Identification of competitive inhibitors of the human taurine transporter TauT in a human kidney cell line

Abstract

Background

The osmolyte and antioxidant taurine plays an important role in regulation of cellular volume, oxidative status and Ca²⁺-homeostasis. Taurine uptake in human cells is regulated by the Na⁺- and Cl⁻-dependent taurine transporter TauT. In order to gain deeper structural insights about the substrate binding pocket of TauT, a HEK293 cell line producing a GFP-TauT fusion protein was generated.

Methods

Transport activity was validated using cell-based [³H]-taurine transport assays. We determined the K_m and IC₅₀ values of taurine, β-alanine and γ-aminobutyrate. Additionally we were able to identify structurally similar compounds as potential new substrates or inhibitors of the TauT transporter. Substrate induced cytotoxicity was analyzed using a cell viability assay.

Results

In this study we show competitive effects of the 3-pyridinesulfonate, 2-aminoethylhydrogen sulfate, 5-aminovalerate, β-aminobutyrate, piperidine-4-sulfonate, 2-aminoethylphosphate and homotaurine. We demonstrate that taurine uptake can be inhibited by a phosphate. Furthermore our studies revealed that piperidine-4-sulfonate interacts with TauT with a higher affinity than γ-aminobutyrate and imidazole-4-acetate.

Conclusion

We propose that piperidine-4-sulfonate may serve as a potential lead structure for the design of novel drug candidates required for specific modulation of the TauT transporter in therapy of neurodegenerative diseases.

Introduction

The transport and homeostasis of bioactive solutes and small compounds is of fundamental importance in human physiology. The conditionally essential β-amino acid taurine (2-aminoethanesulfonate) is one of the most highly accumulated amino acids in the brain and plays a critical role in neural development [1]. The total taurine content in mammals is derived from two sources: mainly by dietary intake and partly also by taurine biosynthesis in liver and kidney [2]. Taurine is involved in fundamental biological processes. For instance, taurine is an indirect antioxidant and plays a key role in osmoregulation, membrane stabilization and modulation of Ca²⁺-signaling [3]. In brain and retinal tissues, the function of taurine as antioxidant and osmolyte is essential for cell volume homeostasis. In this context it was shown that the regulatory effect of taurine on cell volume leads to neuroprotection [4]. Taurine deficiency in humans causes development of retinal disorders, which in severe cases can lead to blindness [5], [6]. The function of solute carriers (SLC) in drug absorption and disposition has increased their clinical importance in recent years [7].

Taurine supply is regulated by the taurine transporter TauT (SLC6A6) [8] in combination with the gamma-aminobutyric acid (GABA) transporter GAT2 (SLC6A13) [9] and the proton-coupled amino acid transporter PAT1 (SLC36A1) [10]. The transport activity of TauT is sodium- and chloride dependent with a stoichiometry of 2:1:1 (Na⁺:Cl⁻: taurine) [11]. As a high-affinity and low-specificity transporter TauT has the capability to transport hypotaurine, β-alanine and γ-aminobutyrate (GABA), apart from taurine [12]. In addition there are several drug candidates that interfere with GABA transporters, but so far there is only little knowledge about the interference of structural analogs with TauT.

In 2016, Rasmussen et al. investigated the influence of basic GABA-analogs like nipecotic acid, guvacine, δ-aminolevulinic acid, vigabatrin und gaboxadol on taurine uptake [13].

The anti-epileptic drug vigabatrin increases the intracellular level of GABA by irreversible inhibition of the GABA-aminotransferase [14]. The intestinal absorption of vigabatrin is mainly regulated by PAT1 with a partial involvement of TauT [15]. Gaboxadol, also known as 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridine-3-ol (THIP), acts as a GABA_A receptor type A (GABA_AR) agonist. GABA_A receptors are Cl⁻-channels, which form the major inhibitory system in the central nervous system. Gaboxadol showed inhibitory properties on the GABA_AR as well as on the TauT-mediated uptake of taurine in SKPT cells and ARPE-19 cells [13], [16].

Valembois et al. demonstrated 2017 that various other GABA analogs (imidazole-4-acetat (I4AA), 4,5,6,7-tetrahydropyrazolo[5,4-c]pyridin-3-ol (Aza-THIP), muscimol and thiomuscimol) also act as competitive inhibitors of taurine transport [16]. In particular the histamine metabolite I4AA significantly inhibits taurine uptake in a concentration-dependent manner. Due to its additional interaction with the GABA_AR, they defined I4AA as a new lead structure for the development of new compounds.

In the meantime, Suárez et al. reported guanidinoethyl sulfonate [17] to mimic the effects of taurine on long-term synaptic potentiation. GES, a natural guanidine-taurine analog, causes taurine depletion by acting as a competitive inhibitor of TauT, PAT1 and GAT2 as well as interfering with taurine biosynthesis [18], [19], [20]. In order to analyze the involvement of taurine transport by TauT in relation to PAT1- and GAT2-mediated taurine uptake, TauT was recombinantly overproduced in this study.

To gain a deeper knowledge about the structural requirements of ligands for interaction with the taurine transporter (TauT), we investigated the competitive inhibition by various GABA- and TauT-analogs. Therefore we selected structural similar compounds containing one acidic and one amino head group separated by a carbon chain and determined their inhibitory properties on taurine uptake (Fig. 1). The compounds analyzed in this study were chosen mostly due to their known physiological effects on either humans or other eukaryotes. Our taurine uptake assays using HEK293-cells producing TauT-GFP indicated that homotaurine, ethanolamine-O-sulfate (EOS), piperidine-4-sulfonate (P4S) and 3-pyridinesulfonate (PYR) act as taurine analogs; 5-aminovalerate (5AVA) and β-aminobutyrate (BABA) as GABA analogs and 2-aminoethylphosphate (2AEP) as competitive inhibitors for TauT with a phosphate group. Here we report a robust in vitro model for studying TauT and the inhibitory potencies of substrate analogs on taurine uptake.