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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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Biophysical characterization defines TcCat as an inward rectifier channel.A. Representative current traces applying a voltage-step protocol from −80 to 80 mV in 20 mV steps. The recordings were obtained under symmetrical conditions in the absence of Mg2+ with bath and pipette solution containing 140 mMKCl, 10 mMHepes-K, pH 7.4. B. Current-voltage relationship under symmetrical conditions described in (A). Data correspond to the unitary currentsrecorded in continuous voltage steps at the indicated holding potentials in the absence (open circles, n = 14) or in the presence of 1 mM MgCl2 in the bath solution (solid squares, n = 13). The non-linear relationship indicates the inward rectification. C. Current traces obtained at the indicated holding potentials showing the functional association of TcCat in clusters under asymmetrical conditions with bath solution 300 mMKCl and pipette solution 140 mMKCl, both containing 10 mMHepes-K, pH 7.4. Doted lines indicate the open state of the two channels (O1 and O2) present in the seal. C indicates the closed state of the channels. Histograms represent the unitary current corresponding to one or 2 channels at the indicated voltages. D. Open probability analysis of the single channel events. Values correspond to mean ± SEM from 14 independent experiments.
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ppat-1002750-g004: Biophysical characterization defines TcCat as an inward rectifier channel.A. Representative current traces applying a voltage-step protocol from −80 to 80 mV in 20 mV steps. The recordings were obtained under symmetrical conditions in the absence of Mg2+ with bath and pipette solution containing 140 mMKCl, 10 mMHepes-K, pH 7.4. B. Current-voltage relationship under symmetrical conditions described in (A). Data correspond to the unitary currentsrecorded in continuous voltage steps at the indicated holding potentials in the absence (open circles, n = 14) or in the presence of 1 mM MgCl2 in the bath solution (solid squares, n = 13). The non-linear relationship indicates the inward rectification. C. Current traces obtained at the indicated holding potentials showing the functional association of TcCat in clusters under asymmetrical conditions with bath solution 300 mMKCl and pipette solution 140 mMKCl, both containing 10 mMHepes-K, pH 7.4. Doted lines indicate the open state of the two channels (O1 and O2) present in the seal. C indicates the closed state of the channels. Histograms represent the unitary current corresponding to one or 2 channels at the indicated voltages. D. Open probability analysis of the single channel events. Values correspond to mean ± SEM from 14 independent experiments.

Mentions: The activity of TcCat was detected in patches excised from cell-size giant liposomes (inside-out configuration) containing the purified recombinant protein (Text S1, Figs. S4 and S5, and Table S1).Currents from liposomes containing only asolectin were recorded as control (Fig. S6A,black squares), showing a significant lower level compared with currents from liposomes containing purified TcCat (Fig. S6A, red circles). Currents were recorded under symmetrical conditions in the absence of Mg2+, unless stated otherwise, with bath and pipette solutions containing 140 mMKCl, 10 mMHepes-K, pH 7.4. Single channel currents were observed when an increasing voltage-pulse protocol between −80 to +80 mV was applied (Fig. 4A). The current-potential relationship for the single channel was not linear in the presence of Mg2+, as expected for an inward rectifier channel. The chord conductance (γ) (Fig. 4B, open circles) calculated under symmetrical KCl in the absence of Mg2+was 77±4 pS and 59±2 pS at −80 and +80 mV, respectively (n = 14) indicating a slight intrinsic rectification. Although no significant reduction in the current was observed at positive potentials in the presence of Mg2+, a significant increase in the inward current was evident at negative potentials in the presence of 1 mM MgCl2 in the bath solution (Fig. 4B, black squares), with unitary conductances of 122±7 pS and 56±3 pS at −80 and +80 mV, respectively (n = 13). These results suggest that the mechanism of blockage by Mg2+ is different from the one described for inward rectifier K+ channels. The unitary level of current was frequently observed in clusters, as shown in Fig. 4C where at least two channels could be detected, opening and closing independently. The histograms showncorrespond to the unitary current of one or two channels at the indicated voltages (Fig. 4C). This recorded activity agrees well with the localization in patches described above. Important variations in the open probability were observed in recordings from different days. When 14 independent experiments were analyzed, the open probability was not significantly sensitive to voltage, with values of 0.26±0.04 and 0.2±0.04 at −80 and +80 mV, respectively (Fig. 4D).


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

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

Biophysical characterization defines TcCat as an inward rectifier channel.A. Representative current traces applying a voltage-step protocol from −80 to 80 mV in 20 mV steps. The recordings were obtained under symmetrical conditions in the absence of Mg2+ with bath and pipette solution containing 140 mMKCl, 10 mMHepes-K, pH 7.4. B. Current-voltage relationship under symmetrical conditions described in (A). Data correspond to the unitary currentsrecorded in continuous voltage steps at the indicated holding potentials in the absence (open circles, n = 14) or in the presence of 1 mM MgCl2 in the bath solution (solid squares, n = 13). The non-linear relationship indicates the inward rectification. C. Current traces obtained at the indicated holding potentials showing the functional association of TcCat in clusters under asymmetrical conditions with bath solution 300 mMKCl and pipette solution 140 mMKCl, both containing 10 mMHepes-K, pH 7.4. Doted lines indicate the open state of the two channels (O1 and O2) present in the seal. C indicates the closed state of the channels. Histograms represent the unitary current corresponding to one or 2 channels at the indicated voltages. D. Open probability analysis of the single channel events. Values correspond to mean ± SEM from 14 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369953&req=5

ppat-1002750-g004: Biophysical characterization defines TcCat as an inward rectifier channel.A. Representative current traces applying a voltage-step protocol from −80 to 80 mV in 20 mV steps. The recordings were obtained under symmetrical conditions in the absence of Mg2+ with bath and pipette solution containing 140 mMKCl, 10 mMHepes-K, pH 7.4. B. Current-voltage relationship under symmetrical conditions described in (A). Data correspond to the unitary currentsrecorded in continuous voltage steps at the indicated holding potentials in the absence (open circles, n = 14) or in the presence of 1 mM MgCl2 in the bath solution (solid squares, n = 13). The non-linear relationship indicates the inward rectification. C. Current traces obtained at the indicated holding potentials showing the functional association of TcCat in clusters under asymmetrical conditions with bath solution 300 mMKCl and pipette solution 140 mMKCl, both containing 10 mMHepes-K, pH 7.4. Doted lines indicate the open state of the two channels (O1 and O2) present in the seal. C indicates the closed state of the channels. Histograms represent the unitary current corresponding to one or 2 channels at the indicated voltages. D. Open probability analysis of the single channel events. Values correspond to mean ± SEM from 14 independent experiments.
Mentions: The activity of TcCat was detected in patches excised from cell-size giant liposomes (inside-out configuration) containing the purified recombinant protein (Text S1, Figs. S4 and S5, and Table S1).Currents from liposomes containing only asolectin were recorded as control (Fig. S6A,black squares), showing a significant lower level compared with currents from liposomes containing purified TcCat (Fig. S6A, red circles). Currents were recorded under symmetrical conditions in the absence of Mg2+, unless stated otherwise, with bath and pipette solutions containing 140 mMKCl, 10 mMHepes-K, pH 7.4. Single channel currents were observed when an increasing voltage-pulse protocol between −80 to +80 mV was applied (Fig. 4A). The current-potential relationship for the single channel was not linear in the presence of Mg2+, as expected for an inward rectifier channel. The chord conductance (γ) (Fig. 4B, open circles) calculated under symmetrical KCl in the absence of Mg2+was 77±4 pS and 59±2 pS at −80 and +80 mV, respectively (n = 14) indicating a slight intrinsic rectification. Although no significant reduction in the current was observed at positive potentials in the presence of Mg2+, a significant increase in the inward current was evident at negative potentials in the presence of 1 mM MgCl2 in the bath solution (Fig. 4B, black squares), with unitary conductances of 122±7 pS and 56±3 pS at −80 and +80 mV, respectively (n = 13). These results suggest that the mechanism of blockage by Mg2+ is different from the one described for inward rectifier K+ channels. The unitary level of current was frequently observed in clusters, as shown in Fig. 4C where at least two channels could be detected, opening and closing independently. The histograms showncorrespond to the unitary current of one or two channels at the indicated voltages (Fig. 4C). This recorded activity agrees well with the localization in patches described above. Important variations in the open probability were observed in recordings from different days. When 14 independent experiments were analyzed, the open probability was not significantly sensitive to voltage, with values of 0.26±0.04 and 0.2±0.04 at −80 and +80 mV, respectively (Fig. 4D).

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