Elsevier

Biochemical Pharmacology

Volume 128, 15 March 2017, Pages 74-82
Biochemical Pharmacology

A novel mode of operation of SLC22A11: Membrane insertion of estrone sulfate versus translocation of uric acid and glutamate

https://doi.org/10.1016/j.bcp.2016.12.020Get rights and content

Abstract

Estrone sulfate alias estrone-3-sulfate (E3S) is considerably larger and much more hydrophobic than typical substrates of SLC22 transporters. It is puzzling that many otherwise unrelated transporters have been reported to transport E3S. Here we scrutinized the mechanism of transport of E3S by SLC22A11 (alias OAT4), by direct comparison with uric acid (UA), an important physiological substrate. Heterologous expression of SLC22A11 in human 293 cells gave rise to a huge unidirectional efflux of glutamate (Glu) and aspartate, as determined by LC–MS/MS. The uptake of E3S was 20-fold faster than the uptake of UA. Yet, the outward transport of Glu was inhibited by extracellular E3S, but not by UA. The release of E3S after preloading was trans-stimulated by extracellular dehydroepiandrosterone sulfate (DHEAS), but neither by UA nor 6-carboxyfluorescein (6CF). The equilibrium accumulation of E3S was enhanced 3-fold by replacement of chloride with gluconate, but the opposite effect was observed for UA. These results establish that SLC22A11 provides entirely different transport mechanisms for E3S and UA. Therefore, E3S must not be used as a substitute for UA to assay the function of SLC22A11. In equilibrium accumulation experiments, the transporter-mediated uptake was a linear function of the concentration of UA and 6CF. By contrast, in the same concentration range the graph for E3S was hyperbolic. This suggests that SLC22A11 inserts E3S into a small volume with limited capacity, the plasma membrane. Our data support the notion that the reverse process, extraction from the membrane, is also catalyzed by the carrier.

Introduction

In humans, normal blood serum levels of uric acid (UA) are much higher than in other mammals (e.g. 30–50 μM in mice) [1]. The upper end of the normal range is 360 μM for women and 400 μM for men. Chronic hyperuricemia promotes the deposition of sodium urate crystals within joints which then causes gout. In developed countries, gout is the most common inflammatory arthritis, affecting 1–2% of adults [2]. With prior serum urate levels ⩾535 μM, the annual incidence rate of gouty arthritis in men was 4.9%, compared with 0.1% for urate levels below 415 μM [3]. Hyperuricemia is not only associated with gout, but also with hypertension, diabetes, and renal and cardiovascular diseases. For example, there is a strong correlation between hyperuricemia and children with primary hypertension [4]. Underexcretion of UA by the kidney is the primary cause of hyperuricemia in about 90% of cases [2].

Renal excretion accounts for more than 70% of the daily uric acid disposal. Glomerular filtration is followed by reabsorption and secretion; these transport processes occur in parallel in the proximal tubule [5]. The overall effect is that in healthy adults only 7–12% of the filtered urate is excreted into urine. At the normal blood pH (= 7.4), UA (effective pKa in blood = 5.75) is present almost completely (98%) as urate anion (see Fig. 1). Urate therefore cannot pass through cell membranes passively, but requires assistance from urate transporters. Clearly, these carriers are important, since altered function (by mutation or drug interaction) can cause hyperuricemia. Indeed, the main causal factors of primary gout seem to be diet and genetic polymorphisms of renal urate transporters [2]. Drugs that raise serum UA levels (= antiuricosuric) include diuretics, pyrazinoate, pyrazinamide, ethambutol, and the NSAIDs aspirin and diclofenac.

Prominent transporters for the reabsorption of urate in the proximal tubule are SLC22A12 (functional name URAT1) [6] in the apical and SLC2A9 (URATv1, GLUT9) [7] in the basolateral domain; however, several other carriers seem to be involved, both in secretion and reabsorption [5], [8]. Among these, SLC22A11 (OAT4) [9] stands out since it exists only in humans and higher primates; this matches exactly the species with high basal UA serum levels. Even more importantly, SLC22A11 must be relevant for serum UA levels, since an association between genetic variants of SLC22A11 and serum UA levels was detected in genome-wide association studies [10], [11].

SLC22A11 mRNA was confined on a Northern blot to kidney and placenta [12]. The protein was detected by immunohistochemistry in the apical membrane of proximal tubules [13] and on the basolateral membrane of the syncytiotrophoblast [14]. A transport function was first demonstrated for SLC22A11 by heterologous expression in Xenopus oocytes where the uptake of radiotracer, compared to control oocytes, was increased about 10-fold for estrone sulfate (E3S) and dehydroepiandrosterone sulfate (DHEAS) [12]. The Km values were very low, at 1 and 0.6 μM, respectively. Later, uptake of 14C-uric acid via SLC22A11 was demonstrated based on the expression in oocytes (5-fold increase; the specific uptake relative to URAT1 was at 38%) and in 293 cells (1.7-fold increase; specific clearance = 0.013 μl min−1 mg protein−1) [9]. Also, 6-carboxyfluorescein (6CF) was introduced as a substrate for uptake here. Uptake of p-aminohippuric acid (PAH) and glutarate into mouse cells stably expressing SLC22A11 [13] was not confirmed in subsequent studies [9] including our own experiments (PAH uptake, radiotracer and LC–MS/MS assays; not shown); however, efflux of PAH and glutarate via SLC22A11 was inferred from trans-stimulation (293 cells) and radiotracer efflux (oocytes) experiments [9]. Because of the much higher transport efficiency and the better signal-to-background ratio, E3S and its congener DHEAS (see Fig. 1 for structures) are usually favored over uric acid as model uptake substrates of SLC22A11.

Because of its vastly hydrophobic structure (log P = 3.1), estrone is hardly soluble in water (limit: 50 or 110 μM). Attachment of sulfate improves solubility about 100 times to 10 mM. Compared to other typical substrates of SLC22 transporters like 1-methyl-4-phenylpyridinium (Mr = 170), PAH (193), ergothioneine (229), carnitine (161), and uric acid (167), E3S is considerably larger (349) and much more hydrophobic. It is puzzling that besides SLC22A11 so many (= 18) transporters from separate families have been reported to take up E3S (Table 1). The aim of this study was to scrutinize the mechanism of transport of E3S by SLC22A11, by direct comparison with uric acid. Amazingly, our results indicate that SLC22A11 provides entirely different transport mechanisms for E3S and UA. We arrive at the unprecedented conclusion that E3S is not translocated into the cytosol. Instead, SLC22A11 catalyzes both the insertion into and the extraction from the plasma membrane of E3S.

Section snippets

Plasmid constructs

The cDNA coded by the SLC22A11 gene from human was generated by RT-PCR, cloned into pUC19, fully sequenced, and inserted into expression vector pEBTetLNC, a derivative of pEBTetD. pEBTetD is an episomal Epstein-Barr plasmid vector for doxycycline-inducible protein expression in human cell lines based on the simple tetracycline repressor [15]. pEBTetLNC contains, in addition, UCOE0.7 (ubiquitous chromatin opening element) [16] upstream of the CMV enhancer/promoter to prolong plasmid maintenance

SLC22A11 catalyzes unidirectional efflux of glutamate and aspartate

Uptake of 0.1 μM 3H-labeled glutamate (Glu) into 293 cells with or without expression of SLC22A11 was examined as a function of time (Fig. 2). Cellular accumulation of radiotracer after prolonged incubation (= the plateau level) was markedly decreased by the expression of SLC22A11 (t-test comparison of values at 20 min: P = 0.04). Detailed analysis revealed a reduction in kin from 47 (95% confidence interval: 40–54) μl min−1 mg protein−1 (expression off) to 34 (31–38) μl min−1 mg protein−1 (expression

Discussion

Heterologous expression of SLC22A11 in human 293 cells gives rise to a huge efflux of glutamate and aspartate (Fig. 3). This transport is unidirectional, there is no uptake of these anionic amino acids (Fig. 2). Apart from the order of transport efficiency (Glu > Asp), this feature is very similar to SLC22A13 (Asp > Glu) [17]. Likewise, other compounds such as uric acid (SLC22A11) and orotic acid (SLC22A13) [18] can be transported in both directions. Our present results are greatly at odds with

Funding

This study was supported by the University Hospital of Cologne. The authors declare no conflict of interest.

Acknowledgments

We thank Samira Boussettaoui, Simone Kalis, and Kathi Krüsemann for their skillful technical assistance.

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