Barrera P et al. (2020), A Novel Calcium-Activated Potassium Channel Con...

XB-ART-56668

Front Cell Infect Microbiol 2020 Jan 15;9:464. doi: 10.3389/fcimb.2019.00464.

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A Novel Calcium-Activated Potassium Channel Controls Membrane Potential and Intracellular pH in Trypanosoma cruzi.

Barrera P , Skorka C , Boktor M , Dave N , Jimenez V .

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Trypanosoma cruzi develops in environments where nutrient availability, osmolarity, ionic concentrations, and pH undergo significant changes. The ability to adapt and respond to such conditions determines the survival and successful transmission of T. cruzi. Ion channels play fundamental roles in controlling physiological parameters that ensure cell homeostasis by rapidly triggering compensatory mechanisms. Combining molecular, cellular and electrophysiological approaches we have identified and characterized the expression and function of a novel calcium-activated potassium channel (TcCAKC). This channel resides in the plasma membrane of all 3 life stages of T. cruzi and shares structural features with other potassium channels. We expressed TcCAKC in Xenopus laevis oocytes and established its biophysical properties by two-electrode voltage clamp. Oocytes expressing TcCAKC showed a significant increase in inward currents after addition of calcium ionophore ionomycin or thapsigargin. These responses were abolished by EGTA suggesting that TcCAKC activation is dependent of extracellular calcium. This activation causes an increase in current and a negative shift in reversal potential that is blocked by barium. As predicted, a single point mutation in the selectivity filter (Y313A) completely abolished the activity of the channels, confirming its potassium selective nature. We have generated knockout parasites deleting one or both alleles of TcCAKC. These parasite strains showed impaired growth, decreased production of trypomastigotes and slower intracellular replication, pointing to an important role of TcCAKC in regulating infectivity. To understand the cellular mechanisms underlying these phenotypic defects, we used fluorescent probes to evaluate intracellular membrane potential, pH, and intracellular calcium. Epimastigotes lacking the channel had significantly lower cytosolic calcium, hyperpolarization, changes in intracellular pH, and increased rate of proton extrusion. These results are in agreement with previous reports indicating that, in trypanosomatids, membrane potential and intracellular pH maintenance are linked. Our work shows TcCAKC is a novel potassium channel that contributes to homeostatic regulation of important physiological processes in T. cruzi and provides new avenues to explore the potential of ion channels as targets for drug development against protozoan parasites.

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Species referenced: Xenopus laevis
Genes referenced: atp2b1 dtl

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	Figure 1. TcCAKC localization. (A) Immunofluorescence analysis of T. cruzi trypomastigotes (T), epimastigotes (E), and amastigotes (A) with polyclonal antibodies against the channel (green). (B) Trypomastigotes immunofluorescence showing TcCAKC (green) colocalization with membrane marker SSP-1 (red), and calcium binding proteins FCaBP and calmodulin. Nuclei and kinetoplasts were DAPI stained. Bar size: 10 μm.
	Figure 2. Electrophysiological characterization of TcCAKC. (A) Current-voltage relationship of oocytes expressing TcCAKC in the absence (black) or presence (red) of 1 μM ionomycin. Control currents are obtained from oocytes injected with DEPC water and treated with 1 μM ionomycin (blue line). Representative traces are shown in the right panel. Values are Mean ± SD of n = 18 oocytes. (B) Current-voltage relationship of oocytes expressing TcCAKC (red) or control (blue) preincubated with thapsigargin (n = 15). TcCAKC currents in absence of thapsigargin are indicated in black. Representative traces are shown in the right panel. All recordings are in ND-96 buffer with a holding potential of −60 and 20 mV step protocol between −80 and 40 mV. (C) Maximum currents at −80 mV (in absolute values) for oocytes under the indicated conditions. Calcium free conditions were achieved by addition of 1 mM EGTA in ND 96 buffer without added CaCl2. Values are Mean ± SD of n = 15 oocytes. p < 0.01 respect to the WT, *p < 0.01 respect to the corresponding condition without EGTA.
	Figure 3. Blockage characteristics of TcCAKC. (A) Current-voltage relationship showing the effect of potassium channel blockers on thapsigargin-elicited TcCAKC currents (red line). One millimolar of extracellular BaCl2 significantly reduced the current (black line) while up to 300 μM 4-AP had no significant effect. Values are Mean ± SD of n = 10 oocytes from 3 independent days or recording. Representative traces are shown in the right panel. (B) Current-voltage relationship of oocytes expressing WT TcCAKC in absence (black line) or presence (red line) of ionomycin or TcCAKC-Y313A mutant (doted green and blue lines). Values are Mean ± SD of n = 15 oocytes from 3 independent experiments. Representative traces are shown in the right panel. (C) Quantification of the reversal potential calculated from the currents obtained under ramp protocols in control oocytes (black), cells expressing TcCAKC (red) or TcCAKC-Y313A (green)Values are Mean ± SD of n = 35 oocytes from 7 independent experiments.
	Figure 4. Effect of TcCAKC knockout in T. cruzi fitness. (A) Schematic representation of the allelic replacement strategy for TcCAKC and genomic DNA screening showing the correct insertion of the drug resistance cassettes, screened with primers P1–P2 (neomycin) and P1–P3 (hygromycin). (B) qPCR analysis of TcCAKC expression levels in epimastigotes wild type (WT), one (sKO) or both (dKO) TcCAKC alleles replaced. Values are expressed as ΔΔCq and normalized using GAPDH and tubulin as housekeeping genes. Mean ± SD of 3 independent experiments. ,p < 0.01 respect to WT. (C) Growth curve of epimastigotes WT (black), sKO (blue) and dKO (red). Mean ± SD of 3 independent experiments. ,p < 0.05 respect to WT at day 5 of growth. (D) Quantification of intracellular amastigotes comparing WT and dKO parasites at 48 h post-infection. At least 100 host cells were counted in 4 coverslips per experiment, 3 independent experiments. Mean ± SD of 3 independent experiments, p < 0.01. (E) Representative images of infections quantified in (D). The cells were fixed and DAPI stained for quantification.
	Figure 5. Membrane potential. (A) Resting membrane potential of epimastigotes WT (black), TcCAKC sKO (blue) and TcCAKC dKO (red) in standard buffer with 1 μM DisBac2(3). (B) Resting membrane potential of epimastigotes in standard buffer (solid bars) or NMDG buffer (striped bars). Values represent Mean ± SD of 5 independent experiments. *p < 0.05 compared with WT, #p < 0.05 compared with the values obtained in standard buffer.
	Figure 6. Intracellular pH and proton extrusion. (A) Intracellular pH measurements of epimastigotes in standard (solid bars) or NMDG buffer (striped bars). Values represent Mean ± SD of 4 independent experiments. # indicates p < 0.05 compared with WT, p < 0.05 compared with the values obtained in standard buffer. (B) Effect of BaCl2 on pH regulation. Values of intracellular pH were compared in standard buffer with (dotted bars) or without (solid bars) 1 mM BaCl2. p < 0.05 from 4 independent experiments. (C) Representative traces showing proton extrusion in epimastigotes WT (black trace) or TcCAKC dKO (red trace) measured with BCECF free acid. (D) Slope analysis for the first 50 s of proton extrusion under standard low buffering solution conditions (WT: black bar, dKO: red bar), K+-free (white bars), Na+ free (gray bars) or NMDG buffer (striped bars). Values are Mean ± SD of 4 independent experiments. p < 0.05 compared with WT in standard buffer, *p < 0.05 for each strain in K+ free compared with the values obtained in standard buffer, #p < 0.05 of Na+ free or NMDG buffer compared with standard conditions.
	Figure 7. Intracellular calcium measurements. (A) Representative traces of Fura-2 AM loaded epimastigotes showing intracellular calcium concentrations for WT (black trace), sKO (blue trace) or dKO (red trace) in BAG. At 50 s of recording, 1.8 mM CaCl2 was added to the extracellular buffer. (B) Quantification of baseline intracellular calcium in BAG prior to extracellular calcium addition. Values are Mean ± SD of 5 independent experiments. p < 0.05 compared with WT. (C,D) Representative traces of intracellular calcium measurements (in relative fluorescence units for comparison purposes) of WT (C) and TcCAKS dKO (D), in the indicated buffer conditions. (E) Intracellular calcium changes (in relative units) upon CaCl2 addition in WT (black), sKO (blue), and dKO (red) epimastigotes in BAG (solid bars), Na+ free (dotted bars), K+ free (horizontal stripe bars) or NMDG buffer (striped bars). Values are Mean ± SD of 5 independent experiments. p < 0.05 compared with BAG, **p < of each condition and strain compared with WT in BAG. Statistical analysis for multiple conditions was done by two-way ANOVA with post-hoc Bonferroni correction.
	Figure 8. Proposed model of ionic homeostasis in T. cruzi. H+ ATPases maintain the gradient of protons that drive the resting membrane potential, which is about −100 mV in epimastigotes. Additionally, Na+/H+ and K+/H+ exchangers contribute to H+ extrusion mobilizing other monovalent ions. Intracellular potassium concentration is balanced through the combined action of channels and exchangers. Given the negative membrane potential beyond the theoretical equilibrium potential for K+ (~80 mV), activation of TcCAKC can mediate K+ efflux until the membrane potential reaches values higher than the equilibrium potential, when the electrochemical gradient will drive K+ out of the cells. Depolarization of the membrane could then activate voltage-gated calcium channels (VGCC), responsible for oscillations of the intracellular Ca2+ concentration. For simplicity purposes, in this model we have omitted PMCA and SERCA-like pumps that participate in intracellular calcium homeostasis.

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