A store-operated Ca²⁺-entry in Trypanosoma equiperdum: Physiological evidences of its presence

Highlights

• A store operated calcium entry is evidenced in Trypanosoma equiperdum.

• Thapsigargin and BHQ induced a Ca²⁺-entry in presence of extracellular Ca²⁺.

• The Ca²⁺-entry induced by SERCA inhibitors is blocked by 2APB.

• Mn²⁺ quenching assays confirm the unidirectional influx of Ca²⁺.

• The parasite possesses sequences for TRP channels, candidates for this SOCE effect.

Abstract

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 Ca²⁺ homeostasis is vital for these parasites, and the mechanism involved in the intracellular Ca²⁺ regulation have been widely described. However, the evidences related to the mechanisms responsible for the Ca²⁺ entry are scarce. Even more, to date the presence of a store-operated Ca²⁺ 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 Ca²⁺-entry (SOCE) in T. equiperdum, using physiological techniques. This Ca²⁺-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.

Introduction

The maintenance of intracellular Ca²⁺ homeostasis is vital for all organisms. High concentrations of this cation usually result in cell death, but low intracellular concentrations are required for signal transduction and the active sites of some enzymes [1,2]. Ca²⁺ gains access into eukaryotic cells through several plasma membrane channels, some of which are under control of receptors (receptor-operated Ca²⁺-channels, ROCs), the potential across the plasma membrane (voltage-gated Ca²⁺-channels, VGCCs), and the content of intracellular Ca²⁺-stores (store-operated Ca²⁺-channels, SOCs) [1]. Another class of channels is the “Transient Receptor Potential” (TRP) family, which can also generate changes in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) [3].

SOCE is an ubiquitous and singular mechanism for Ca²⁺ influx activated in response to Ca²⁺ release from endoplasmic reticulum (ER), which is triggered physiologically through stimulation of diverse surface receptors. The basic components of this signaling pathway include a mechanism for discharging Ca²⁺ stores commonly (but not exclusively) phospholipase C (PLC) and inositol 1,4,5-trisphosphate (IP₃), consisting of a sensor at the ER (such as STIM1−2), which is able to communicate the ER Ca²⁺ levels to plasma membrane Ca²⁺ channels, and also serves as an activator of the SOCs [4]. At least two types of SOCs are distinguished electro-physiologically and now molecularly [5]. The first type are the Ca²⁺ release-activated Ca²⁺ (CRAC) channels, where the Orai family of proteins has been established as the pore-forming component of CRAC channels, interacting with STIM1, in order to activate the channels. The second type are the TRP channels, especially TRPC subfamily, that contribute to SOCE in several cell types [5].

Classically, the use of SERCA inhibitors and SOC modulators demonstrates and validates the presence of the SOCE pathway. TG and BHQ, specific inhibitors of the ER Ca²⁺-ATPase (SERCA) [6,7], provide a non-invasive technique for manipulating ER Ca²⁺ pools in intact cells while within the main modulators of SOC, the most extensively used is 2-aminoethoxydiphenyl borate (2APB), but its effect is not very specific. This compound at high concentrations (20–100 μM) completely inhibits SOCE [8].

In trypanosomatids, Ca²⁺ is also an important second messenger, which contributes to the cellular signaling and the mechanisms involving its regulation have been widely described [2,9,10]. This parasite family encompasses a large number of unicellular organisms, many of which are causative agents of several tropical diseases that affect humans and animals. Livestock tripanosomosis caused by T. brucei, T. equiperdum and T. evansi, has a significant socio-economic impact and limit animal protein productivity throughout the intertropical zones of the world [11]. Like other eukaryotic cells, trypanosomatids use two sources of Ca²⁺ for generating signals: Ca²⁺ release from intracellular stores and Ca²⁺-entry across the plasma membrane [10]. Even though there is scant information regarding the mechanism of Ca²⁺-entry, the presence of a Ca²⁺-channel in the plasma membrane is warranted, since these parasites can significantly change the [Ca²⁺]_i depending on different conditions, for example, during cell invasion [12]. Ca²⁺-channels regulate many crucial processes within cells and their abnormal activity can be damaging to cell survival. In this sense, several Ca²⁺ channel blockers have been tested in vitro against trypanosomatids, demonstrating that they inhibit parasite growth, which suggests that Ca²⁺ channels might represent attractive therapeutic targets for the treatment of Trypanosomosis [13,9].

Regardless of the need for the existence of Ca²⁺-channels in trypanosomatids, the mechanisms responsible for Ca²⁺-entry remain unclear. To date, there are only reports on the presence of a sphingosine-activated VGCC in Leishmania mexicana [14], Leishmania donovani [15] and T. cruzi [16], which are blocked by nifedipine and verapamil and stimulated by the agonist Bay K8644. At molecular level, some genes encoding homologous of VGCCs (similar to dihydropyridine-sensitive l-type Ca²⁺-channels) and TRP channels have been described in trypanosomatids [10,17,18,9]. Nevertheless, the genes encoding homologous for Ca²⁺ channels such as the components of SOC complex, the pore-forming entity (Orai) at the plasma membrane and the ER Ca²⁺ sensor protein (STIM), have not yet been identified so far [10,[17], [18], [19]].

In the present work, it is demonstrated for the first time the presence of a SOCE in T. equiperdum. This Ca²⁺-entry is induced by TG and BHQ, which was previously established that inhibits SERCA in this parasite [20,21], and inhibited by the classic drug 2APB, used for distinguishing SOC from other channels [8,22]. Additionally, using bioinformatics tools, we found homologous sequences to Ca²⁺ channels in T. equiperdum, which may possibly mediate the SOCE demonstrated in this study.