Limits...
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.

Show MeSH

Related in: MedlinePlus

Functional yeast complementation with TcCat.A–D. TcCat expression, as analyzed by immunofluorescence, at different times after induction. Yeast were collected at the indicated times and incubated with anti-TcCat antibodies (green) and anti-vacuolar H+ ATPase (red) as a control for proper permeabilization. Nuclei were DAPI stained (blue). Left panels are DIC images, right panels are anti-TcCat stained cells and central panels are merge immunofluorescence images. Bars = 10 µm. E. Western blot analysis of yeast homogenate with specific anti-TcCat antibody. Lanes, 1: control non-complemented mutant yeast, 2: wild-type strain complemented with TcCat, 3: mutant strain complemented with TcCat, 4: TcCat recombinant protein. F. Growth-assay of complemented yeast in SC ura- galactose agar plates. Serial dilutions of initial cultures at OD600 = 0.6 were incubated for 72 h at 30°C. WT corresponds to wild type strain, MpYES2 is the mutant transformed with the empty vector pYES2, MC represents the mutant strain transformed with TcCat-pYES2 construct.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3369953&req=5

ppat-1002750-g003: Functional yeast complementation with TcCat.A–D. TcCat expression, as analyzed by immunofluorescence, at different times after induction. Yeast were collected at the indicated times and incubated with anti-TcCat antibodies (green) and anti-vacuolar H+ ATPase (red) as a control for proper permeabilization. Nuclei were DAPI stained (blue). Left panels are DIC images, right panels are anti-TcCat stained cells and central panels are merge immunofluorescence images. Bars = 10 µm. E. Western blot analysis of yeast homogenate with specific anti-TcCat antibody. Lanes, 1: control non-complemented mutant yeast, 2: wild-type strain complemented with TcCat, 3: mutant strain complemented with TcCat, 4: TcCat recombinant protein. F. Growth-assay of complemented yeast in SC ura- galactose agar plates. Serial dilutions of initial cultures at OD600 = 0.6 were incubated for 72 h at 30°C. WT corresponds to wild type strain, MpYES2 is the mutant transformed with the empty vector pYES2, MC represents the mutant strain transformed with TcCat-pYES2 construct.

Mentions: Potassium uptake defective S. cerevisiae mutants (Δtrk1, Δtrk2, Δtok1) were used to investigate the K+ influx ability of TcCat (see Text S1). These mutants depend on high extracellular K+ concentration for their growth as they only have the non-specific cation uptake mechanism, termed NSC1, for growth [25]. Mutants were kept in defined medium (SC ura-) supplemented with 100 mMKCl, pH 5.8. TcCat expression was induced by switching the carbon source and the channel was rapidly detected on the yeast surface by immunofluorescence analysis with anti-TcCat antibodies (Fig. 3B). After 2 h (Fig. 3C), yeasts were collected by centrifugation and placed in standard SC ura-medium without KCl. Otherwise the presence of high K+ concentration was toxic upon induction of TcCat expression. The channel was expressed on the yeast surface for up to 72 h at high levels, although, at 24 h, some labeling could be observed in the periphery of the yeast vacuole, probably due to recycling or degradation (Fig. 3D). Control cells were transformed with empty vector pYES2. A monoclonal antibody against the 69-kDa subunit of the vacuolar H+-ATPase was used as a control of proper permeabilization (Fig. 3A–D).


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

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

Functional yeast complementation with TcCat.A–D. TcCat expression, as analyzed by immunofluorescence, at different times after induction. Yeast were collected at the indicated times and incubated with anti-TcCat antibodies (green) and anti-vacuolar H+ ATPase (red) as a control for proper permeabilization. Nuclei were DAPI stained (blue). Left panels are DIC images, right panels are anti-TcCat stained cells and central panels are merge immunofluorescence images. Bars = 10 µm. E. Western blot analysis of yeast homogenate with specific anti-TcCat antibody. Lanes, 1: control non-complemented mutant yeast, 2: wild-type strain complemented with TcCat, 3: mutant strain complemented with TcCat, 4: TcCat recombinant protein. F. Growth-assay of complemented yeast in SC ura- galactose agar plates. Serial dilutions of initial cultures at OD600 = 0.6 were incubated for 72 h at 30°C. WT corresponds to wild type strain, MpYES2 is the mutant transformed with the empty vector pYES2, MC represents the mutant strain transformed with TcCat-pYES2 construct.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002750-g003: Functional yeast complementation with TcCat.A–D. TcCat expression, as analyzed by immunofluorescence, at different times after induction. Yeast were collected at the indicated times and incubated with anti-TcCat antibodies (green) and anti-vacuolar H+ ATPase (red) as a control for proper permeabilization. Nuclei were DAPI stained (blue). Left panels are DIC images, right panels are anti-TcCat stained cells and central panels are merge immunofluorescence images. Bars = 10 µm. E. Western blot analysis of yeast homogenate with specific anti-TcCat antibody. Lanes, 1: control non-complemented mutant yeast, 2: wild-type strain complemented with TcCat, 3: mutant strain complemented with TcCat, 4: TcCat recombinant protein. F. Growth-assay of complemented yeast in SC ura- galactose agar plates. Serial dilutions of initial cultures at OD600 = 0.6 were incubated for 72 h at 30°C. WT corresponds to wild type strain, MpYES2 is the mutant transformed with the empty vector pYES2, MC represents the mutant strain transformed with TcCat-pYES2 construct.
Mentions: Potassium uptake defective S. cerevisiae mutants (Δtrk1, Δtrk2, Δtok1) were used to investigate the K+ influx ability of TcCat (see Text S1). These mutants depend on high extracellular K+ concentration for their growth as they only have the non-specific cation uptake mechanism, termed NSC1, for growth [25]. Mutants were kept in defined medium (SC ura-) supplemented with 100 mMKCl, pH 5.8. TcCat expression was induced by switching the carbon source and the channel was rapidly detected on the yeast surface by immunofluorescence analysis with anti-TcCat antibodies (Fig. 3B). After 2 h (Fig. 3C), yeasts were collected by centrifugation and placed in standard SC ura-medium without KCl. Otherwise the presence of high K+ concentration was toxic upon induction of TcCat expression. The channel was expressed on the yeast surface for up to 72 h at high levels, although, at 24 h, some labeling could be observed in the periphery of the yeast vacuole, probably due to recycling or degradation (Fig. 3D). Control cells were transformed with empty vector pYES2. A monoclonal antibody against the 69-kDa subunit of the vacuolar H+-ATPase was used as a control of proper permeabilization (Fig. 3A–D).

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