Molecular, immunological, and physiological evidences of a sphingosine-activated plasma membrane Ca2+-channel in Trypanosoma equiperdum

Abstract

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, 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.

Keywords: Trypanosoma equiperdum; Ca2+ channel; Sphingosine; Verapamil.