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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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Ion selectivity of TcCat.A. Representative current traces recorded under a voltage-ramp protocol between −80 and +80 mV. Back line corresponds to symmetrical conditions in the absence of Mg2+ (bath and pipette solution 140 mMKCl, 10 mMHepes-K, pH 7.4). Grey line represents the current under non-symmetrical conditions, replacing the bath solution for 140 mM NMDG-Cl, 10 mMHepes-K, pH 7.4. Red line shows the current trace when the bath solution contains 140 mM K-gluconate, 10 mMHepes-K, pH 7.4. Arrows indicate the shift in the reversal potential of the current for asymmetrical conditions. B. Current traces recorded applying a voltage-ramp protocol between −80 to +80 mV under symmetrical conditions described in (A) (black line) or replacing the bath solution for 140 mMXCl, 10 mMHepes-K, pH 7.4, X being Na+ (red line), Cs+ (green line), NH4+ (blue line) or NMDG (grey line). C. Relative permeability ratios for monovalent cations (X) respect to K+ (PX/PK). ΔVrev corresponds to the difference between the reversal potential of the current for the control and the experimental shift in reversal potential when replacing the monovalent cation in the bath solution. Values are expressed as mean ± SEM. N indicates independent experiment. Each experimental value is the average of 5 measurements for each experiment.
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ppat-1002750-g005: Ion selectivity of TcCat.A. Representative current traces recorded under a voltage-ramp protocol between −80 and +80 mV. Back line corresponds to symmetrical conditions in the absence of Mg2+ (bath and pipette solution 140 mMKCl, 10 mMHepes-K, pH 7.4). Grey line represents the current under non-symmetrical conditions, replacing the bath solution for 140 mM NMDG-Cl, 10 mMHepes-K, pH 7.4. Red line shows the current trace when the bath solution contains 140 mM K-gluconate, 10 mMHepes-K, pH 7.4. Arrows indicate the shift in the reversal potential of the current for asymmetrical conditions. B. Current traces recorded applying a voltage-ramp protocol between −80 to +80 mV under symmetrical conditions described in (A) (black line) or replacing the bath solution for 140 mMXCl, 10 mMHepes-K, pH 7.4, X being Na+ (red line), Cs+ (green line), NH4+ (blue line) or NMDG (grey line). C. Relative permeability ratios for monovalent cations (X) respect to K+ (PX/PK). ΔVrev corresponds to the difference between the reversal potential of the current for the control and the experimental shift in reversal potential when replacing the monovalent cation in the bath solution. Values are expressed as mean ± SEM. N indicates independent experiment. Each experimental value is the average of 5 measurements for each experiment.

Mentions: The cationic nature of the TcCat conductive properties was verified applying a voltage-ramp protocol from −80 to +80 mV under symmetrical conditions(Fig. 5A, black line) or replacing the bath solution for a non-permeantcation (140 mM NMDG-Cl, 10 mMHepes-K, pH 7.4 (Fig. 5A, gray line). A shift in the reversal potential of the current (ΔVrev) was observed from 0 mV to −54±6 mV (n = 4), close to the theoretical Vrev calculated for K+ under those conditions (−70 mV). Replacement of the bath solution for buffered 340 mMKCl induced a ΔVrev of +39±1 mV (n = 5) with a theoretical calculated Vrev of +22 mV. These results suggest that TcCat preferentially permeates K+. To calculate the selectivity for cations over anions of TcCat, we applied similar voltage ramp protocols but replacing the bath solution for a non-permeant anion (140 mM K-gluconate, 10 mMHepes-K, pH 7.4). A ΔVrev of −8.7±0.4 (n = 10) was measured under asymmetrical conditions (Fig. 5A, red line). Based on the bi-ionic equation (see Equations under Text S1), the calculated permeability ratio for K+ over Cl− was 5.9±0.5 (n = 10), indicating a preferential cation permeability but with weak selectivity filter, in agreement with the sequence data.


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

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

Ion selectivity of TcCat.A. Representative current traces recorded under a voltage-ramp protocol between −80 and +80 mV. Back line corresponds to symmetrical conditions in the absence of Mg2+ (bath and pipette solution 140 mMKCl, 10 mMHepes-K, pH 7.4). Grey line represents the current under non-symmetrical conditions, replacing the bath solution for 140 mM NMDG-Cl, 10 mMHepes-K, pH 7.4. Red line shows the current trace when the bath solution contains 140 mM K-gluconate, 10 mMHepes-K, pH 7.4. Arrows indicate the shift in the reversal potential of the current for asymmetrical conditions. B. Current traces recorded applying a voltage-ramp protocol between −80 to +80 mV under symmetrical conditions described in (A) (black line) or replacing the bath solution for 140 mMXCl, 10 mMHepes-K, pH 7.4, X being Na+ (red line), Cs+ (green line), NH4+ (blue line) or NMDG (grey line). C. Relative permeability ratios for monovalent cations (X) respect to K+ (PX/PK). ΔVrev corresponds to the difference between the reversal potential of the current for the control and the experimental shift in reversal potential when replacing the monovalent cation in the bath solution. Values are expressed as mean ± SEM. N indicates independent experiment. Each experimental value is the average of 5 measurements for each experiment.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369953&req=5

ppat-1002750-g005: Ion selectivity of TcCat.A. Representative current traces recorded under a voltage-ramp protocol between −80 and +80 mV. Back line corresponds to symmetrical conditions in the absence of Mg2+ (bath and pipette solution 140 mMKCl, 10 mMHepes-K, pH 7.4). Grey line represents the current under non-symmetrical conditions, replacing the bath solution for 140 mM NMDG-Cl, 10 mMHepes-K, pH 7.4. Red line shows the current trace when the bath solution contains 140 mM K-gluconate, 10 mMHepes-K, pH 7.4. Arrows indicate the shift in the reversal potential of the current for asymmetrical conditions. B. Current traces recorded applying a voltage-ramp protocol between −80 to +80 mV under symmetrical conditions described in (A) (black line) or replacing the bath solution for 140 mMXCl, 10 mMHepes-K, pH 7.4, X being Na+ (red line), Cs+ (green line), NH4+ (blue line) or NMDG (grey line). C. Relative permeability ratios for monovalent cations (X) respect to K+ (PX/PK). ΔVrev corresponds to the difference between the reversal potential of the current for the control and the experimental shift in reversal potential when replacing the monovalent cation in the bath solution. Values are expressed as mean ± SEM. N indicates independent experiment. Each experimental value is the average of 5 measurements for each experiment.
Mentions: The cationic nature of the TcCat conductive properties was verified applying a voltage-ramp protocol from −80 to +80 mV under symmetrical conditions(Fig. 5A, black line) or replacing the bath solution for a non-permeantcation (140 mM NMDG-Cl, 10 mMHepes-K, pH 7.4 (Fig. 5A, gray line). A shift in the reversal potential of the current (ΔVrev) was observed from 0 mV to −54±6 mV (n = 4), close to the theoretical Vrev calculated for K+ under those conditions (−70 mV). Replacement of the bath solution for buffered 340 mMKCl induced a ΔVrev of +39±1 mV (n = 5) with a theoretical calculated Vrev of +22 mV. These results suggest that TcCat preferentially permeates K+. To calculate the selectivity for cations over anions of TcCat, we applied similar voltage ramp protocols but replacing the bath solution for a non-permeant anion (140 mM K-gluconate, 10 mMHepes-K, pH 7.4). A ΔVrev of −8.7±0.4 (n = 10) was measured under asymmetrical conditions (Fig. 5A, red line). Based on the bi-ionic equation (see Equations under Text S1), the calculated permeability ratio for K+ over Cl− was 5.9±0.5 (n = 10), indicating a preferential cation permeability but with weak selectivity filter, in agreement with the sequence data.

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