Molecular and Electrophysiological Characterization of a Novel Cation Channel of Trypanosoma cruzi

Abstract

We report the identification, functional expression, purification, reconstitution and electrophysiological characterization of a novel cation channel (TcCat) from Trypanosoma cruzi, the etiologic agent of Chagas disease. This channel is potassium permeable and shows inward rectification in the presence of magnesium. Western blot analyses with specific antibodies indicated that the protein is expressed in the three main life cycle stages of the parasite. Surprisingly, the parasites have the unprecedented ability to rapidly change the localization of the channel when they are exposed to different environmental stresses. TcCat rapidly translocates to the tip of the flagellum when trypomastigotes are submitted to acidic pH, to the plasma membrane when epimastigotes are submitted to hyperosmotic stress, and to the cell surface when amastigotes are released to the extracellular medium. Pharmacological block of TcCat activity also resulted in alterations in the trypomastigotes ability to respond to hyperosmotic stress. We also demonstrate the feasibility of purifying and reconstituting a functional ion channel from T. cruzi after recombinant expression in bacteria. The peculiar characteristics of TcCat could be important for the development of specific inhibitors with therapeutic potential against trypanosomes.

Polyphosphate and acidocalcisomes

“…In support of this model treatment of epimastigotes with HgCl 2 , a known inhibitor of T. cruzi aquaporin 1 (TcAQP1), or knockdown of TcAQP1 expression reduces the intensity of shrinking after hyperosmotic stress while overexpression of TcAQP1 increased shrinking, suggesting that the contractile vacuole mediates water efflux during hyperosmotic challenge. Shrinking is also favored by cation elimination through a cation channel (TcCAT) that is translocated to the plasma membrane of epimastigotes submitted to hyperosmotic stress [55]. Inhibitors of TcCAT (BaCl 2 , 4-aminopyridine) inhibit shrinking of trypomastigotes under hyperosmotic stress [55].…”

“…Shrinking is also favored by cation elimination through a cation channel (TcCAT) that is translocated to the plasma membrane of epimastigotes submitted to hyperosmotic stress [55]. Inhibitors of TcCAT (BaCl 2 , 4-aminopyridine) inhibit shrinking of trypomastigotes under hyperosmotic stress [55]. Synthesis of polyP and inorganic ions sequestration in acidocalcisomes prevents the deleterious effects of a cellular increase in ionic strength.…”

Rab32 is essential for maintaining functional acidocalcisomes, and for growth and infectivity of Trypanosoma cruzi

“…Labeled epimastigotes were then placed onto glass-bottomed petri dishes that had been previously treated with polylysine. Cells were allowed to settle in the dish for 2 min, and loose parasites were washed away with isosmotic buffer (Jimenez and Docampo, 2012). Live-cell imaging was started in isosmotic conditions at 1 frame/s, and after 30 s hyposmotic buffer was added to the cells to reach a final osmolarity of 117 mosm/l.…”

New Insights into Roles of Acidocalcisomes and Contractile Vacuole Complex in Osmoregulation in Protists

“…Shrinking is also probably due to cation elimination through a cation channel (TcCAT) that is translocated to the plasma membrane of epimastigotes submitted to hyperosmotic stress (Jimenez and Docampo, 2012). Inhibitors of TcCAT (BaCl 2 , 4-aminopyridine) inhibit shrinking of trypomastigotes under hyperosmotic stress (Jimenez and Docampo, 2012).…”

“…Shrinking is also probably due to cation elimination through a cation channel (TcCAT) that is translocated to the plasma membrane of epimastigotes submitted to hyperosmotic stress (Jimenez and Docampo, 2012). Inhibitors of TcCAT (BaCl 2 , 4-aminopyridine) inhibit shrinking of trypomastigotes under hyperosmotic stress (Jimenez and Docampo, 2012). Early synthesis of polyP and sequestration of inorganic ions in acidocalcisomes of epimastigotes and the simultaneous increase in compatible osmolytes prevents the deleterious effects of a cellular increase in ionic strength.…”