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Molecular and electrophysiological characterization of a novel cation channel of Trypanosoma cruzi.

Jimenez V, Docampo R - PLoS Pathog. (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, Georgia, United States of America. vjimen@uga.edu

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.

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Changes in TcCat localization during differentiation.TcCat immunolocalization (green) at different time points after mammalian cell infection (A–D) or during in vitro differentiation of trypomastigotes to amastigotes at acidic pH (E–H). Yellow arrows indicate trypomastigote-like morphology and red arrows indicate amastigote-like forms at 5 h post-infection. Nuclei were DAPI stained (blue). Bars = 10 µm.
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ppat-1002750-g002: Changes in TcCat localization during differentiation.TcCat immunolocalization (green) at different time points after mammalian cell infection (A–D) or during in vitro differentiation of trypomastigotes to amastigotes at acidic pH (E–H). Yellow arrows indicate trypomastigote-like morphology and red arrows indicate amastigote-like forms at 5 h post-infection. Nuclei were DAPI stained (blue). Bars = 10 µm.

Mentions: We evaluated the change in the localization of TcCat by IFA during the differentiation in vivo. At 5 h post-infection of mammalian cells, TcCat is already detected at a single intracellular spot both in parasites with trypomastigote-like morphology (Fig. 2A, yellow arrows) and in rounded amastigote-like cells (Fig. 2B, red arrows). At 24 and 48 h post-infection (Figs. 2C and 2D) TcCat remains intracellular in the replicating amastigotes, close to the flagellar pocket. In extracellular trypomastigotes, 96 h post-infection, TcCat was always localized at the plasma membrane (Fig. 1A).


Molecular and electrophysiological characterization of a novel cation channel of Trypanosoma cruzi.

Jimenez V, Docampo R - PLoS Pathog. (2012)

Changes in TcCat localization during differentiation.TcCat immunolocalization (green) at different time points after mammalian cell infection (A–D) or during in vitro differentiation of trypomastigotes to amastigotes at acidic pH (E–H). Yellow arrows indicate trypomastigote-like morphology and red arrows indicate amastigote-like forms at 5 h post-infection. Nuclei were DAPI stained (blue). Bars = 10 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369953&req=5

ppat-1002750-g002: Changes in TcCat localization during differentiation.TcCat immunolocalization (green) at different time points after mammalian cell infection (A–D) or during in vitro differentiation of trypomastigotes to amastigotes at acidic pH (E–H). Yellow arrows indicate trypomastigote-like morphology and red arrows indicate amastigote-like forms at 5 h post-infection. Nuclei were DAPI stained (blue). Bars = 10 µm.
Mentions: We evaluated the change in the localization of TcCat by IFA during the differentiation in vivo. At 5 h post-infection of mammalian cells, TcCat is already detected at a single intracellular spot both in parasites with trypomastigote-like morphology (Fig. 2A, yellow arrows) and in rounded amastigote-like cells (Fig. 2B, red arrows). At 24 and 48 h post-infection (Figs. 2C and 2D) TcCat remains intracellular in the replicating amastigotes, close to the flagellar pocket. In extracellular trypomastigotes, 96 h post-infection, TcCat was always localized at the plasma membrane (Fig. 1A).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, Georgia, United States of America. vjimen@uga.edu

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.

Show MeSH
Related in: MedlinePlus