhibitor-1 is associated with calmodulin binding and ion homeostasis

Cell death suppressor Bax inhibitor-1 (BI-1), an endoplasmic reticulum membrane protein, exists in a wide range of organisms. The split-ubiquitin system, overlay assay, and bimolecular fluorescence complementation analysis demonstrated that Arabidopsis (Arabidopsis thaliana) BI-1 (AtBI-1) interacted with calmodulin in yeast (Saccharomyces cerevisiae) and in plant cells. Furthermore, AtBI-1 failed to rescue yeast mutants lacking Ca2+ ATPase (Pmr1 or Spf1) from Bax-induced cell death. Pmr1 and Spf1, p-type ATPases localized at the inner membrane, are believed to be involved in transmembrane movement of calcium ions in yeast. Thus, the presence of intact Ca2+ ATPases was essential for AtBI-1-mediated cell death suppression in yeast. To investigate the effect of AtBI-1 on calcium homeostasis, we evaluated sensitivity against cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase in AtBI-1-overexpressing or knock-down transgenic Arabidopsis plants. These plants demonstrated altered CPA or ion stress sensitivity. Furthermore, AtBI-1-overexpressing cells demonstrated an attenuated rise in cytosolic calcium following CPA or H2O2 treatment, suggesting that AtBI-1 affects ion homeostasis in plant cell death regulation.     

ATPase activity and Ca2+ transport by reconstituted tryptic fragments of the Ca2+ pump of the erythrocyte plasma membrane

The purified Ca2+ ATPase of the erythrocyte plasma membrane has been submitted to controlled trypsin proteolysis under conditions that favor either its (putative) E1 or E2 configurations. The former configuration has been forced by treating the enzyme with Ca2+-saturated calmodulin, the latter with vanadate and Mg2+. The E1 conformation leads to the accumulation of a polypeptide of Mr 85 KDa which still binds calmodulin, the E2 conformation to the accumulation of one of Mr 81 KDa which does not. Both fragments arise from the hydrolysis of a transient 90 KDa product which has Ca2+-calmodulin dependent ATPase activity, and which retains the ability to pump Ca2+ in reconstituted liposomes. Highly enriched preparations of the 85 and 81 KDa fragments have been obtained and reconstituted into liposomes. The former has limited ATPase and Ca2+ transport ability and is not stimulated by calmodulin. The latter has much higher ATPase and Ca2+ transport activity. It is proposed that the Ca2+ pumping ATPase of erythrocytes plasma membrane contains a 9 KDa domain which is essential for the interaction of the enzyme with calmodulin and for the full expression of the hydrolytic and transport activity. This putative 9 KDa sequence contains a 4 KDa "inhibitory" domain which limits the activity of the ATPase. In the presence of this 4 KDa sequence, i.e., when the enzyme is degraded to the 85 KDa product, calmodulin can still be bound, but no longer stimulates ATPase and Ca2+ transport.     

Calcium regulation of calmodulin binding to and dissociation from the myo1c regulatory domain

Myo1c is an unconventional myosin involved in cell signaling and membrane dynamics. Calcium binding to the regulatory-domain-associated calmodulin affects myo1c motor properties, but the kinetic details of this regulation are not fully understood. We performed actin gliding assays, ATPase measurements, fluorescence spectroscopy, and stopped-flow kinetics to determine the biochemical parameters that define the calmodulin-regulatory-domain interaction. We found calcium moderately increases the actin-activated ATPase activity and completely inhibits actin gliding. Addition of exogenous calmodulin in the presence of calcium fully restores the actin gliding rate. A fluorescently labeled calmodulin mutant (N111C) binds to recombinant peptides containing the myo1c IQ motifs at a diffusion-limited rate in the presence and absence of calcium. Measurements of calmodulin dissociation from the IQ motifs in the absence of calcium show that the calmodulin bound to the IQ motif adjacent to the motor domain (IQ1) has the slowest dissociation rate (0.0007 s-1), and the IQ motif adjacent to the tail domain (IQ3) has the fastest dissociation rate (0.5 s-1). When the complex is equilibrated with calcium, calmodulin dissociates most rapidly from IQ1 (60 s-1). However, this increased rate of dissociation is limited by a slow calcium-induced conformational change (3 s-1). Fluorescence anisotropy decay of fluorescently labeled N111C bound to myo1c did not depend appreciably on Ca2+. Our data suggest that the calmodulin bound to the IQ motif adjacent to the motor domain is rapidly exchangeable in the presence of calcium and is responsible for regulation of myo1c ATPase and motile activity.     

The effects of felodipine and bepridil on calcium-stimulated calmodulin binding and calcium pumping ATPase of cardiac sarcolemma before and after removal of endogenous calmodulin

Summary The Ca²⁺ channel blockers felodipine and bepridil are known to affect selectively functions of calmodulin. We studied their effects on calmodulin binding and ATPase activities of calmodulin-containing and calmodulin-depleted rabbit heart sarcolemma. Both drugs as well as the specific anti-calmodulin drug calmidazolium at a concentration of 50 µM, inhibited the Ca²⁺-stimulated calmodulin binding to calmodulin-depleted sarcolemma. Within the concentration range of 3 to 100 µM all three drugs also progressively inhibited Ca²⁺ pumping ATPase in calmodulin containing sarcolemma, although the enzyme was assayed at saturating Ca²⁺ (100 µM). The inhibitory potency of calmidazolium and bepridil, but not that of felodipine, increased when the membrane protein concentration in the ATPase assay was lowered. At low membrane protein concentration 30 µM calmidazolium completely blocked calmodulin-dependent Ca²⁺ pumping ATPase, whereas the inhibition caused by 30 µM felodipine or bepridil remained partially. A similar inhibition pattern of the drugs was found in the calmodulin binding experiments. Within a concentration range of 3 to 30 µM, all three drugs had negligible effects on the basal Ca²⁺ pumping ATPase which was measured in calmodulin-depleted sarcolemma. In conclusion, the characteristics of the anti-calmodulin action of felodipine on the rabbit heart sarcolemmal Ca²⁺ pumping ATPase are not different from those of bepridil. Both drugs may inhibit the enzyme by interference with the Ca²⁺-stimulated binding of calmodulin.Abbreviations Ca²⁺ pumping ATPase Ca²⁺ stimulated Mg²⁺-dependent ATP hydrolyzing activity - Na⁺ pumping ATPase Na⁺-stimulated K⁺- and Mg²⁺-dependent ATP hydrolyzing activity - Tris-maleate tris (hydroxymethyl) aminomethane hydrogen maleate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Mes 2-(N-morpholino) ethane sulfonic acid and Egta, ethylene glycol bis (p-amino ethylether)-N,N,N,N tetraacetic acid     

The partial reactions in the catalytic cycle of the calcium-dependent adenosine triphosphatase purified from erythrocyte membranes

A preparation of purified erythrocyte membrane ATPase whose activation by Ca2+ is or is not dependent on calmodulin depending on the enzyme dilution was used in the low dilution state for these studies. In appropriate conditions, the purified ATPase in the absence of calmodulin exhibited a Ca2+ concentration dependence identical to that of the native enzyme in the erythrocyte membrane ghost in the presence of calmodulin. Accordingly, an apparent Kd approximately equal to 1 X 10(-7) M was derived for cooperative calcium binding to the activating and transport sites of the nonphosphorylated enzyme. The kinetics of enzyme phosphorylation in the transient state following addition of ATP to enzyme activated with calcium were then resolved by rapid kinetic methods, demonstrating directly that phosphoenzyme formation precedes Pi production, consistent with the phosphoenzyme role as an intermediate in the catalytic cycle. Titration of a low affinity site (Kd approximately equal to 2 X 10(-3) M) with calcium produced inhibition of phosphoenzyme cleavage and favored reversal of the catalytic cycle, indicating that calcium dissociation from the transport sites precedes hydrolytic cleavage of the phosphoenzyme. The two different calcium dissociation constants of the nonphosphorylated and phosphorylated enzyme demonstrate that a phosphorylation-induced reduction of calcium affinity is the basic coupling mechanism of catalysis and active transport, with an energy expenditure of approximately 6 kcal/mol of calcium in standard conditions. From the kinetic point of view, a rate-limiting step is identified with the slow dissociation of calcium from the phosphoenzyme; another relatively slow step following hydrolytic cleavage and preceding recycling of the enzyme is suggested by the occurrence of a presteady state phosphoenzyme overshoot.     

The Membrane-Bound Forms of the Mitochondrial ATPase of Turnip (Brassica napus L.) and Mung Bean (Phaseolus aureus): Characterization and Investigation of Factors Controlling Activity

Isolated intact plant mitochondria, including those from turnipand mung bean, show low endogenous Mg²⁺-ATPase activity and,unlike mammalian mitochondria, lack significant uncoupler-stimulatedATPase activity. In contrast, the rates of respiration-drivenATP synthesis are comparable to those in mammalian mitochondria,suggesting the presence of an ATPase inhibitor. Disruption ofintact turnip mitochondria only results in limited increasesin ATPase activity, indicating that a permeability barrier toATP transport is not primarily responsible for the low endogenousactivity. The ATPase activity of turnip mitochondria and membraneparticles can be increased up to 50-fold when assayed underoptimum conditions. Time-dependent increases in activity inducedby ageing, exposure to salts and trypsin treatment, are allconsistent with an inhibitor protein being responsible for thelow endogenous activity and lack of uncoupler-stimulation. TheATPase activity of particles under optimum conditions and afterageing is sufficient to account for the rates of ATP synthesis.After activation, turnip mitochondrial ATPase activity is similarto the mammalian enzyme in inhibitor sensitivity, pH optimum,bivalent cation requirement, and sensitivity to ‘activatinganions’. In mung bean mitochondria, a permeability barrierto ATP is only partly responsible for the low endogenous ATPaseactivity, together with the inhibitory factor. On the basisof variation in the relative Ca²⁺ and Mg²⁺ -ATPase activitiesafter various treatments, a Ca²⁺-regulatory site which affectsATPase activity is proposed to exist in the F₁ATPase complex. Key words: Plant mitochondrial ATPase, calcium/magnesium -ATPase, inhibitor+ nucleotide specificity, cation/anion effects     

Control of erythrocyte shape by calmodulin

Erythrocytes are deformable cells whose shapes can be altered by treatments with a variety of drugs. The forms the erythrocyte may assume vary continuously from the spiny "echinocytes" or crenated cells at one extreme to highly folded and dented "cupped" cells at the other extreme. Examination of 39 compounds for cup-forming activity revealed a remarkable correlation between their ability to form cupped cells and their inhibitory activity against the calcium regulatory protein, calmodulin. Calmodulin is known to interact with several erythrocyte proteins including spectrin, spectrin kinase, and the Ca++ ATPase calcium pump of the membrane. These proteins regulate the form of the cytoskeleton as well as intracellular calcium and ATP levels. It is proposed that calmodulin is required to maintain normal erythrocyte morphology and that in the presence of calmodulin inhibitors, the cell assumes a cupped shape.     

Calcium depletion and calmodulin inhibition affect the import of nuclear-encoded proteins into plant mitochondria

Many metabolic processes essential for plant viability take place in mitochondria. Therefore, mitochondrial function has to be carefully balanced in accordance with the developmental stage and metabolic requirements of the cell. One way to adapt organellar function is the alteration of protein composition. Since most mitochondrial proteins are nuclear encoded, fine-tuning of mitochondrial protein content could be achieved by the regulation of protein translocation. Here we present evidence that the import of nuclear-encoded mitochondrial proteins into plant mitochondria is influenced by calcium and calmodulin. In pea mitochondria, the calmodulin inhibitor ophiobolin A as well as the calcium ionophores A23187 and ionomycin inhibit translocation of nuclear-encoded proteins in a concentration-dependent manner, an effect that can be countered by the addition of external calmodulin or calcium, respectively. Inhibition was observed exclusively for proteins translocating into or across the inner membrane but not for proteins residing in the outer membrane or the intermembrane space. Ophiobolin A and the calcium ionophores further inhibit translocation into mitochondria with disrupted outer membranes, but their effect is not mediated via a change in the membrane potential across the inner mitochondrial membrane. Together, our results suggest that calcium/calmodulin influences the import of a subset of mitochondrial proteins at the inner membrane. Interestingly, we could not observe any influence of ophiobolin A or the calcium ionophores on protein translocation into mitochondria of yeast, indicating that the effect of calcium/calmodulin on mitochondrial protein import might be a plant-specific trait.     

Activation of the Ca2+-ATPase of human erythrocyte membrane by an endogenous Ca2+-dependent neutral protease

Limited proteolysis of the plasma membrane calcium transport ATPase (Ca2+-ATPase) from human erythrocytes by trypsin produces a calmodulin-like activation of its ATP hydrolytic activity and abolishes its calmodulin sensitivity. We now demonstrate a similar kind of activation of the human erythrocyte membrane Ca2+-ATPase by calpain (calcium-dependent neutral protease) isolated from the human red cell cytosol. Upon incubation of red blood cell membranes with purified calpain in the presence of Ca2+ the membrane-bound Ca2+-ATPase activity was increased and its sensitivity to calmodulin was lost. In contrast to the action of other proteases tested, proteolysis by calpain favors activation over inactivation of the Ca2+-ATPase activity, except at calpain concentrations more than 2 orders of magnitude higher. Exogenous calmodulin protects the Ca2+-ATPase against calpain-mediated activation at concentrations which also activate the Ca2+-ATPase activity. Calcium-dependent proteolytic modification of the Ca2+-ATPase could provide a mechanism for the irreversible activation of the membrane-bound enzyme.     

Calmodulin Stimulation of Ca2+-Dependent ATP Hydrolysis and ATP-Dependent Ca2+ Transport in Synaptic Membranes

We report here characterization of calmodulin-stimulated Ca2+ transport activities in synaptic plasma membranes (SPM). The calcium transport activity consists of a Ca2+-stimulated, Mg2+-dependent ATP hydrolysis coupled with ATP-dependent Ca2+ uptake into membraneous sacs on the cytosolic face of the synaptosomal membrane. These transport activities have been found in synaptosomal subfractions to be located primarily in SPM-1 and SPM-2. Both Ca2+-ATPase and ATP-dependent Ca2+ uptake require calmodulin for maximal activity (KCm for ATPase = 60 nM; KCm for uptake = 50 nM). In the reconstituted membrane system, KCa was found to be 0.8 microM for Ca2+-ATPase and 0.4 microM for Ca2+ uptake. These results demonstrate for the first time the calmodulin requirements for the Ca2+ pump in SPM when Ca2+ ATPase and Ca2+ uptake are assayed under functionally coupled conditions. They suggest that calmodulin association with the membrane calcium pump is regulated by the level of free Ca2+ in the cytoplasm. The activation by calmodulin, in turn, regulates the cytosolic Ca2+ levels in a feedback process. These studies expand the calmodulin hypothesis of synaptic transmission to include activation of a high-affinity Ca2+ + Mg2+ ATPase as a regulator for cytosolic Ca2+.     

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.     

Phenothiazines and related compounds disrupt mitochondrial energy production by a calmodulin-independent reaction

Phenothiazines and related compounds bind to mitochondrial membranes in approximate proportion to their affinities for calmodulin. Penfluridol (16 microM), pimozide (20 microM), or trifluoperazine (66 microM) completely inhibit ADP-stimulated respiration in isolated rat liver mitochondria, but exert no effect on either uncoupler- or Ca2+-stimulated respiration. The inhibition of ADP-stimulated respiration results from inhibition of the oligomycin-sensitive ATPase. Inhibition of the ATPase does not involve interaction of phenothiazine with calmodulin. The addition of calmodulin with or without calcium to mitochondrial inner membrane preparations has no effect on ATPase activity. The addition of EGTA and the ionophore A23187 prior to the addition of phenothiazine does not prevent the phenothiazine-induced inhibiton of the ATPase. Measurements of inner membrane calmodulin content by gel electrophoresis or cyclic nucleotide phosphodiesterase activation are negative. Despite the absence of calmodulin in the inner membrane preparations, 12.5 nmol trifluoperazine bind per 100 microgram of membrane protein with an association constant, K, of 6.5 . 10(4) M-1. We conclude that calmodulin-binding neuroleptic agents, when added to whole cells, have the potential to disrupt mitochondrial energy production by a reaction which apparently does not involve a phenothiazine-calmodulin interaction.