Calcium-dependent protein kinase from Plasmodium falciparum (PfCPK) is a multidomain protein composed of an N-terminal kinase domain connected via a linker region to a C-terminal CaM-like calcium-binding domain. The kinase can be activated by Ca2+ alone and associates with 45Ca2+. Here we describe the calcium-binding properties of the kinase and the significance of the individual calcium-binding sites with respect to enzymatic activation, as well as the Ca(2+)-induced conformational change as detected by circular dichroism. As predicted from the cDNA sequence, the kinase has four EF-hand calcium-binding sites in the C-terminal domain. To understand the roles of the individual calcium-binding sites, two series of mutations were generated at the individual EF-hand motifs. The highly conserved glutamic acid residue at position 12 in each calcium-binding loop was mutated to either lysine or glutamine, and therefore a total of eight mutants were generated. Either of these mutations (to lysine or glutamine) is sufficient to eliminate calcium binding at the mutated site. Sites I and II appear to be crucial for both Ca(2+)-induced conformational change and enzymatic activation. Whereas mutations at site II almost completely abolish kinase activity, mutations at site I are also deleterious and dramatically reduce the sensitivity of the Ca(2+)-induced conformational change and the Ca(2+)-dependent activation. Mutations at sites III and IV have minor effects.

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A P-glycoprotein homologue (Pgh1) is believed to play a role in modulating levels of chloroquine resistance in Plasmodium falciparum. To study the role of Pgh1 in the mechanism of chloroquine (CQ) resistance, antisera were raised against this protein. There was no direct association between the level of Pgh1 expression and chloroquine sensitivity. We also failed to detect phosphorylation of Pgh1 in the food vacuole (FV), suggesting that other mechanisms regulate the chloroquine-resistant (CQR) phenotype. Therefore, high levels of expression of Pgh1 or phosphorylation of this protein in the FV could not account for CQ sensitivity. In addition, the lack of inhibition of CQ accumulation by anti-Pgh1 antibodies suggests that Pgh1 is not involved as a CQ transporter in the plasma membrane of P. falciparum. Furthermore, resistance reversers do not appear to act at the plasma membrane level.