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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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Effect of different blockers on TcCat currents.A. Representative current traces recorded under a voltage-ramp protocol between −80 and + 80 mV, under control symmetrical conditions (black line) or in the presence of 10 mM BaCl2 (red line) or CaCl2 (green line). After applying BaCl2, the seal was washed and once the current was back to the levels recorded under control conditions, CaCl2 was applied. B. Voltage-current relationship obtained from total currents of the seal recorded applying a voltage-step protocol from −80 to +80 mV under symmetrical control conditions for K+ (black squares), or in the presence of 1 mM BaCl2 (red circles), 1 mM CaCl2 (green triangles) or 1 mM MgCl2. C. Normalized total currents respect to the total current of the seal recorded at the indicated holding potentials in the presence of blockers: 1 mM BaCl2, 1 mM CaCl2, 1 mM MgCl2, 1 µM 4-AP, 10 mM TEA. A significant reduction in the current was found in the presence of the indicated blockers (p<0.001, n = 3 independent experiments). D. Current traces recorded applying a pulse protocol at the indicated holding potentials in control conditions (140 mMKCl, 10 mMHepes-K, pH 7.4), in the presence of pre-immune serum, or in the presence of specific anti-TcCat antibody in the bath solution at a concentration of 0.12 mg/ml.
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ppat-1002750-g006: Effect of different blockers on TcCat currents.A. Representative current traces recorded under a voltage-ramp protocol between −80 and + 80 mV, under control symmetrical conditions (black line) or in the presence of 10 mM BaCl2 (red line) or CaCl2 (green line). After applying BaCl2, the seal was washed and once the current was back to the levels recorded under control conditions, CaCl2 was applied. B. Voltage-current relationship obtained from total currents of the seal recorded applying a voltage-step protocol from −80 to +80 mV under symmetrical control conditions for K+ (black squares), or in the presence of 1 mM BaCl2 (red circles), 1 mM CaCl2 (green triangles) or 1 mM MgCl2. C. Normalized total currents respect to the total current of the seal recorded at the indicated holding potentials in the presence of blockers: 1 mM BaCl2, 1 mM CaCl2, 1 mM MgCl2, 1 µM 4-AP, 10 mM TEA. A significant reduction in the current was found in the presence of the indicated blockers (p<0.001, n = 3 independent experiments). D. Current traces recorded applying a pulse protocol at the indicated holding potentials in control conditions (140 mMKCl, 10 mMHepes-K, pH 7.4), in the presence of pre-immune serum, or in the presence of specific anti-TcCat antibody in the bath solution at a concentration of 0.12 mg/ml.

Mentions: The effect of divalent cations was evaluated by adding controlled concentrations of Ba2+, Ca2+or Mg2+to the bath solution. Fig. 6A shows that in the presence of 10 mM BaCl2 (red line) or 10 mM CaCl2 (green line) a significant decrease in the total current can be observed compared with the control (black line). No important shift in the Vrev was recorded, indicating the low permeability for these ions. A consistent decrease in the total current was observed when a voltage-step protocol was applied (Fig. 6B) in the presence of lower concentrations of the divalent cations, with a more remarkable effect for Mg2+ (blue inverted triangles). A concentration-dependent effect was observed for Ba2+ and Ca2+ when applying a voltage-step protocol in the presence of controlled concentrations of both ions (Fig. S6 C and D). The effect of Ba2+ over the leak through asolectin vesicles was evaluated comparing the normalized current in the presence of different concentrations of the divalent ion on empty liposomes (Fig. S6B,black circles) or liposomes containing TcCat (Fig. S6B,black squares). In the presence of 1 mM BaCl2 the residual current at −80 mV (Fig. S6B,upper panel) is about 55% in empty liposomes while it is close to 30% in liposomes containing TcCat, indicating that a percentage of the current through the channel is sensitive to the presence of the divalent cation. Similar results are obtained at +80 mV (Fig. S6B,lower panel).Based on the dose-dependent blockage, the calculated inhibition constants (Ki) for Ba2+ were (in mM): 0.54±0.08 and 0.63±0.06 at −80 and +80 mV, respectively, with no significant dependency on the applied voltage (n = 3 independent experiments).


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

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

Effect of different blockers on TcCat currents.A. Representative current traces recorded under a voltage-ramp protocol between −80 and + 80 mV, under control symmetrical conditions (black line) or in the presence of 10 mM BaCl2 (red line) or CaCl2 (green line). After applying BaCl2, the seal was washed and once the current was back to the levels recorded under control conditions, CaCl2 was applied. B. Voltage-current relationship obtained from total currents of the seal recorded applying a voltage-step protocol from −80 to +80 mV under symmetrical control conditions for K+ (black squares), or in the presence of 1 mM BaCl2 (red circles), 1 mM CaCl2 (green triangles) or 1 mM MgCl2. C. Normalized total currents respect to the total current of the seal recorded at the indicated holding potentials in the presence of blockers: 1 mM BaCl2, 1 mM CaCl2, 1 mM MgCl2, 1 µM 4-AP, 10 mM TEA. A significant reduction in the current was found in the presence of the indicated blockers (p<0.001, n = 3 independent experiments). D. Current traces recorded applying a pulse protocol at the indicated holding potentials in control conditions (140 mMKCl, 10 mMHepes-K, pH 7.4), in the presence of pre-immune serum, or in the presence of specific anti-TcCat antibody in the bath solution at a concentration of 0.12 mg/ml.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369953&req=5

ppat-1002750-g006: Effect of different blockers on TcCat currents.A. Representative current traces recorded under a voltage-ramp protocol between −80 and + 80 mV, under control symmetrical conditions (black line) or in the presence of 10 mM BaCl2 (red line) or CaCl2 (green line). After applying BaCl2, the seal was washed and once the current was back to the levels recorded under control conditions, CaCl2 was applied. B. Voltage-current relationship obtained from total currents of the seal recorded applying a voltage-step protocol from −80 to +80 mV under symmetrical control conditions for K+ (black squares), or in the presence of 1 mM BaCl2 (red circles), 1 mM CaCl2 (green triangles) or 1 mM MgCl2. C. Normalized total currents respect to the total current of the seal recorded at the indicated holding potentials in the presence of blockers: 1 mM BaCl2, 1 mM CaCl2, 1 mM MgCl2, 1 µM 4-AP, 10 mM TEA. A significant reduction in the current was found in the presence of the indicated blockers (p<0.001, n = 3 independent experiments). D. Current traces recorded applying a pulse protocol at the indicated holding potentials in control conditions (140 mMKCl, 10 mMHepes-K, pH 7.4), in the presence of pre-immune serum, or in the presence of specific anti-TcCat antibody in the bath solution at a concentration of 0.12 mg/ml.
Mentions: The effect of divalent cations was evaluated by adding controlled concentrations of Ba2+, Ca2+or Mg2+to the bath solution. Fig. 6A shows that in the presence of 10 mM BaCl2 (red line) or 10 mM CaCl2 (green line) a significant decrease in the total current can be observed compared with the control (black line). No important shift in the Vrev was recorded, indicating the low permeability for these ions. A consistent decrease in the total current was observed when a voltage-step protocol was applied (Fig. 6B) in the presence of lower concentrations of the divalent cations, with a more remarkable effect for Mg2+ (blue inverted triangles). A concentration-dependent effect was observed for Ba2+ and Ca2+ when applying a voltage-step protocol in the presence of controlled concentrations of both ions (Fig. S6 C and D). The effect of Ba2+ over the leak through asolectin vesicles was evaluated comparing the normalized current in the presence of different concentrations of the divalent ion on empty liposomes (Fig. S6B,black circles) or liposomes containing TcCat (Fig. S6B,black squares). In the presence of 1 mM BaCl2 the residual current at −80 mV (Fig. S6B,upper panel) is about 55% in empty liposomes while it is close to 30% in liposomes containing TcCat, indicating that a percentage of the current through the channel is sensitive to the presence of the divalent cation. Similar results are obtained at +80 mV (Fig. S6B,lower panel).Based on the dose-dependent blockage, the calculated inhibition constants (Ki) for Ba2+ were (in mM): 0.54±0.08 and 0.63±0.06 at −80 and +80 mV, respectively, with no significant dependency on the applied voltage (n = 3 independent experiments).

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