The genetic Ca2+ sensor GCaMP3 reveals multiple Ca2+ stores differentially coupled to Ca2+ entry in the human malaria parasite Plasmodium falciparum

Abstract

Cytosolic Ca²⁺ regulates multiple steps in the host-cell invasion, growth, proliferation, and egress of blood-stage <i>Plasmodium falciparum</i>, yet our understanding of Ca²⁺ signaling in this endemic malaria parasite is incomplete. By using a newly generated transgenic line of <i>P. falciparum</i> (PfGCaMP3) that expresses constitutively the genetically encoded Ca²⁺ indicator GCaMP3, we have investigated the dynamics of Ca²⁺ release and influx elicited by inhibitors of the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase pumps, cyclopiazonic acid (CPA), and thapsigargin (Thg). Here we show that in isolated trophozoite phase parasites: (i) both CPA and Thg release Ca²⁺ from intracellular stores in <i>P. falciparum</i> parasites; (ii) Thg is able to induce Ca²⁺ release from an intracellular compartment insensitive to CPA; (iii) only Thg is able to activate Ca²⁺ influx from extracellular media, through a mechanism resembling store-operated Ca²⁺ entry, typical of mammalian cells; and (iv) the Thg-sensitive Ca²⁺ pool is unaffected by collapsing the mitochondria membrane potential with the uncoupler carbonyl cyanide <i>m</i>-chlorophenyl hydrazone or the release of acidic Ca²⁺ stores with nigericin. These data suggest the presence of two Ca²⁺ pools in <i>P. falciparum</i> with differential sensitivity to the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase pump inhibitors, and only the release of the Thg-sensitive Ca²⁺ store induces Ca²⁺ influx. Activation of the store-operated Ca²⁺ entry-like Ca²⁺ influx may be relevant for controlling processes such as parasite invasion, egress, and development mediated by kinases, phosphatases, and proteases that rely on Ca²⁺ levels for their activation.