Melanie S. Joy2
1 Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ, USA
2 Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, Aurora, CO, USA
3.1 INTRODUCTION
Cationic compounds undergo transepithelial secretion through a two‐step process: entry across the basolateral membrane followed by exit across the apical surface [1, 2]. In 1994, the organic cation transporter 1 (Oct1) was cloned from rat kidneys, which provided initial insight into how positively charged molecules gain access into cells [3]. By 1999, the bacterial NorM transporter was classified as a “multidrug and toxic compound extrusion (MATE)” protein due to its ability to mediate resistance to cationic dyes and antibacterial drugs [4]. The sequence of NorM did not align with the three previously recognized classes of bacterial multidrug efflux proteins leading to the designation of a new family of MATE transporters.
By 2005, the human and mouse orthologs of the prokaryotic MATE transporters were first identified and characterized as the final step in the excretion of organic cations [5]. Screening the human genome yielded two orthologs on chromosome 17, denoted as hMATE1 (SLC47A1) and hMATE2 (SLC47A2) that were approximately 20% identical to the NorM antiporter. This discovery opened the field to exploring how cationic compounds exit mammalian cells through a multispecific transporter.
3.2 TISSUE AND SUBCELLULAR DISTRIBUTION
3.2.1 Tissue Distribution
Human MATE1 (denoted as hMATE1) is enriched in the kidneys, liver, adrenal glands, choroid plexus, and skeletal muscle [5–7]. Similarly, hMATE1 has been detected in salivary glands [8], adipocytes [9], retinal pigment epithelial cells [10], lung bronchial and bronchiolar epithelial cells [11], and dermal fibroblasts [12]. Within the brain, hMATE1 mRNA localizes to capillary endothelial cells [13]. Initial studies of the tissue distribution of mouse Mate1 (denoted as mMate1) revealed predominant expression in kidneys and liver [5, 14]. Additional profiling across mouse tissues extended the distribution of mMate1 protein to include heart, stomach, small intestine, bladder, thyroid, gland, testes, and adrenal gland [15]. Rat Mate1 (denoted as rMate1) was cloned in 2006 and shown to be highly expressed in kidneys, as well as liver, placenta, pancreas, and spleen [16–19].
In contrast, hMATE2 is largely restricted to the kidneys [5]. An alternative kidney splice variant (denoted as hMATE2‐K) was cloned and shown to lack 108 bp [6]. This region codes for an additional 36 amino acids within a predicted intracellular loop—a region that is not observed in mMate2 or rabbit Mate2‐K [20]. Overall, there is 94% identity between hMATE2 and hMATE2‐K and ~50% amino acid identity with hMATE1. Both hMATE2 and hMATE2‐K are expressed within human kidneys [21]. When purified and reconstituted in proteoliposomes, hMATE2 and hMATE2‐K exhibit similar kinetic properties [21]. In addition, a lesser described hMATE2‐B variant has been found in the brain, but this isoform appears to lack transport activity [6].
While there is moderate‐to‐high sequence identity among MATE1/Mate1 orthologs, there appears to be greater divergence for MATE2 with low sequence identity between hMATE2 and mMate2 (38%) [22]. This becomes more evident when viewing the phylogenetic tree for mammalian MATE‐type transporters [22](Fig. 3.1). MATE1 isoforms are found in Class I, whereas primate MATE2 and dog Mate2 are considered members of Class II. Mouse and rat Mate2 are found in Class III [22].
3.2.2 Subcellular Localization
In polarized cells, Mate/MATE transporters localize to apical surfaces including hepatic canalicular and tubular brush border membranes (Fig. 3.2) [5, 6, 23]. This localization coupled with the lower pH of luminal spaces leads to the secretion of organic cations in vivo. Sequestration of organic cations in intracellular vesicles or endosomes can also be accomplished by MATE1 working in concert with the V‐type H+‐ATPase [24]. While these MATE1‐expressing endosomes may alter the intracellular distribution of organic cations, they do not appear to influence the subsequent apical efflux. In other cell types, such as serous and mucous acinar cells and duct cells, MATE1 protein can be observed on both the apical and basolateral membranes [8].
FIGURE 3.1 Mate transporters across species and localization. (a) Phylogenic tree of MATE transporters in mammals according to three subclasses: class I (red), class II (black), and class III (blue).
Adapted from [22].
(b) Transepithelial transport by OCT and MATE proteins in liver (left) and kidneys (right).
3.3 TRANSPORT ACTIVITY
While MATE transporters are the final step in organic cation secretion in renal tubules and hepatocytes, most studies evaluating MATE activity and function have been performed in overexpressing cells that operate in an “uptake mode.” This is accomplished by creating a proton gradient that favors MATE uptake of organic cations from the external face of the plasma membrane. Evaluation of MATE transport in an uptake mode overcomes limitations with quantifying efflux from cells that may not be otherwise permeable to organic cation entry. The energetics and kinetics of transport by MATEs are considered similar while operating in either the uptake or efflux mode [25].
FIGURE 3.2 Examples of MATE inhibitors. Structures of chemical inhibitors were downloaded from public database. https://www.ebi.ac.uk/chebi/init.do.
3.3.1 Energetics of Transport
Tetraethylammonium (TEA) and 1‐methyl‐4‐phenylpyridinium (MPP) are routinely used as substrates of MATE in vitro and in preclinical studies. Heterologous expression of MATE transporters in cells has revealed that the uptake of TEA, metformin, MPP, cimetidine, and procainamide is saturable and dependent upon pH [5, 6]. MATE1 utilizes an outwardly directed H+ gradient to enable antiport uptake of 14C [TEA]. Notably, lower extracellular pH (~6.0) reduces activity of hMATE1, mMate1, and rMate1, and maximal activity is observed between pH values of 8.0 and 8.5 [5, 15, 16]. Similarly, extracellular pH between 6.0 and 9.0 increases TEA uptake by hMATE2‐K and rbMate2‐K [6, 20]. Further analysis confirmed that the H+ gradient, and not just the environmental pH, is the driving force required for rMate1 activity [26]. Using membrane vesicles isolated from rMate1‐expressing cells, it was demonstrated that a high intravesicular H+ concentration stimulated 14C‐TEA uptake that was not observed in HEK‐pcDNA control cells [26].
Using inhibitors that disrupt proton conduction and pH gradients, it was demonstrated that hMATE1 utilizes H+/TEA antiport exchange [5]. Incubation of mMate1‐expressing cells and rMate1‐expressing membrane vesicles with inhibitors of membrane depolarization (e.g., valinomycin) had no effect on transporter activity [15, 26]. Taken together, these findings suggested that the exchange of cations and protons did not involve a net flux of electric charge and was electroneutral [15]. This observation contrasted
