Identification and electrophysiological properties of a sphingosine‐dependent plasma membrane Ca²⁺ channel in Trypanosoma cruzi

Abstract

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 Ca2+ regulation has been postulated as a possible target, due to differences found between host cells and the parasite. The mechanisms involved in the intracellular Ca2+ regulation of T. cruzi have been partially elucidated. However, nothing is known about a putative channel responsible for the Ca2+ entry into this parasite. In contrast, in Leishmania spp., a closely related hemoflagelate, a sphingosine‐activated plasma membrane Ca2+ channel has been recently described. The latter resembles the L‐type voltage‐gated Ca2+ 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 Ca2+ 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, Ba2+, Ca2+ and Sr2+ was similar, the parasite channel appears not to be voltage dependent. A gene that presents homology in critical amino acids with its human ortholog Ca2+ channel was identified.

Disruption of Intracellular Calcium Homeostasis as a Therapeutic Target Against Trypanosoma cruzi

“…The mechanism responsible for Ca 2+ entry in T. cruzi has been discovered just recently (Figure 1). A sphingosinestimulated plasma membrane Ca 2+ -channel similar to that described in L. mexicana (Benaim et al, 2013) has been reported (Rodriguez-Duran et al, 2019), and characterized electrophysiologically by Patch Clamp techniques (Figure 2). This Ca 2+ channel shares some characteristics with the human L-Type Voltage gated Ca 2+ channel (VGCC), including inhibition by the VGCC blocker nifedipine, a dihydropiridine, and activation by Bay K8644, but differs from the L-type VGCC in its activation by sphingosine.…”

“…Another relevant drug against Chagas disease is the alkyllysophospholipid miltefosine (Figure 3), the only approved oral formulation against visceral leishmaniasis, the lethal form of the leishmania disease spectrum (Croft and Coombs, 2003), which has also shown promising anti-Trypanosomal activity (Luna et al, 2009;Saraiva et al, 2009;Rodriguez-Duran et al, 2019). Known mechanisms of action of miltefosine include inhibition of the synthesis of phosphatidylcholine, mitochondrial injury, and inhibition of the parasite cytochrome c oxidase (Pinto-Martinez et al, 2018b).…”

“…In T. cruzi, this drug inhibits the biosynthesis of phosphatidylcholine 10-20 times more potently when compared to mammalian cells (Urbina, 2017). More recently, the spectrum of its mechanism of action has broadened, reporting a direct action on the disruption of the parasites Ca 2+ homeostasis (Pinto-Martinez et al, 2018b;Rodriguez-Duran et al, 2019).…”

“…Amiodarone Mitochondria, Acidocalcisomes, Ergosterol synthesis Benaim et al, 2006;Serrano-Martín et al, 2009a,b;Benaim and Paniz-Mondolfi, 2012 Dronedarone Mitochondria, Acidocalcisomes, Ergosterol synthesis Benaim and Paniz-Mondolfi, 2012;SQ109 Mitochondria, Acidocalcisomes, Ergosterol synthesis Veiga-Santos et al, 2015García-García et al, 2016;Gil et al, 2020 Amioder (Benzofuran derivative) Mitochondria, Acidocalcisomes, Pinto-Martinez et al, 2018a;Martinez-Sotillo et al, 2019 Miltefosine Sph-activated Plasma membrane Ca 2+ -Channel, Mitochondria, Acidocalcisomes Pinto-Martinez et al, 2018b;Rodriguez-Duran et al, 2019 Posaconazole Elevation of intracellular Ca 2+ Benaim et al, 2006 channel (Rodriguez-Duran et al, 2019) which allows the opening of Ca 2+ currents in a similar fashion to the physiological activator of the channel sphingosine. Concomitantly, miltefosine has also been demonstrated to collapse the mitochondrial electrochemical membrane potential ( φ), and to induce a rapid alkalinization of the parasites acidocalcisomes through direct action (Pinto-Martinez et al, 2018b).…”

A Novel Calcium-Activated Potassium Channel Controls Membrane Potential and Intracellular pH in Trypanosoma cruzi

“…They are also validated targets for treatment of highly prevalent diseases such as cardiovascular pathologies (Gill et al, 1992;Turley et al, 2016) and are currently being re-evaluated as potential drug targets against parasitic infections (Meier et al, 2018). In protozoans, ion channel characterization lags behind the general progress of the field, mostly due to technical limitations for direct electrophysiological recordings in motile cells, but in recent years we have gained insight into the role of calcium channels in trypanosomes (Chiurillo et al, 2017;Huang and Docampo, 2018;Potapenko et al, 2019;Rodriguez-Duran et al, 2019).…”