Figure 3. The role of TgTRPPL-2 in PM Ca²⁺ influx.

(A) Scheme showing the mechanism of Ca²⁺ influx and how cytosolic Ca²⁺ may activate the PM channel (Ca²⁺-activated calcium entry). NSC: nifedipine-sensitive channel; PKG: protein kinase G; PDE: phosphodiesterase; Thap: thapsigargin; Zap: Zaprinast.(B) Cytosolic Ca²⁺ measurements of Fura-2 loaded tachyzoites of the parental (RH), ΔTgTRPPL-2 and ΔTgTRPPL-2-trppl2 lines. The buffer contains 100 μM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) to chelate contaminating Ca²⁺ and at 300 s, 1.8 mM Ca²⁺ were added to the suspension. The pink box indicates the area used for the quantification presented in (C). (C) Quantification and statistical analysis of the change in cytosolic Ca²⁺ during the first 20 s after addition of extracellular Ca²⁺. ***p<0.0002. (D) Constitutive Ca²⁺ influx into the cytosol of parasites suspended in a buffer with 1.8 mM Ca²⁺. (E) Quantification and statistical analysis of the slopes from (D). ****p<0.0001. (F) Cytosolic Ca²⁺ increase after adding Thap (1 µM) followed by Ca²⁺ influx after the addition of 1.8 mM extracellular Ca²⁺ at 400 s. The pink boxes indicate the area used for the quantification presented in (G) and (H). (G) Quantification and statistical analysis of the change in cytosolic Ca²⁺(Δ[Ca²⁺]_cyt) at 50 s after the addition of Thap. (H) Quantification of the Δ[Ca²⁺]_cyt 20 s after the addition of 1.8 mM of Ca²⁺. ***p<0.0008, ****p<0.0001. (I) Cytosolic Ca²⁺ increase stimulated by Zaprinast (100 µM) in the presence of 1.8 mM extracellular Ca²⁺. (J) Quantification and statistical analysis of the Δ[Ca²⁺]_cyt during the first 15 s after adding Zaprinast (100 µM) (pink box, in I). **p<0.001, ****p<0.0001. (K) Quantification and statistical analysis of the Δ[Ca²⁺]_cyt during the 20 s after adding Ca²⁺ without additions (ND) or after adding Thap or Zap. *p<0.02, **p<0.005, ***p<0.0008. Statistical analysis for all experiments was done from a minimum of three independent trials using Student’s t-test.

Figure 3—figure supplement 1. TgTRPPL-2 regulates Ca²⁺in T. gondii.

(A) Cytosolic Ca²⁺ measurements of Fura-2-loaded tachyzoites of the parental and ΔTgTRPPL-2 mutant after addition of 1 mM extracellular Ca²⁺ at 300 s. (B) Cytosolic Ca²⁺ measurement after the addition of Zaprinast (100 µM) at 100 s and Ca²⁺ influx stimulated by the addition of 1.8 mM extracellular Ca²⁺ at 400 s. (C) Change in cytosolic Ca²⁺ 15 s after the addition of Zaprinast (labeled Zaprinast) and 20 s after the addition of 1.8 mM of extracellular Ca²⁺ (labeled Ca²⁺after Zaprinast). Values are means ± SEM, n = 3, **p<0.007, ****p<0.0001. Measurements of the complemented strain are not shown as the concentration of calcium goes outside the Fura 2 range.