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ABSTRACT: Background
Calcium (Ca²?) signalling is fundamental for host cell invasion, motility, in vivo synchronicity and sexual differentiation of the malaria parasite. Consequently, cytoplasmic free Ca²? is tightly regulated through the co-ordinated action of primary and secondary Ca²? transporters. Identifying selective inhibitors of Ca²? transporters is key towards understanding their physiological role as well as having therapeutic potential, therefore screening systems to facilitate the search for potential inhibitors are a priority. Here, the methodology for the expression of a Calcium membrane transporter that can be scaled to high throughputs in yeast is presented.Methods
The Plasmodium falciparum Ca²?/H? antiporter (PfCHA) was expressed in the yeast Saccharomyces cerevisiae and its activity monitored by the bioluminescence from apoaequorin triggered by divalent cations, such as calcium, magnesium and manganese.Results
Bioluminescence assays demonstrated that PfCHA effectively suppressed induced cytoplasmic peaks of Ca²?, Mg²? and Mn²? in yeast mutants lacking the homologue yeast antiporter Vcx1p. In the scalable format of 96-well culture plates pharmacological assays with a cation antiporter inhibitor allowed the measurement of inhibition of the Ca²? transport activity of PfCHA conveniently translated to the familiar concept of fractional inhibitory concentrations. Furthermore, the cytolocalization of this antiporter in the yeast cells showed that whilst PfCHA seems to locate to the mitochondrion of P. falciparum, in yeast PfCHA is sorted to the vacuole. This facilitates the real-time Ca²?-loading assays for further functional and pharmacological studies.Discussion
The functional expression of PfCHA in S. cerevisiae and luminescence-based detection of cytoplasmic cations as presented here offer a tractable system that facilitates functional and pharmacological studies in a high-throughput format. PfCHA is shown to behave as a divalent cation/H? antiporter susceptible to the effects of cation/H? inhibitors such as KB-R7943. This type of gene expression systems should advance the efforts for the screening of potential inhibitors of this type of divalent cation transporters as part of the malaria drug discovery initiatives and for functional studies in general.Conclusion
The expression and activity of the PfCHA detected in yeast by a bioluminescence assay that follows the levels of cytoplasmic Ca²? as well as Mg²? and Mn²? lend itself to high-throughput and quantitative settings for pharmacological screening and functional studies.
SUBMITTER: Salcedo-Sora JE
PROVIDER: S-EPMC3488005 | biostudies-literature | 2012 Aug
REPOSITORIES: biostudies-literature
Malaria journal 20120801
<h4>Background</h4>Calcium (Ca²⁺) signalling is fundamental for host cell invasion, motility, in vivo synchronicity and sexual differentiation of the malaria parasite. Consequently, cytoplasmic free Ca²⁺ is tightly regulated through the co-ordinated action of primary and secondary Ca²⁺ transporters. Identifying selective inhibitors of Ca²⁺ transporters is key towards understanding their physiological role as well as having therapeutic potential, therefore screening systems to facilitate the sear ...[more]