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The hemoparasite Trypanosoma equiperdum belongs to the Trypanozoon subgenus and includes several species that are pathogenic to animals and humans in tropical and subtropical areas across the world. As with all eukaryotic organisms, Ca 2+ is essential for these parasites to perform cellular processes thus ensuring their survival across their life cycle. Despite the established paradigm to study proteins related to Ca 2+ homeostasis as potential drug targets, so far little is known about Ca 2+ entry into trypanosomes. Therefore, in the present study, the presence of a plasma membrane Ca 2+ -channel in T. equiperdum (TeCC), activated by sphingosine and inhibited by verapamil, is described. The TeCC was cloned and analyzed using bioinformatic resources, which confirmed the presence of several domains, motifs, and a topology similar to the Ca 2+ channels found in higher eukaryotes. Biochemical and confocal microscopy assays using antibodies raised against an internal region of human L-type Ca 2+ channels indicate the presence of a protein with similar predicted molar mass to the sequence analyzed, located at the plasma membrane of T. equiperdum. Physiological assays based on Fura-2 signals and Mn 2+ quenching performed on whole parasites showed a unidirectional Ca 2+ entry, which is activated by sphingosine and blocked by verapamil, with the distinctive feature of insensitivity to nifedipine and Bay K 8644. This suggests a second Ca 2+ entry for T. equiperdum, different from the store-operated Ca 2+ entry (SOCE) previously described. Moreover, the evidence presented here for the TeCC indicates molecular and pharmacological differences with their mammal counterparts, which deserve further studies to evaluate the potential of this channel as a drug target., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Trypanosoma cruzi is the causative agent of Chagas disease. The only two drugs accepted for the treatment of this infection are benznidazole and nifurtimox, which are of limited use in the predominant chronic phase. On the search for new drugs, the intracellular Ca 2+ regulation has been postulated as a possible target, due to differences found between host cells and the parasite. The mechanisms involved in the intracellular Ca 2+ regulation of T. cruzi have been partially elucidated. However, nothing is known about a putative channel responsible for the Ca 2+ entry into this parasite. In contrast, in Leishmania spp., a closely related hemoflagelate, a sphingosine-activated plasma membrane Ca 2+ channel has been recently described. The latter resembles the L-type voltage-gated Ca 2+ channel present in humans, but with distinct characteristics. This channel is one of the main targets concerning the mechanism of action of miltefosine, the unique oral drug approved against leishmaniasis. In the present work, we describe for the first time, the electrophysiological characterization of a sphingosine-activated Ca 2+ channel of T. cruzi by reconstituting plasma membrane vesicles into giant liposomes and patch clamp. This channel shares some characteristic as activation by Bay K8644 and inhibition by channel blockers such as nifedipine. However, the T. cruzi channel differs from the L-type VGCC in its activation by sphingosine and/or miltefosine. Albeit the conductance for each, Ba 2+ , Ca 2+ and Sr 2+ was similar, the parasite channel appears not to be voltage dependent. A gene that presents homology in critical amino acids with its human ortholog Ca 2+ channel was identified., (© 2019 Federation of European Biochemical Societies.)

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.)

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 is a key enzyme in the regulation of the intracellular Ca(2+) concentration. On the other hand, sphingolipids have been recognized recently as important second messengers, acting in many systems in combination with Ca(2+). In view of the fact that the Ca(2+)-ATPase is stimulated by ethanol, and since sphingolipids possess free hydroxy groups, we decided to study the possible effect of ceramide and sphingosine on this calcium pump. Here we show that ceramide stimulates the Ca(2+)-ATPase in a dose-dependent manner and additively to the activation observed in the presence of calmodulin or ethanol, when compared with any of these effectors added alone. Ceramide affects both the affinity for Ca(2+) and the V(max) of the enzyme. Furthermore, this second messenger also stimulates Ca(2+) transport in inside-out plasma-membrane vesicles from erythro cytes. Conversely, sphingosine, which is reported to act in many systems antagonistically with ceramide, showed an inhibitory effect on Ca(2+)-ATPase activity. This inhibition was also observed on the calmodulin-stimulated enzyme. These results, taken together, suggest that ceramide and sphingosine act antagonistically on the plasma-membrane Ca(2+)-ATPase. This is in accordance with the frequently reported opposite effect of these sphingolipids on intracellular Ca(2+) concentration.

The hemoparasite Trypanosomaequiperdum belongs to the Trypanozoon subgenus and includes several species that are pathogenic to animals and humans in tropical and subtropical areas across the world. As with all eukaryotic organisms, Ca2+ is essential for these parasites to perform cellular processes thus ensuring their survival across their life cycle. Despite the established paradigm to study proteins related to Ca2+ homeostasis as potential drug targets, so far little is known about Ca2+ entry into trypanosomes. Therefore, in the present study, the presence of a plasma membrane Ca2+-channel in T. equiperdum (TeCC), activated by sphingosine and inhibited by verapamil, is described. The TeCC was cloned and analyzed using bioinformatic resources, which confirmed the presence of several domains, motifs, and a topology similar to the Ca2+ channels found in higher eukaryotes. Biochemical and confocal microscopy assays using antibodies raised against an internal region of human L-type Ca2+ channels indicate the presence of a protein with similar predicted molar mass to the sequence analyzed, located at the plasma membrane of T. equiperdum. Physiological assays based on Fura-2 signals and Mn2+ quenching performed on whole parasites showed a unidirectional Ca2+ entry, which is activated by sphingosine and blocked by verapamil, with the distinctive feature of insensitivity to nifedipine and Bay K 8644. This suggests a second Ca2+ entry for T. equiperdum, different from the store-operated Ca2+ entry (SOCE) previously described. Moreover, the evidence presented here for the TeCC indicates molecular and pharmacological differences with their mammal counterparts, which deserve further studies to evaluate the potential of this channel as a drug target. [ABSTRACT FROM AUTHOR]