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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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Osmotic stress effect of TcCat localization.TcCat immunolocalization in T. cruzi epimastigotes (A) and trypomastigotes (B) under isosmotic (Iso), hyperosmotic (Hyper) or hyposmotic (Hypo) conditions. TcCat was detected with purified specific antibody and secondary anti-rabbit Alexa-488 conjugated (green). DNA was stained with DAPI (blue). Bars = 10 µm. C. Quantification of the TcCat label intensity in trypomastigotes under isosmotic or hyperosmotic conditions. Values are expressed in arbitrary units (AU) as mean ± SEM of n = 3 independent experiments. For each experiment and treatment, the pixel intensity of 75 parasites was measured. *P<0.01 respect to the isosmotic condition. **P<0.01 respect to hyperosmotic in the absence of the blockers. D. Relative change in trypomastigotes cell volume under hyperosmotic stress (control, open diamonds). TcCat blockers significantly reduce the shrinkage (1 mM BaCl2open triangles; 100 µM 4-AP, black squares). Values are mean ± SEM of n = 3 independent experiments.
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ppat-1002750-g007: Osmotic stress effect of TcCat localization.TcCat immunolocalization in T. cruzi epimastigotes (A) and trypomastigotes (B) under isosmotic (Iso), hyperosmotic (Hyper) or hyposmotic (Hypo) conditions. TcCat was detected with purified specific antibody and secondary anti-rabbit Alexa-488 conjugated (green). DNA was stained with DAPI (blue). Bars = 10 µm. C. Quantification of the TcCat label intensity in trypomastigotes under isosmotic or hyperosmotic conditions. Values are expressed in arbitrary units (AU) as mean ± SEM of n = 3 independent experiments. For each experiment and treatment, the pixel intensity of 75 parasites was measured. *P<0.01 respect to the isosmotic condition. **P<0.01 respect to hyperosmotic in the absence of the blockers. D. Relative change in trypomastigotes cell volume under hyperosmotic stress (control, open diamonds). TcCat blockers significantly reduce the shrinkage (1 mM BaCl2open triangles; 100 µM 4-AP, black squares). Values are mean ± SEM of n = 3 independent experiments.

Mentions: As mentioned before, an inward-rectifier K+ channel seems to be involved in the maintenance of plasma membrane potential, intracellular pH and osmoregulation [5], [9]. To assess the role of TcCat on some of these processes we evaluated the localization of the protein in T. cruzi epimastigotes and trypomastigotes under osmotic stress. Under isosmotic conditions, the channel is localized at the plasma membrane, in a punctuate pattern, with some intracellular staining more evident in epimastigotes (Fig. 7A and B, Iso). When epimastigotes were placed under hyperosmotic stress, maintaining the same ionic concentrations, TcCat almost completely translocated to the plasma membrane (Fig. 7A, Hyper). Remarkably, in trypomastigotes, after 30 sec under hyperosmotic stress, TcCat disappeared from the cell surface of the parasites (Fig. 7B, Hyper). No intracellular accumulation of the protein was observed suggesting that the protein is released to the extracellular medium, probably by shedding mechanisms previously described for other T. cruzi surface proteins [27]. To prove this hypothesis, the supernatant of parasites under different osmotic conditions were precipitated and evaluated by western blot analysis. In trypomastigotes under hyperosmotic stress TcCat was detected in the supernatants (Fig. S7A). That was not the case for trypomastigotes under isosmotic or hyposmotic conditions (Fig. S7A) or for epimastigotes under similar treatments (Fig. S7B). Parasites overexpressing GFP were used as a control to rule out lysis of the parasites as a mechanism of release of TcCat(Fig. S7A).


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

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

Osmotic stress effect of TcCat localization.TcCat immunolocalization in T. cruzi epimastigotes (A) and trypomastigotes (B) under isosmotic (Iso), hyperosmotic (Hyper) or hyposmotic (Hypo) conditions. TcCat was detected with purified specific antibody and secondary anti-rabbit Alexa-488 conjugated (green). DNA was stained with DAPI (blue). Bars = 10 µm. C. Quantification of the TcCat label intensity in trypomastigotes under isosmotic or hyperosmotic conditions. Values are expressed in arbitrary units (AU) as mean ± SEM of n = 3 independent experiments. For each experiment and treatment, the pixel intensity of 75 parasites was measured. *P<0.01 respect to the isosmotic condition. **P<0.01 respect to hyperosmotic in the absence of the blockers. D. Relative change in trypomastigotes cell volume under hyperosmotic stress (control, open diamonds). TcCat blockers significantly reduce the shrinkage (1 mM BaCl2open triangles; 100 µM 4-AP, black squares). Values are mean ± SEM of n = 3 independent experiments.
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Related In: Results  -  Collection

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ppat-1002750-g007: Osmotic stress effect of TcCat localization.TcCat immunolocalization in T. cruzi epimastigotes (A) and trypomastigotes (B) under isosmotic (Iso), hyperosmotic (Hyper) or hyposmotic (Hypo) conditions. TcCat was detected with purified specific antibody and secondary anti-rabbit Alexa-488 conjugated (green). DNA was stained with DAPI (blue). Bars = 10 µm. C. Quantification of the TcCat label intensity in trypomastigotes under isosmotic or hyperosmotic conditions. Values are expressed in arbitrary units (AU) as mean ± SEM of n = 3 independent experiments. For each experiment and treatment, the pixel intensity of 75 parasites was measured. *P<0.01 respect to the isosmotic condition. **P<0.01 respect to hyperosmotic in the absence of the blockers. D. Relative change in trypomastigotes cell volume under hyperosmotic stress (control, open diamonds). TcCat blockers significantly reduce the shrinkage (1 mM BaCl2open triangles; 100 µM 4-AP, black squares). Values are mean ± SEM of n = 3 independent experiments.
Mentions: As mentioned before, an inward-rectifier K+ channel seems to be involved in the maintenance of plasma membrane potential, intracellular pH and osmoregulation [5], [9]. To assess the role of TcCat on some of these processes we evaluated the localization of the protein in T. cruzi epimastigotes and trypomastigotes under osmotic stress. Under isosmotic conditions, the channel is localized at the plasma membrane, in a punctuate pattern, with some intracellular staining more evident in epimastigotes (Fig. 7A and B, Iso). When epimastigotes were placed under hyperosmotic stress, maintaining the same ionic concentrations, TcCat almost completely translocated to the plasma membrane (Fig. 7A, Hyper). Remarkably, in trypomastigotes, after 30 sec under hyperosmotic stress, TcCat disappeared from the cell surface of the parasites (Fig. 7B, Hyper). No intracellular accumulation of the protein was observed suggesting that the protein is released to the extracellular medium, probably by shedding mechanisms previously described for other T. cruzi surface proteins [27]. To prove this hypothesis, the supernatant of parasites under different osmotic conditions were precipitated and evaluated by western blot analysis. In trypomastigotes under hyperosmotic stress TcCat was detected in the supernatants (Fig. S7A). That was not the case for trypomastigotes under isosmotic or hyposmotic conditions (Fig. S7A) or for epimastigotes under similar treatments (Fig. S7B). Parasites overexpressing GFP were used as a control to rule out lysis of the parasites as a mechanism of release of TcCat(Fig. S7A).

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