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Originally developed as a chemotherapeutic agent, miltefosine (hexadecylphosphocholine) is an inhibitor of phosphatidylcholine synthesis with proven antiparasitic effects. It is the only oral drug approved for the treatment of Leishmaniasis and American Trypanosomiasis (Chagas disease). Although its precise mechanisms are not yet fully understood, miltefosine exhibits broad-spectrum anti-parasitic effects primarily by disrupting the intracellular Ca 2+ homeostasis of the parasites while sparing the human hosts. In addition to its inhibitory effects on phosphatidylcholine synthesis and cytochrome c oxidase, miltefosine has been found to affect the unique giant mitochondria and the acidocalcisomes of parasites. Both of these crucial organelles are involved in Ca 2+ regulation. Furthermore, miltefosine has the ability to activate a specific parasite Ca 2+ channel that responds to sphingosine, which is different to its L-type VGCC human ortholog. Here, we aimed to provide an overview of recent advancements of the anti-parasitic mechanisms of miltefosine. We also explored its multiple molecular targets and investigated how its pleiotropic effects translate into a rational therapeutic approach for patients afflicted by Leishmaniasis and American Trypanosomiasis. Notably, miltefosine's therapeutic effect extends beyond its impact on the parasite to also positively affect the host's immune system. These findings enhance our understanding on its multi-targeted mechanism of action. Overall, this review sheds light on the intricate molecular actions of miltefosine, highlighting its potential as a promising therapeutic option against these debilitating parasitic diseases.

The Trypanosomatidae family encompasses many unicellular organisms responsible of several tropical diseases that affect humans and animals. Livestock tripanosomosis caused by Trypanosoma brucei brucei (T. brucei), Trypanosoma equiperdum (T. equiperdum) and Trypanosoma evansi (T. evansi), have a significant socio-economic impact and limit animal protein productivity throughout the intertropical zones of the world. Similarly, to all organisms, the maintenance of Ca 2+ homeostasis is vital for these parasites, and the mechanism involved in the intracellular Ca 2+ regulation have been widely described. However, the evidences related to the mechanisms responsible for the Ca 2+ entry are scarce. Even more, to date the presence of a store-operated Ca 2+ channel (SOC) has not been reported. Despite the apparent absence of Orai and STIM-like proteins in these parasites, in the present work we demonstrate the presence of a store-operated Ca 2+ -entry (SOCE) in T. equiperdum, using physiological techniques. This Ca 2+ -entry is induced by thapsigargin (TG) and 2,5-di-t-butyl-1,4-benzohydroquinone (BHQ), and inhibited by 2-aminoethoxydiphenyl borate (2APB). Additionally, the use of bioinformatics techniques allowed us to identify putative transient receptor potential (TRP) channels, present in members of the Trypanozoon family, which would be possible candidates responsible for the SOCE described in the present work in T. equiperdum., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The plasma membrane Ca 2+ -ATPase (PMCA) from trypanosomatids lacks a classical calmodulin (CaM) binding domain, although CaM stimulated activities have been detected by biochemical assays. Recently we proposed that the Trypanosoma equiperdum CaM-sensitive PMCA (TePMCA) contains a potential 1-18 CaM-binding motif at the C-terminal region of the pump. In the present study, we evaluated the potential CaM-binding motifs using CaM from Trypanosoma cruzi and either the recombinant full length TePMCA C-terminal sequence (P14) or synthetic peptides comprising different regions of the C-terminal domain. We demonstrated that P14 and a synthetic peptide corresponding to residues 1037-1062 (which contains the predicted 1-18 binding motif) competed efficiently for binding to TcCaM, exhibiting similar IC 50 s of 200 nM. A stable complex of this peptide and TcCaM was formed in the presence of Ca 2+ , as determined by native-polyacrylamide gel electrophoresis. A predicted structure obtained by molecular docking showed an interaction of the 1-18 binding motif with the Ca 2+ /CaM complex. Moreover, when the peptide was incubated with CaM and Ca 2+ , a blue shift in the tryptophan fluorescence spectrum (from 350 to 329 nm) was observed. Substitutions at W 1039 and F 1056 , strongly decreased both CaM-peptide interaction and the complex assembly. Our results demonstrated the presence of a functional 1-18 motif at the TePMCA C-terminal domain. Furthermore, on the basis of spectrofluorometric assays and the resulting structure modeled by docking we propose that the L 1042 and W 1060 residues might also participate as anchors to form a 1-4-18-22 motif., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The increase in the intracellular Ca(2+) concentration ([Ca(2+)]i) is the key variable for many different processes, ranging from regulation of cell proliferation to apoptosis. In this work we demonstrated that the sphingolipid sphingosine (Sph) increases the [Ca(2+)]i by inhibiting the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), in a similar manner to thapsigargin (Tg), a specific inhibitor of this Ca(2+) pump. The results showed that addition of sphingosine produced a release of Ca(2+) from the endoplasmic reticulum followed by a Ca(2+) entrance from the outside mileu. The results presented in this work support that this sphingolipid could control the activity of the SERCA, and hence sphingosine may participate in the regulation of [Ca(2+)]I in mammalian cells., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Src family non-receptor tyrosine kinases play a prominent role in multiple cellular processes, including: cell proliferation, differentiation, cell survival, stress response, and cell adhesion and migration, among others. And when deregulated by mutations, overexpression, and/or the arrival of faulty incoming signals, its hyperactivity contributes to the development of hematological and solid tumors. c-Src is a prototypical member of this family of kinases, which is highly regulated by a set of phosphorylation events. Other factor contributing to the regulation of Src activity appears to be mediated by the Ca2+ signal generated in cells by different effectors, where the Ca2+-receptor protein calmodulin (CaM) plays a key role. In this report we demonstrate that CaM directly interacts with Src in both Ca2+-dependent and Ca2+-independent manners in vitro and in living cells, and that the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibits the activation of this kinase induced by the upstream activation of the epidermal growth factor receptor (EGFR), in human carcinoma epidermoide A431 cells, and by hydrogen peroxide-induced oxidative stress, in both A431 cells and human breast adenocarcinoma SK-BR-3 cells. Furthermore, we show that the Ca2+/CaM complex strongly activates the auto-phosphorylation and tyrosine kinase activity of c-Src toward exogenous substrates, but most relevantly and for the first time, we demonstrate that Ca2+-free CaM (apo-CaM) exerts a far higher activatory action on Src auto-phosphorylation and kinase activity toward exogenous substrates than the one exerted by the Ca2+/CaM complex. This suggests that a transient increase in the cytosolic concentration of free Ca2+ is not an absolute requirement for CaM-mediated activation of Src in living cells, and that a direct regulation of Src by apo-CaM could be inferred.

The disruption of the intracellular Ca(2+) homeostasis of Leishmania mexicana represents a major target for the action of drugs, such as amiodarone and miltefosine. However, little is known about the mechanism of Ca(2+) entry to these cells. Here we show the presence of a Ca(2+) channel in the plasma membrane of these parasites. This channel has many characteristics similar to the human L-type voltage-gated Ca(2+) channel. Thus, Ca(2+) entry is blocked by verapamil, nifedipine and diltiazem while Bay K 8644 opened this channel. However, different to its human counterpart, sphingosine was able to open this channel, while other well known sphingolipids had no effect. This fact could have important pharmacological implications., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The plasma membrane Ca(2+)-ATPase (PMCA) plays a key role in the regulation of the intracellular Ca(2+) concentration. Ethanol stimulates this Ca(2+) pump in an isoform-specific manner. On search for a physiological molecule that could mimic the effect of ethanol, we have previously demonstrated that some sphingolipids containing free "hydroxyl" groups, like ceramide, are able to stimulate the PMCA. Since diacylglycerol (DAG) structurally shares some characteristics with ceramide, we evaluate its effect on the PMCA. We demonstrated that DAG is a potent stimulator of this enzyme. The activation induced is additive to that produced by calmodulin, protein-kinase C and ethanol, which implies that DAG interacts with the PMCA through a different mechanism. Additionally, by different fluorescent approaches, we demonstrated a direct binding between PMCA and DAG. The results obtained in this work strongly suggest that DAG is a novel effector of the PMCA, acting by a direct interaction.

Leishmaniasis represents a serious public health problem worldwide. The first line of treatment is based on glucantime and pentostan, which generate toxic effects in treated patients. We have recently shown that amiodarone, frequently used as an antiarrhythmic, possesses activity against Trypanosoma cruzi through the disruption of mitochondrial Ca(2+) homeostasis and the inhibition of parasite ergosterol biosynthesis, specifically at the level of oxidosqualene cyclase activity (G. Benaim, J. Sanders, Y. Garcia-Marchan, C. Colina, R. Lira, A. Caldera, G. Payares, C. Sanoja, J. Burgos, A. Leon-Rossell, J. Concepcion, A. Schijman, M. Levin, E. Oldfield, and J. Urbina, J. Med. Chem. 49:892-899, 2006). Here we show that at therapeutic concentrations, amiodarone has a profound effect on the viability of Leishmania mexicana promastigotes. Additionally, its effect on the viability of the parasite was greater against intracellular amastigotes than against promastigotes, and it did not affect the host cell. Using fluorimetric and confocal microscopy techniques, we also demonstrated that the mechanism of action of amiodarone was related to the disruption of intracellular Ca(2+) homeostasis through a direct action not only on the mitochondria but also on the acidocalcisomes. On the other hand, analysis of the free sterols in promastigotes incubated with amiodarone showed that this drug also affected the biosynthesis of 5-dehydroepisterol, which results in squalene accumulation, thus suggesting that amiodarone inhibits the squalene epoxidase activity of the parasite. Taken together, the results obtained in the present work point to a more general effect of amiodarone in trypanosomatids, opening potential therapeutic possibilities for this infectious disease.

We have previously demonstrated that rat cerebellar Type-1 astrocytes express a very active genistein sensitive Na(+)/Ca(2+) exchanger, which accounts for most of the total plasma membrane Ca(2+) fluxes and for the clearance of loads induced by physiological agonists. In this work, we have explored the mechanism by which the reverse Na(+)/Ca(2+) exchange is involved in agonist-induced Ca(2+) signaling in rat cerebellar astrocytes. Microspectrofluorometric measurements of Cai(2+) with Fluo-3 demonstrate that the Cai(2+) signals associated long (> 20 s) periods of reverse operation of the Na(+)/Ca(2+) exchange are amplified by a mechanism compatible with calcium-calcium release, while those associated with short (< 20 s) pulses are not amplified. This was confirmed by pharmacological experiments using ryanodine receptors agonist (4-chloro-m-cresol) and the endoplasmic reticulum ATPase inhibitor (thapsigargin). Confocal microscopy demonstrates a high co-localization of immunofluorescent labeled Na(+)/Ca(2+) exchanger and RyRs. Low (< 50 micromol/L) or high (> 500 micromol/L) concentrations of L-glutamate (L-Glu) or L-aspartate causes a rise in which is completely blocked by the Na(+)/Ca(2+) exchange inhibitors KB-R7943 and SEA0400. The most important novel finding presented in this work is that L-Glu activates the reverse mode of the Na(+)/Ca(2+) exchange by inducing Na(+) entry through the electrogenic Na(+)-Glu-co-transporter and not through the ionophoric L-Glu receptors, as confirmed by pharmacological experiments with specific blockers of the ionophoric L-Glu receptors and the electrogenic Glu transporter.

Sphingolipids comprise a very important class of second messengers involved in cell growth, differentiation, and apoptosis, among other different functions. Recently, these lipids have been implicated in calcium mobilization in different cell lines, including Jurkat T-lymphocytes. However, the effect of each particular sphingolipid appears to be cell-line specific. Among them, the least studied is ceramide-1-P (Cer-1-P). Here, we show that Cer-1-P increased the intracellular Ca(2+) concentration in Jurkat T-cells. Furthermore, laser-scanning confocal microscopy indicated that Ca(2+) is released from the endoplasmic reticulum. An effect on store-operated Ca(2+) channels was evidenced by whole-cell "patch clamp" measurements after Cer-1-P induced Ca(2+) store depletion. The mechanism of action of Cer-1-P resembles that of the Jurkat anti-TCR antibody, but differs from that of ceramide, since Cer-1-P induced an increase in Ins(1,4,5)-P(3).

The effect of ceramide on the cytoplasmic Ca2+ concentration ([Ca2+]i) varies depending on the cell type. We have found that in Jurkat human T cells ceramide increases the [Ca2+]i from a thapsigargin-sensitive calcium pool and the subsequent activation of a capacitative Ca2+ entry. This effect occurs both in the presence and in the absence of extracellular calcium. Addition of ceramine, a non-hydrolysable analogue of ceramide, reproduced its effect on the [Ca2+]i ruling out that this is due to the conversion of ceramide to sphingosine. The effect of ceramide was additive to that obtained by sphingosine, but not to the Jurkat T cells specific antibody OKT3. However, different to the latter, ceramide do not induced an elevation of InsP3. The opening of a store operated Ca2+ channel by ceramide was corroborated by experiments of Fura-2 quenching, using Mn2+ as a surrogate for Ca2+ and confirmed by whole-cell recording patch clamp techniques.

Ca2+ plays an important role in the regulation of several important activities in different trypanosomatids. These parasites possess a Ca2+ transport system in the endoplasmic reticulum (ER) involved in Ca2+ homeostasis, which has been reported to be insensitive to thapsigargin, a classical inhibitor of the sarcoplasmic-ER Ca2+ adenosine triphosphatase (ATPase) (SERCA) in most eukaryotic cells. However, currently there is a controversy regarding the existence of a thapsigargin-sensitive ER Ca2+ store in these parasites. Therefore, we decided to explore the effect of this inhibitor using different methodological approaches. First, we selected Trypanosoma evansi as a parasite model to warrant the homogeneity of the population because this parasite has only a single life cycle, i.e., bloodstream-form trypomastigotes. Second, we compared the thapsigargin effect on Ca2+ homeostasis by spectrophotometrical Ca2+ measurements using 3 different approaches: whole-cell populations, cells that have been permeabilized by treatment with digitonin, and intact single cells. Our results demonstrate that a low concentration of thapsigargin induces Ca2+ release from intracellular Ca2+ stores in this parasite, which can be observed independently of the method used. Furthermore, the addition of thapsigargin before or after nigericin did not abolish its effect, showing that thapsigargin acts specifically on the ER. In conclusion, our results indicate the presence of a nonmitochondrial thapsigargin-sensitive Ca2+ store in T. evansi.

We have demonstrated previously that crystal violet induces a rapid, dose-related collapse of the inner mitochondrial membrane potential of Trypanosoma cruzi epimastigotes. In this work, we show that crystal violet-induced dissipation of the membrane potential was accompanied by an efflux of Ca2+ from the mitochondria. In addition, crystal violet inhibited the ATP-dependent, oligomycin-, and antimycin A-insensitive Ca2+ uptake by digitonin-permeabilized epimastigotes. Crystal violet also induced Ca2+ release from the mitochondria and endoplasmic reticulum of digitonin-permeabilized trypomastigotes. Furthermore, crystal violet inhibited Ca2+ uptake and the (Ca(2+)-Mg2+)-ATPase of a highly enriched plasma membrane fraction of epimastigotes, thus indicating an inhibition of other calcium transport mechanisms of the cells. Disruption of Ca2+ homeostasis by crystal violet may be a key process leading to trypanosome cell injury by this drug.

The intracellular Ca2+ concentration in all eukaryotic cells so far studied is 4 orders of magnitude lower than in the extracellular milieu. In trypanosomatids, the intracellular concentration of this cation is around 50 nM, even lower than in higher eukaryotics. This differential concentration is maintained by diverse transport systems at the plasma membrane level and at certain intracellular organelles. In the case of trypanosomatids it have been identified the presence of an electrophoretic uniporter at the internal membrane of the unique giant mitochondrion of these parasites, showing identical kinetics properties to the homologous system of higher eukaryotics. Thus, the low Ca2+ affinity of this system is not compatible with its putative function of maintaining intracellular Ca2+ at the submicromolar level. Contrary to previous reports, we have identified a Ca(2+)-ATPase in the plasma membrane of Leishmania braziliensis, Leishmania mexicana, Trypanosoma cruzi and Trypanosoma brucei. The enzyme possesses a high Ca2+ affinity (Km Ca2+ = 0.5 microM), is Mg(2+)-dependent and activatable by calmodulin purified from these hemoflagellates. The Ca(2+)-ATPase is sensitive to vanadate (Ki = 1 microM), thus typifying it as a "P" type ion pump. Vesicles from the plasma membrane of these parasites are able to accumulate Ca2+ against a concentration gradient. The kinetic properties of both transport and ATPase are essentially the same, thus suggesting the same molecular entity. The above results strongly suggest that the Ca(2+)-ATPase is the mechanism responsible for the long-term fine-tuning of intracellular Ca2+ at the submicromolar lever in trypanosomatids.

The intracellular Ca2+ concentration in different trypanosomatids is about 50 nanomolar, which concentration in different trypanosomatids is about 50 nanomolar, which is 4 orders of magnitude lower than in the extracellular milieu. This fact implies the existence of well developed mechanisms for the maintenance of such a high calcium gradient. In higher eukaryotics a number of different structures have been implicated in this function. Some of them are located in intracellular organelles, and others in the plasma membrane. Since intracellular organelles are limited by their storage capacity, long-term Ca2+ homeostasis resides solely in the plasma membrane. In higher eukaryotics, a calcium pump or Ca(2+)-ATPase located in the plasma membrane, because of its high Ca2+ affinity, has been proposed as the structure responsible for the maintenance of the cytoplasmic Ca2+ concentration at the submicromolar level. The presence of a Ca(2+)-ATPase in trypanosomatids has been debated. While some groups have reported its absence, others have reported the existence of an enzyme which is Mg(2+)-independent or even inhibited by Mg2+. On the other hand, in none of these reports any correlation was shown between the Ca(2+)-ATPase activity observed and the Ca2+ transport function attributed to this enzyme. We have previously shown that a calmodulin-stimulated Mg(2+)-dependent Ca(2+)-ATPase is present in the plasma membrane of Leishmania braziliensis and of Trypanosoma cruzi. Plasma membrane vesicles from these parasites are able to accumulate Ca2+ in the presence of the ATP-Mg complex. The similarities found between the kinetics parameters and other properties of the Ca(2+)-ATPase and the Ca2+ transport activity strongly suggest a common molecular entity. The stoichiometry calculated from these parameters approaches the 1:1 stoichiometry for Ca2+ and ATP, as reported for the Ca2+ pump from higher eukaryotic cells. In this report we show that plasma membrane vesicles from Leishmania mexicana possess a Ca(2+)-ATPase with characteristics which are similar to that reported by us for other trypanosomatids. Thus, the enzyme has a high Ca2+ affinity which is further increased upon addition of calmodulin. The maximal velocity is also increased by calmodulin. As it has been found in the Ca(2+)-ATPase from human erythrocytes, trypsin proteolysis stimulates the ATPase activity mimicking the effect of calmodulin. On the other hand, antibodies raised against the isolated Ca(2+)-ATPase from human erythrocytes are effective in recognizing the enzyme from Leishmania mexicana, thus supporting a stronger homology between both Ca(2+)-ATPases.

High-affinity Ca(2+)-activated ATPases that do not show any demonstrable dependence on Mg2+ have been reported in the plasma membranes of different trypanosomatids, and it has been suggested [McLaughlin (1985) Mol. Biochem. Parasitol. 15, 189-201; Ghosh, Ray, Sarkar & Bhaduri (1990) J. Biol. Chem. 265, 11345-11351] that these enzymes may have a role in Ca2+ transport by the plasma membrane and in the regulation of intracellular Ca2+ in these parasites. In this report we investigated Ca2+ transport by Trypanosoma cruzi plasma membrane vesicles using Arsenazo III as a Ca2+ indicator. These vesicles accumulated Ca2+ upon addition of ATP only when Mg2+ was present and released it in response to the Ca2+ ionophore A23187, but were insensitive to inositol 1,4,5-trisphosphate. Ca2+ transport was insensitive to antimycin A, oligomycin and carbonyl cyanide p-trifluorophenylhydrazone, ruling out any mitochondrial contamination. Staurosporine and phorbol myristate acetate had no effect on this activity, while low concentrations of vanadate (10 microM) completely inhibited it. In addition, we describe a high-affinity vanadate-sensitive (Ca(2+)-Mg2+)-ATPase in the highly enriched plasma membrane fraction of T. cruzi. Kinetic studies indicated that the apparent Km for free Ca2+ was 0.3 microM. On the other hand, Ca(2+)-ATPase activity and Ca2+ transport were both stimulated by bovine brain calmodulin and by endogenous calmodulin purified from these cells. In addition, trifluoperazine and calmidazolium, at concentrations in the range in which they normally exert anti-calmodulin effects, inhibited the calmodulin-stimulated Ca(2+)-ATPase activity. These observations support the notion that a Mg(2+)-dependent plasma membrane Ca2+ pump is present in these parasites.

A subcellular fraction highly enriched in plasma membrane vesicles was prepared from Leishmania promastigotes. This fraction showed (Ca2+ + Mg2+)-ATPase activity. This, however, represented a small fraction (about 25%) of the overall ATPase activity. The Ca2(+)-ATPase showed general characteristics common to plasma membrane ATPases involved in Ca2+ transport. Thus, the Ca2(+)-ATPase was activated by Ca2+ with a high affinity (Km about 0.7 microM), saturating at about 5 microM Ca2+. Furthermore, it was stimulated by calmodulin (about 70-80% with 5 micrograms/ml) and almost fully inhibited by trifluoperazine (100 microM). The above vesicles accumulated Ca2+ against a concentration gradient and released it after the addition of A23187, as shown independently by 45Ca2+ and Arsenazo III studies. The transport mechanism showed the same kinetics parameters as described for the enzyme, indicating a single molecular entity. In addition, Ca2(+)-ATPase activity and Ca2+ uptake were completely inhibited by vanadate (20 microM), indicating that an E1-E2 type mechanism is involved. The results clearly demonstrate the presence of a Ca2+ pump in the plasma membrane of Leishmania which is capable of maintaining a low cytoplasmic Ca2+ concentration.

Leishmania braziliensis maintained very low (50 +/- 20 nM) intracellular concentrations of calcium ions under normal conditions, as shown by the fluorimetric indicator QUIN2. Digitonin-permeabilized cells liberated large amounts of calcium ions in the presence of the ionophore A23187, indicating the presence of a large intracellular reservoir for this ion. Given the extraordinary extension of the single giant mitochondrion of Kinetoplastida and the known capacity of mitochondria from other sources to accumulate calcium, we tested the capacity of this organelle to accumulate calcium ions in Leishmania. Coupled mitochondrial vesicles, five-fold enriched in succinate-cytochrome c oxidoreductase, were obtained from promastigotes by gentle grinding (45 s) with glass beads in hypertonic buffer solution, followed by differential centrifugation. These vesicles had a respiratory control ratio of 1.82 +/- 0.15, and two phosphorylation sites (sites II and III) using succinate as electron donor, and were capable of calcium uptake in the presence of several respiratory substrates; this uptake was enhanced in the presence of ADP and Pi and was blocked by classical electron transport inhibitors. Uncouplers such as carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP) and the calcium ionophore A23187 released previously accumulated calcium ions, suggesting that the driving force for the calcium uptake by the vesicles is the respiratory generated electrochemical potential gradient of protons. A study of the affinity of this system for calcium showed that even at 90 microM free calcium, succinate-induced calcium uptake is not saturated while approaching a level of 200 nmol min-1 (mg protein)-1, indicating a low-affinity, large-capacity system.(ABSTRACT TRUNCATED AT 250 WORDS)

The mode of action of the polyene antibiotic amphotericin B (AmB), the drug of choice for the treatment of systemic fungal infections and visceral leishmaniasis, is still unclear. An increase in intracellular Ca2+ concentration [( Ca2+]i), toxic in many cases, has been postulated as a possible lethal mechanism for AmB. Cell permeabilization to ethidium bromide (EB) was used as a criterion of viability. Kinetics of the DNA-EB fluorescent complex formation was studied in ergosterol-containing Leishmania promastigotes. Intracellular Ca2+ concentration was measured using quin-2 fluorescence in parallel aliquots. It is shown in this work that AmB can act as an efficient Ca2+ ionophore. However, the rapid permeabilization effect induced by AmB on these cells was not dependent on an increase in [Ca2+]i. On the contrary, it was found that leishmanicidal effect of AmB was enhanced in the absence of external calcium. Furthermore, A23187 a Ca2+ ionophore did not provoke cell permeabilization to EB.

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.