A yeast expression system for functional and pharmacological studies of the malaria parasite Ca 2+ /H + antiporter
Abstract Background Calcium (Ca 2+ ) signalling is fundamental for host cell invasion, motility, in vivo synchronicity and sexual differentiation of the malaria parasite. Consequently, cytoplasmic free Ca 2+ is tightly regulated through the co-ordinated action of primary and secondary Ca 2+ transpor...
| Published in: | Malaria Journal |
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| Main Authors: | , , |
| Format: | Article in Journal/Newspaper |
| Language: | English |
| Published: |
BMC
2012
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| Subjects: | |
| Online Access: | https://doi.org/10.1186/1475-2875-11-254 https://doaj.org/article/29169cbb5c0d43309f26865baaef57b6 |
| Summary: | Abstract Background Calcium (Ca 2+ ) signalling is fundamental for host cell invasion, motility, in vivo synchronicity and sexual differentiation of the malaria parasite. Consequently, cytoplasmic free Ca 2+ is tightly regulated through the co-ordinated action of primary and secondary Ca 2+ transporters. Identifying selective inhibitors of Ca 2+ 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 Ca 2+ /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 Ca 2+ , Mg 2+ and Mn 2+ 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 Ca 2+ 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 Ca 2+ -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 ... |
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