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

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.

Figure 1. TcCat expression and localization in T. cruzi life stages.

TcCat immunolocalization (green) in T. cruzi trypomastigotes (A), epimastigotes (B), extracellular amastigotes (C), intracellular amastigotes (D) and metacyclictrypomastigotes (E). Nuclei were DAPI stained. Bars = 10 µm. F, G. Immunoelectron microscopy localization of TcCat in trypomastigotes with purified anti-TcCat and secondary anti-rabbit gold-labeled antibody. Bars: F = 0.5 µm, G = 0.2 µm. H. Western blot analysis of TcCat expression in T. cruzi homogenates. Lanes: M: molecular weight markers in kDa (MagicMark XP, Invitrogen), T: trypomastigotes, E: epimastigotes, A: extracellular amastigotes. Bottom: membranes were stripped and re-incubated with anti-tubulin antibody as a loading control. I. Densitometry of TcCat detection by western blot analysis in arbitrary units (AU). T: trypomastigotes, E: epimastigotes, A: extracellular amastigotes. Values in arbitrary units (AU) correspond to mean ± SEM from 3 independent experiments.

Figure 2. Changes in TcCat localization during differentiation.

TcCat immunolocalization (green) at different time points after mammalian cell infection (A–D) or during in vitro differentiation of trypomastigotes to amastigotes at acidic pH (E–H). Yellow arrows indicate trypomastigote-like morphology and red arrows indicate amastigote-like forms at 5 h post-infection. Nuclei were DAPI stained (blue). Bars = 10 µm.

Figure 3. 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.

Figure 4. 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.

Figure 5. 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⁺ (P_X/P_K). 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.

Figure 6. 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.

Figure 7. 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 BaCl₂ open triangles; 100 µM 4-AP, black squares). Values are mean ± SEM of n = 3 independent experiments.