Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK.
Malar J. 2012 Aug 1;11:254. doi: 10.1186/1475-2875-11-254.
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.
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.
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.
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.
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.
钙（Ca²⁺）信号对于宿主细胞入侵、运动、疟原虫体内同步性和有性分化至关重要。因此，细胞质游离 Ca²⁺通过初级和次级 Ca²⁺转运体的协调作用进行严格调节。鉴定 Ca²⁺转运体的选择性抑制剂对于理解其生理作用以及具有治疗潜力至关重要，因此筛选系统以促进潜在抑制剂的寻找是当务之急。在这里，介绍了在酵母中表达可以大规模应用于高通量的钙膜转运蛋白的方法。
在酵母酿酒酵母中表达了恶性疟原虫 Ca²⁺/H⁺反向转运蛋白（PfCHA），并通过二价阳离子（如钙、镁和锰）触发的脱辅基 apoaequorin 的生物发光来监测其活性。
生物发光测定表明，PfCHA 可有效抑制缺乏同源酵母反向转运蛋白 Vcx1p 的酵母突变体中诱导的细胞质 Ca²⁺、Mg²⁺和 Mn²⁺峰。在可扩展的 96 孔培养板格式中，使用阳离子反向转运体抑制剂进行药理学测定，方便地测量 PfCHA 的 Ca²⁺转运活性的抑制作用，方便地转化为熟悉的分数抑制浓度概念。此外，该反向转运体在酵母细胞中的细胞定位表明，虽然 PfCHA 似乎定位于恶性疟原虫的线粒体，但在酵母中 PfCHA 被分拣到液泡中。这为进一步的功能和药理学研究提供了实时 Ca²⁺加载测定。
本文介绍了 PfCHA 在酿酒酵母中的功能表达和基于细胞质阳离子的发光检测，提供了一种易于处理的系统，可在高通量格式中进行功能和药理学研究。PfCHA 表现为一种二价阳离子/H⁺反向转运体，易受阳离子/H⁺抑制剂（如 KB-R7943）的影响。这种基因表达系统应有助于筛选这种类型的二价阳离子转运体的潜在抑制剂，作为疟疾药物发现计划的一部分，并用于一般的功能研究。
PfCHA 在酵母中的表达和活性通过生物发光测定来检测，该测定通过细胞质 Ca²⁺以及 Mg²⁺和 Mn²⁺的水平来检测，适合高通量和定量的药理学筛选和功能研究。
