Dataset Information

Ca²⁺ entry at the plasma membrane and uptake by acidic stores is regulated by the activity of the V-H⁺ -ATPase in Toxoplasma gondii.

ABSTRACT: Ca²⁺ is a universal intracellular signal that regulates many cellular functions. In Toxoplasma gondii, the controlled influx of extracellular and intracellular Ca²⁺ into the cytosol initiates a signaling cascade that promotes pathogenic processes like tissue destruction and dissemination. In this work, we studied the role of proton transport in cytosolic Ca²⁺ homeostasis and the initiation of Ca²⁺ signaling. We used a T. gondii mutant of the V-H⁺ -ATPase, a pump previously shown to transport protons to the extracellular medium, and to control intracellular pH and membrane potential and we show that proton gradients are important for maintaining resting cytosolic Ca²⁺ at physiological levels and for Ca²⁺ influx. Proton transport was also important for Ca²⁺ storage by acidic stores and, unexpectedly, the endoplasmic reticulum. Proton transport impacted the amount of polyphosphate (polyP), a phosphate polymer that binds Ca²⁺ and concentrates in acidocalcisomes. This was supported by the co-localization of the vacuolar transporter chaperone 4 (VTC4), the catalytic subunit of the VTC complex that synthesizes polyP, with the V-ATPase in acidocalcisomes. Our work shows that proton transport regulates plasma membrane Ca²⁺ transport and control acidocalcisome polyP and Ca²⁺ content, impacting Ca²⁺ signaling and downstream stimulation of motility and egress in T. gondii.

SUBMITTER: Stasic AJ

PROVIDER: S-EPMC9142151 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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Publications

Ca2+ entry at the plasma membrane and uptake by acidic stores is regulated by the activity of the V-H+ -ATPase in Toxoplasma gondii.

Stasic Andrew J AJ Dykes Eric J EJ Cordeiro Ciro D CD Vella Stephen A SA Fazli Mojtaba S MS Quinn Shannon S Docampo Roberto R Moreno Silvia N J SNJ

Molecular microbiology 20210419 5

Ca2+ is a universal intracellular signal that regulates many cellular functions. In Toxoplasma gondii, the controlled influx of extracellular and intracellular Ca2+ into the cytosol initiates a signaling cascade that promotes pathogenic processes like tissue destruction and dissemination. In this work, we studied the role of proton transport in cytosolic Ca2+ homeostasis and the initiation of Ca2+ signaling. We used a T. gondii mutant of the V-H+< ...[more]

PMID: 33793004

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Project description:AimsPhospholamban (PLB) is the key regulator of the cardiac Ca2+ pump (SERCA2a)-mediated sarcoplasmic reticulum Ca2+ stores. We recently reported that PLB is highly concentrated in the nuclear envelope (NE) from where it can modulate perinuclear Ca2+ handling of the cardiomyocytes (CMs). Since inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) mediates nuclear Ca2+ release, we examined whether the nuclear pool of PLB regulates IP3-induced nuclear Ca2+ handling.Methods and resultsFluo-4 based confocal Ca2+ imaging was performed to measure Ca2+ dynamics across both nucleus and cytosol in saponin-permeabilized CMs isolated from wild-type (WT) or PLB-knockout (PLB-KO) mice. At diastolic intracellular Ca2+ ([Ca2+]i = 100 nM), the Fab fragment of the monoclonal PLB antibody (anti-PLB Fab) facilitated the formation and increased the length of spontaneous Ca2+ waves (SCWs) originating from the nuclear region in CMs from WT but not from PLB-KO mice. We next examined nuclear Ca2+ activities at basal condition and after sequential addition of IP3, anti-PLB Fab, and the IP3R inhibitor 2-aminoethoxydiphenyl borate (2-APB) at a series of [Ca2+]i. In WT mice, at 10 nM [Ca2+]i where ryanodine receptor (RyR2) based spontaneous Ca2+ sparks rarely occurred, IP3 increased fluorescence amplitude (F/F0) of overall nuclear region to 1.19 ± 0.02. Subsequent addition of anti-PLB Fab significantly decreased F/F0 to 1.09 ± 0.02. At 50 nM [Ca2+]i, anti-PLB Fab not only decreased the overall nuclear F/F0 previously elevated by IP3, but also increased the amplitude and duration of spark-like nuclear Ca2+ release events. These nuclear Ca2+ releases were blocked by 2-APB. At 100 nM [Ca2+]i, IP3 induced short SCWs originating from nucleus. Anti-PLB Fab transformed those short waves into long SCWs with propagation from the nucleus into the cytosol. In contrast, neither nuclear nor cytosolic Ca2+ dynamics was affected by anti-PLB Fab in CMs from PLB-KO mice in all these conditions. Furthermore, in WT CMs pretreated with RyR2 blocker tetracaine, IP3 and anti-PLB Fab still increased the magnitude of nuclear Ca2+ release but failed to regenerate SCWs. Finally, anti-PLB Fab increased low Ca2+ affinity mag-fluo 4 fluorescence intensity in the lumen of NE of nuclei isolated from WT but not in PLB-KO mice.ConclusionPLB regulates nuclear Ca2+ handling. By increasing Ca2+ uptake into lumen of the NE and perhaps other perinuclear membranes, the acute reversal of PLB inhibition decreases global Ca2+ concentration at rest in the nucleoplasm, and increases Ca2+ release into the nucleus, through mechanisms involving IP3R and RyR2 in the vicinity.

Dataset Information

Ca²⁺ entry at the plasma membrane and uptake by acidic stores is regulated by the activity of the V-H⁺ -ATPase in Toxoplasma gondii.

Publications

Ca<sup>2+</sup> entry at the plasma membrane and uptake by acidic stores is regulated by the activity of the V-H<sup>+</sup> -ATPase in Toxoplasma gondii.

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