8 research outputs found
Influence of growth conditions on physicochemical surface characteristics of two gramnegative and two grampositive bacteria
A yeast expression system for functional and pharmacological studies of the malaria parasite Ca<sup>2+</sup>/H<sup>+</sup> antiporter
<p>Abstract</p> <p>Background</p> <p>Calcium (Ca<sup>2+</sup>) signalling is fundamental for host cell invasion, motility, <it>in vivo</it> synchronicity and sexual differentiation of the malaria parasite. Consequently, cytoplasmic free Ca<sup>2+</sup> is tightly regulated through the co-ordinated action of primary and secondary Ca<sup>2+</sup> transporters. Identifying selective inhibitors of Ca<sup>2+</sup> 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.</p> <p>Methods</p> <p>The <it>Plasmodium falciparum</it> Ca<sup>2+</sup>/H<sup>+</sup> antiporter (PfCHA) was expressed in the yeast <it>Saccharomyces cerevisiae</it> and its activity monitored by the bioluminescence from apoaequorin triggered by divalent cations, such as calcium, magnesium and manganese.</p> <p>Results</p> <p>Bioluminescence assays demonstrated that PfCHA effectively suppressed induced cytoplasmic peaks of Ca<sup>2+</sup>, Mg<sup>2+</sup> and Mn<sup>2+</sup> 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<sup>2+</sup> 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 <it>P. falciparum</it>, in yeast PfCHA is sorted to the vacuole. This facilitates the real-time Ca<sup>2+</sup>-loading assays for further functional and pharmacological studies.</p> <p>Discussion</p> <p>The functional expression of PfCHA in <it>S. cerevisiae</it> 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<sup>+</sup> antiporter susceptible to the effects of cation/H<sup>+</sup> 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.</p> <p>Conclusion</p> <p>The expression and activity of the PfCHA detected in yeast by a bioluminescence assay that follows the levels of cytoplasmic Ca<sup>2+</sup> as well as Mg<sup>2+</sup> and Mn<sup>2+</sup> lend itself to high-throughput and quantitative settings for pharmacological screening and functional studies.</p
Heteromeric Solute Carriers: Function, Structure, Pathology and Pharmacology
Solute carriers form one of three major superfamilies of membrane transporters in humans, and include uniporters, exchangers and symporters. Following several decades of molecular characterisation, multiple solute carriers that form obligatory heteromers with unrelated subunits are emerging as a distinctive principle of membrane transporter assembly. Here we comprehensively review experimentally established heteromeric solute carriers: SLC3-SLC7 amino acid exchangers, SLC16 monocarboxylate/H+ symporters and basigin/embigin, SLC4A1 (AE1) and glycophorin A exchanger, SLC51 heteromer Ost α-Ost β uniporter, and SLC6 heteromeric symporters. The review covers the history of the heteromer discovery, transporter physiology, structure, disease associations and pharmacology - all with a focus on the heteromeric assembly. The cellular locations, requirements for complex formation, and the functional role of dimerization are extensively detailed, including analysis of the first complete heteromer structures, the SLC7-SLC3 family transporters LAT1-4F2hc, b0,+AT-rBAT and the SLC6 family heteromer B0AT1-ACE2. We present a systematic analysis of the structural and functional aspects of heteromeric solute carriers and conclude with common principles of their functional roles and structural architecture