ENT4

ENT4 (equlibrative nucleoside transporter 4)

Alias: PMAT
Gene name: Solute carrier family 29 member 4 (SLC29A4)

Summary

Equilibrative nucleoside transporters (ENTs) play important roles in the disposition as well as the physiological and pharmacological activities of nucleosides and their analogues [1]. The four isoforms (ENT1-4) of the ENT family are encoded by genes of the SLC29 family, share a proposed 11-transmembrane (TM) helix topology and the ability to transport adenosine, but differ in their abilities to transport other nucleosides and nucleobases [2]. ENT4 (SLC29A4), a ~58 kDa membrane protein, is a polyspecific cation transporter highly expressed in the brain, with a substrate profile more similar to OCTs (SLC22s) than to the other SLC29 family members. Since typical endogenous substrates of ENT4 are monoamine neurotransmitters (e.g. dopamine, serotonin), Engel et al. proposed a functional name, plasma membrane monoamine transporter (PMAT), for SLC29A4 [3]. In addition to its presumed function in monoamine uptake, and a putative role in transporting neurotoxins associated with Parkinsonism ENT4 transports adenosine [4].

Localization

ENT4 was cloned from a human kidney cDNA library by Engel et al [3]. ENT4 is predominantly expressed on the plasma membrane, and it has a broad tissue distribution. In the brain, ENT4 is localized to the apical membrane of the choroid plexus [5], and is also detected in many brain regions (cortex, hippocampus, etc.) [6]. Outside the central nervous system, ENT4 is expressed on the luminal membrane of small intestinal enterocytes [7], and it is also found, albeit in small amounts, in renal proximal tubular cells and podocytes, the latter playing an essential role in the function of the filtration barrier [8]. Further, ENT4 is also present in skeletal muscle, [3] liver, and heart [9].

Function, physiology, and clinically significant polymorphisms

Although ENT4 is localized to the plasma membrane, it does not function as a typical nucleoside transporter, in that it fails to interact significantly with nucleosides or nucleoside analogs, nucleotides or nucleobases, except for a weak activity toward adenosine [3]. Whether at pH 7.4 or 6.6, ENT4 transports adenosine with an efficiency about tenfold lower than it does 1-methyl-4-phenylpyridinium (MPP+) or serotonin [10]. Since ENT4 transports adenosine with such low affinity and activity, it was thought not to be a major contributor to physiological adenosine uptake. It is only during hypoxia or ischemia, with extracellular adenosine concentrations rising way above physiologically normal brain levels, that ENT4 may play an important role in adenosine uptake in the brain and in the heart [4, 10]. Adenosine transport by ENT4 has recently been revisited using 2-chloroadenosine, a stable analog of adenosine, and showed biphasic kinetics with a high affinity / low capacity and a low affinity / high capacity component [11], as well as positive cooperativity at acidic pH values. ENT4-mediated adenosine uptake at pH 6.0 displayed a Km of 50 µM that was similar to Km values for adenosine transport by other ENTs [11]. As extracellular adenosine plays a cardioprotective role [12] and given the acidic pH values in ischemic heart, inhibition of ENT4 may provide a cardioprotective mechanism in ischemia-reperfusion [9].
Rather than accepting nucleoside-like substrates, ENT4 is most well-known for transporting monoamine neurotransmitters including 5-hydroxytryptamine and dopamine, noradrenaline and adrenaline, as well as the naturally occurring trace amine tyramine [3]. ENT4 as a Na+-independent, electrogenic, polyspecific cation transporter, also mediates the transport of a variety of structurally diverse (but generally small, polar and aliphatic) xenobiotic cations such as the protonated neurotoxin MPP+, the K+ channel-blocker tetraethylammonium (TEA), or the biguanidine antidiabetic drug metformin [7]. ENT4-mediated transport processes are potential- and pH-sensitive: hyperpolarization and extracellular acidification stimulate ENT4-mediated uptake [10, 13]. Several 3D pharmacophore models have been employed to examine substrate or inhibitor characteristics important for binding to, and transport by, ENT4. Hydrophobicity, along with a well-defined distance between the positive ionizable site and the hydrophobic aromatic site, were identified to be important descriptors for ENT4 affinity [14]. Data obtained from site-directed mutagenesis strongly suggest that TM5 is essential for the cation selectivity of ENT4 [15]. Similar molecular features are known to be important for high affinity interactions with the OCTs. ENT4 is not a mainstream therapeutic target. However, pharmacological inhibition of ENT4 for ischemic heart disease has been suggested [9], and synthesis as well as characterization of ENT4-specific inhibitors has been published [16].
Since ENT4 transports monoamine neurotransmitters, tissue-dependent high expression of ENT4 in tissues such as neurons and the choroid plexus may participate in the regulation of interstitial and intracellular concentrations of monoamine neurotransmitters and cationic drugs. In human astrocyte cultures, inhibition and gene knockdown studies supported that ENT4 may play a pivotal role in monoamine transport [17, 18], although in vivo results to clarify the functions of ENT4 in neurophysiology are still lacking. 

Clinical significance

The ENT4 substrate MPP+ produces Parkinson's disease in humans and animal models. Beta-carbolines, natural analogs of MPP+, have been suggested as an environmental risk factor for Parkinson's disease. Several β-carbolines are transported by ENT4 [14]. ENT4 may be involved in the intestinal absorption of metformin; however, albeit metformin is transported by ENT4 in vitro currently there is no in vivo data to confirm an impact of ENT4 on metformin pharmacokinetics [7].

Regulatory requirements

Due to the scarcity of data on the clinical relevance of ENT4, investigation of this transporter is not currently included in regulatory guidelines.

References

1.    Cass, C.E., J.D. Young, and S.A. Baldwin, Recent advances in the molecular biology of nucleoside transporters of mammalian cells. Biochem Cell Biol, 1998. 76(5): p. 761-70.
2.    Baldwin, S.A., et al., The equilibrative nucleoside transporter family, SLC29. Pflugers Arch, 2004. 447(5): p. 735-43.
3.    Engel, K., M. Zhou, and J. Wang, Identification and characterization of a novel monoamine transporter in the human brain. J Biol Chem, 2004. 279(48): p. 50042-9.
4.    Barnes, K., et al., Distribution and functional characterization of equilibrative nucleoside transporter-4, a novel cardiac adenosine transporter activated at acidic pH. Circ Res, 2006. 99(5): p. 510-9.
5.    Duan, H. and J. Wang, Impaired monoamine and organic cation uptake in choroid plexus in mice with targeted disruption of the plasma membrane monoamine transporter (Slc29a4) gene. J Biol Chem, 2013. 288(5): p. 3535-44.
6.    Dahlin, A., et al., Expression and immunolocalization of the plasma membrane monoamine transporter in the brain. Neuroscience, 2007. 146(3): p. 1193-211.
7.    Zhou, M., L. Xia, and J. Wang, Metformin transport by a newly cloned proton-stimulated organic cation transporter (plasma membrane monoamine transporter) expressed in human intestine. Drug Metab Dispos, 2007. 35(10): p. 1956-62.
8.    Xia, L., et al., Podocyte-specific expression of organic cation transporter PMAT: implication in puromycin aminonucleoside nephrotoxicity. Am J Physiol Renal Physiol, 2009. 296(6): p. F1307-13.
9.    Yang, C. and G.P. Leung, Equilibrative Nucleoside Transporters 1 and 4: Which One Is a Better Target for Cardioprotection Against Ischemia-Reperfusion Injury? J Cardiovasc Pharmacol, 2015. 65(6): p. 517-21.
10.    Zhou, M., et al., Adenosine transport by plasma membrane monoamine transporter: reinvestigation and comparison with organic cations. Drug Metab Dispos, 2010. 38(10): p. 1798-805.
11.    Tandio, D., G. Vilas, and J.R. Hammond, Bidirectional transport of 2-chloroadenosine by equilibrative nucleoside transporter 4 (hENT4): Evidence for allosteric kinetics at acidic pH. Sci Rep, 2019. 9(1): p. 13555.
12.    Dubey, R.K., et al., Smooth muscle cell-derived adenosine inhibits cell growth. Hypertension, 1996. 27(3 Pt 2): p. 766-73.
13.    Itagaki, S., et al., Electrophysiological characterization of the polyspecific organic cation transporter plasma membrane monoamine transporter. Drug Metab Dispos, 2012. 40(6): p. 1138-43.
14.    Ho, H.T., et al., Molecular analysis and structure-activity relationship modeling of the substrate/inhibitor interaction site of plasma membrane monoamine transporter. J Pharmacol Exp Ther, 2011. 339(2): p. 376-85.
15.    Zhou, M., et al., Molecular determinants of substrate selectivity of a novel organic cation transporter (PMAT) in the SLC29 family. J Biol Chem, 2007. 282(5): p. 3188-95.
16.    Wang, C., et al., Dipyridamole analogs as pharmacological inhibitors of equilibrative nucleoside transporters. Identification of novel potent and selective inhibitors of the adenosine transporter function of human equilibrative nucleoside transporter 4 (hENT4). Biochem Pharmacol, 2013. 86(11): p. 1531-40.
17.    Yoshikawa, T., et al., Molecular mechanism of histamine clearance by primary human astrocytes. Glia, 2013. 61(6): p. 905-16.
18.    Naganuma, F., et al., Predominant role of plasma membrane monoamine transporters in monoamine transport in 1321N1, a human astrocytoma-derived cell line. J Neurochem, 2014. 129(4): p. 591-601.