Published On: Mon, Apr 1st, 2019

Simultaneous expression of ClopHensor and SLC26A3 reveals the nature of endogenous oxalate transport in CHO cells [METHODS AND TECHNIQUES]

INTRODUCTION

The fluorescent fusion protein, ClopHensor, has previously been reported as a promising tool for simultaneous measurement of intracellular chloride and pH in live cells (Arosio et al., 2010; Mukhtarov et al., 2013). This sensor has a modified green fluorescent protein, E2GFP, whose green fluorescence is sensitive to pH, and both green and cyan fluorescence are sensitive to chloride concentration (Arosio et al., 2007). Measuring the green-to-cyan fluorescence ratio precludes the influence of chloride on pH measurement, as chloride affects both signals equally, whereas pH affects only the green signal. This has been described as static quenching, wherein chloride binding to E2GFP completely inhibits fluorescence and thus prevents alterations in the ratiometric measurement of pH (Arosio et al., 2010). ClopHensor also possesses a red fluorescent protein, monomeric DsRed, whose signal intensity is not affected by pH or chloride. Therefore, after constructing chloride standard curves at different pH values, one can measure the absolute chloride concentration using the appropriate chloride-pH curve, dictated by the pH calculated from green:cyan fluorescence. Moreover, a critical benefit of the DsRed monomer (or any fused fluorophore not affected by pH or chloride) is that it provides an internal normalizer, so that variations in cell number, or magnitude of expression, from well to well do not produce variations in the signal ratio. This is an advantage over single-fluorophore sensors, which have been successfully used in high-throughput assays, but necessarily preclude ratiometric measurements (Haggie et al., 2018). Other major advantages of genetically encoded fluorophores include resistance to photobleaching, absence of permeation/loss across the plasma membrane, and synthesis by the cell rather than exogenous administration. Alternative methods generally employ small-molecule dyes that have transient residence in the cytosol, and must be washed out of the extracellular fluid before analysis (Chub et al., 2006; Haggie et al., 2016; Ikeuchi et al., 2018; Untiet et al., 2017). Modern multi-well plate reader type fluorometers come with many advantages over microscopy. For example, they can be used for automated kinetic assays, possess on-board temperature regulators, can measure multiple excitation and emission wavelength pairs over relatively short durations, and can be used to calculate average fluorescent signals within a confluent well, which minimizes the influence of artefactual signals that can be found in single-cell microscopy. Furthermore, a multi-well format is often necessary for high-throughput screening of potential ligands or substrates for transporters and receptors.

SLC26A3, or DRA (downregulated in adenoma), is a transporter expressed in mammals, including rodents and humans, that exchanges chloride for bicarbonate. Its predominant role appears to be in the colon, where loss of function leads to severe congenital chloride losing diarrhea (Höglund et al., 1996). However, SLC26A3 has been proposed to bear another role in pathophysiology, as deletion in mice decreased serum oxalate by 60% and 24 h urinary oxalate excretion by 70% (Freel et al., 2013). Oxalate is a component of approximately 80% of kidney stones, giving this simple divalent anion a major role in renal disease (Sakhaee, 2009). There is ongoing debate about the relevance of SLC26A3 to colonic oxalate absorption, especially in humans. One study found a significant, but modest (50%), increase in oxalate absorption in Xenopus laevis oocytes expressing hSLC26A3 (Chernova et al., 2003) and investigators deemed the transport weak. However, it was not clear in the study if chloride, a substrate, and hence competitor, was excluded from the extracellular transport buffer. Moreover, in the aforementioned mouse study by Freel et al., the reduction in colonic mucosal to serosal flux of oxalate in Slc26a3 knockout mice was only 41%, despite a very clear influence of the transporter on urinary oxalate. SLC26A3 does not appear to be expressed in kidney, indicating that urinary oxalate was altered by a change in colonic absorption, and hence, the blood concentration. Therefore, the relevance of SLC26A3 to oxalate absorption cannot be fully determined, or ruled out, solely on in vitro evidence, as a 41% decrease in transport may be very clinically significant if hSLC26A3 is the sole carrier mediating colonic oxalate absorption. Indeed, this has been proposed (Whittamore and Hatch, 2017).

Chinese hamster ovary (CHO) cells are the most widely utilized mammalian cell type in the pharmaceutical industry for production of therapeutic proteins (Butler and Spearman, 2014). CHO cells are also widely used in the academic research setting. Their extensive use stems from their relatively simple handling requirements, suspension and adherent growth, simple medium, and their ability to assimilate and express foreign genes with protein glycosylation patterns similar to human (Butler and Spearman, 2014). The entire CHO cell genome has been sequenced and published (Dahodwala and Sharfstein, 2017). CHO cells can be engineered to stably and constitutively express genes, but are also amenable to inducible expression systems, such as various forms of tetracycline-on and tetracycline-off systems.

Here, we have employed CHO cells stably transfected with constitutively expressed ClopHensor, along with stably inserted tetracycline-inducible hSLC26A3 (SLC26A3-ClopHensor-CHO) to simultaneously determine the role of hSLC26A3 in oxalate transport, and gain some mechanistic insight about the strong endogenous oxalate transport function that we have discovered in our untransfected CHO cells. Employing these tools, we have achieved the following outcomes. (1) We confirmed that excellent chloride and pH standard curves could be generated with ClopHensor in a 96-well format, with pH-dependent chloride affinity values close to those reported using single-cell fluorescence microscopy. (2) We determined that live SLC26A3-ClopHensor-CHO cells could be effectively used to measure chloride transport and intracellular pH, and that bicarbonate exchange for chloride on SLC26A3 could be reliably and rapidly measured in this 96-well format. (3) We determined that an endogenous transport function mediating oxalate influx into CHO cells exists, and it is saturable, strong and sensitive to the inhibitor, niflumic acid. (4) We revealed that the endogenous oxalate transporter was unable to transport chloride, or specifically, was unable to exchange chloride for bicarbonate, unlike SLC26A3. The nature of the oxalate transport is intriguing, as niflumic acid is traditionally used to inhibit chloride transporters that, in some cases, also transport oxalate. In this case, CHO cells appear to express an oxalate transporter that is niflumate-sensitive, but that may not transport chloride. To date, all investigations on ClopHensor and derivatives (e.g. ClopHensorN) have used single cells with microscopy. Here, we report the successful application of ClopHensor in a 96-well assay using live adherent CHO cells.

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