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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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TcCat expression and localization in T. cruzi life stages.TcCat immunolocalization (green) in T. cruzi trypomastigotes (A), epimastigotes (B), extracellular amastigotes (C), intracellular amastigotes (D) and metacyclictrypomastigotes (E). Nuclei were DAPI stained. Bars = 10 µm. F, G. Immunoelectron microscopy localization of TcCat in trypomastigotes with purified anti-TcCat and secondary anti-rabbit gold-labeled antibody. Bars: F = 0.5 µm, G = 0.2 µm. H. Western blot analysis of TcCat expression in T. cruzi homogenates. Lanes: M: molecular weight markers in kDa (MagicMark XP, Invitrogen), T: trypomastigotes, E: epimastigotes, A: extracellular amastigotes. Bottom: membranes were stripped and re-incubated with anti-tubulin antibody as a loading control. I. Densitometry of TcCat detection by western blot analysis in arbitrary units (AU). T: trypomastigotes, E: epimastigotes, A: extracellular amastigotes. Values in arbitrary units (AU) correspond to mean ± SEM from 3 independent experiments.
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ppat-1002750-g001: TcCat expression and localization in T. cruzi life stages.TcCat immunolocalization (green) in T. cruzi trypomastigotes (A), epimastigotes (B), extracellular amastigotes (C), intracellular amastigotes (D) and metacyclictrypomastigotes (E). Nuclei were DAPI stained. Bars = 10 µm. F, G. Immunoelectron microscopy localization of TcCat in trypomastigotes with purified anti-TcCat and secondary anti-rabbit gold-labeled antibody. Bars: F = 0.5 µm, G = 0.2 µm. H. Western blot analysis of TcCat expression in T. cruzi homogenates. Lanes: M: molecular weight markers in kDa (MagicMark XP, Invitrogen), T: trypomastigotes, E: epimastigotes, A: extracellular amastigotes. Bottom: membranes were stripped and re-incubated with anti-tubulin antibody as a loading control. I. Densitometry of TcCat detection by western blot analysis in arbitrary units (AU). T: trypomastigotes, E: epimastigotes, A: extracellular amastigotes. Values in arbitrary units (AU) correspond to mean ± SEM from 3 independent experiments.

Mentions: TcCat localization was analysed by indirect immunofluorescence using affinity-purified antibodies against the recombinant protein. In trypomastigotes, the channel has a clearly defined punctuate pattern along the flagellum (Fig. 1A). In epimastigotes (Fig. 1B), TcCat also has a peripheral punctuated pattern with some apparently intracellular labeling. To further evaluate whether the punctuate localization could be due to labeling of patches of plasma membrane and not intracellular vesicles we performed immunolocalization in permeabilized and non-permeabilized cells. In both trypomastigotes and epimastigotes TcCat was detected, at least in part, exposed to the cell surface (Fig. S2). In amastigotes that were spontaneously released to the supernatant of infected L6E9 myoblasts the channel showed plasma membrane localization (Fig. 1C). However, in intracellular amastigotes TcKCat seems to be confined to a spot that could be the remaining short flagellum (Fig. 1D). This change in localization is consistent with a role of TcCat in K+ uptake, which would become less important in the intracellular environment rich in K+. In agreement with a developmental regulation of TcCat expression, labeling decreased considerably in metacyclictrypomastigotes (Fig. 1E). Immunoelectron microscopy analysis confirmed the patched distribution of TcCat in tissue culture-derived trypomastigotes along the flagellar attachment zone (Figs. 1F and 1G). The association of ion channels in clusters has been described previously [21]–[23] and seems to be related with preferential targeting to specific membrane lipid microdomains or lipid rafts, which are known to be more abundant in the flagellar membrane of trypanosomes [24].


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

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

TcCat expression and localization in T. cruzi life stages.TcCat immunolocalization (green) in T. cruzi trypomastigotes (A), epimastigotes (B), extracellular amastigotes (C), intracellular amastigotes (D) and metacyclictrypomastigotes (E). Nuclei were DAPI stained. Bars = 10 µm. F, G. Immunoelectron microscopy localization of TcCat in trypomastigotes with purified anti-TcCat and secondary anti-rabbit gold-labeled antibody. Bars: F = 0.5 µm, G = 0.2 µm. H. Western blot analysis of TcCat expression in T. cruzi homogenates. Lanes: M: molecular weight markers in kDa (MagicMark XP, Invitrogen), T: trypomastigotes, E: epimastigotes, A: extracellular amastigotes. Bottom: membranes were stripped and re-incubated with anti-tubulin antibody as a loading control. I. Densitometry of TcCat detection by western blot analysis in arbitrary units (AU). T: trypomastigotes, E: epimastigotes, A: extracellular amastigotes. Values in arbitrary units (AU) correspond to mean ± SEM from 3 independent experiments.
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ppat-1002750-g001: TcCat expression and localization in T. cruzi life stages.TcCat immunolocalization (green) in T. cruzi trypomastigotes (A), epimastigotes (B), extracellular amastigotes (C), intracellular amastigotes (D) and metacyclictrypomastigotes (E). Nuclei were DAPI stained. Bars = 10 µm. F, G. Immunoelectron microscopy localization of TcCat in trypomastigotes with purified anti-TcCat and secondary anti-rabbit gold-labeled antibody. Bars: F = 0.5 µm, G = 0.2 µm. H. Western blot analysis of TcCat expression in T. cruzi homogenates. Lanes: M: molecular weight markers in kDa (MagicMark XP, Invitrogen), T: trypomastigotes, E: epimastigotes, A: extracellular amastigotes. Bottom: membranes were stripped and re-incubated with anti-tubulin antibody as a loading control. I. Densitometry of TcCat detection by western blot analysis in arbitrary units (AU). T: trypomastigotes, E: epimastigotes, A: extracellular amastigotes. Values in arbitrary units (AU) correspond to mean ± SEM from 3 independent experiments.
Mentions: TcCat localization was analysed by indirect immunofluorescence using affinity-purified antibodies against the recombinant protein. In trypomastigotes, the channel has a clearly defined punctuate pattern along the flagellum (Fig. 1A). In epimastigotes (Fig. 1B), TcCat also has a peripheral punctuated pattern with some apparently intracellular labeling. To further evaluate whether the punctuate localization could be due to labeling of patches of plasma membrane and not intracellular vesicles we performed immunolocalization in permeabilized and non-permeabilized cells. In both trypomastigotes and epimastigotes TcCat was detected, at least in part, exposed to the cell surface (Fig. S2). In amastigotes that were spontaneously released to the supernatant of infected L6E9 myoblasts the channel showed plasma membrane localization (Fig. 1C). However, in intracellular amastigotes TcKCat seems to be confined to a spot that could be the remaining short flagellum (Fig. 1D). This change in localization is consistent with a role of TcCat in K+ uptake, which would become less important in the intracellular environment rich in K+. In agreement with a developmental regulation of TcCat expression, labeling decreased considerably in metacyclictrypomastigotes (Fig. 1E). Immunoelectron microscopy analysis confirmed the patched distribution of TcCat in tissue culture-derived trypomastigotes along the flagellar attachment zone (Figs. 1F and 1G). The association of ion channels in clusters has been described previously [21]–[23] and seems to be related with preferential targeting to specific membrane lipid microdomains or lipid rafts, which are known to be more abundant in the flagellar membrane of trypanosomes [24].

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