for hMATE1 was developed [63]. This model positions hMATE1 in two bundles of six transmembrane helices (N lobe: transmembrane domains 1–6, C lobe: transmembrane domains 7–12) with an internal cavity of ~4,000 Å that is open to the extracellular space [63]. A relatively short cytoplasmic loop between domains 6 and 7 is positioned to connect the two halves and is consistent with hydropathy plots for N or M. Evaluation of the crystal structure of a MATE transporter from Arabidopsis thaliana (2.6 Å), known as AtDTX14, has also provided insights into hMATE1 structure and function [65]. The amino acid sequence identity between hMATE1 and AtDTX14 is 32%. A key hydrogen bonding network in the C‐lobe demonstrated in AtDTX14 is conserved in hMATE1 and is considered to be the substrate‐binding site [65]. While key insights into the likely structure of hMATE1 (and orthologs) have been made, there is little insight into similarities and differences for MATE2/2‐K, as well as a need for a MATE structure from crystallography or cryo‐electron microscopy.
FIGURE 3.3 Predicted structure of the human MATE1 transporter. The membrane topology of the human MATE1 transporter has been adapted from [128].
3.4.3 Structural Features
Treatment of cells expressing rMate1 with p‐chloromercuribenzene sulfonate, an organic mercurial chemical, significantly reduced uptake of TEA. This inhibition of transport could be rescued by dithiothreitol suggesting that reduced sulfhydryl groups are important for the activity of rMate1 [17]. Subsequent site‐directed mutagenesis identified key residues [66]. One histidine (rMate1: His‐385, hMATE1: His‐386, hMATE2‐K: His‐382) and two cysteine residues (rMate1: Cys‐62 and Cys‐126, hMATE1: Cys‐63 and Cys‐127, hMATE2‐K: Cys‐59 and Cys‐123) were essential for transport activity. The impaired function of mutants in these residues was not due to improper trafficking or reduced expression as all localized to the plasma membrane to similar degrees as wild‐type proteins [66]. Interestingly, unlabeled TEA was able to protect against the transport inhibition achieved by the sulfhydryl reagent PCMBS suggesting that rMate1 substrates interact directly with sulfhydryl‐containing Cys‐62 and Cys‐126 [66]. By comparison, unlabeled TEA had no effect on the ability of the histidine residue modifier DEPC to block rMate1 activity in vitro.
There has also been interest in elucidating the role of negatively charged glutamates in the recognition of substrates that are largely cationic in nature. Mutation of the glutamate residue at position 273 to a glycine reduced hMATE1 function without altering its insertion into the plasma membrane [5]. Notably, this glutamate residue is conserved across rMate1, mMate1, and hMATE2‐K. Substitution of glutamate at position 273 with alanine resulted in the absence of hMATE1 protein, whereas amino acid changes at the remaining glutamates differentially altered activity and affinity for cimetidine and TEA. For example, aspartate mutants at each glutamate residue decreased overall transporter activity; however, Glu273Asp increased affinity for cimetidine but reduced affinity for TEA [67]. Purified MATE1 protein containing glutamine at position 273 (rather than glutamate) further support this residue as a binding site for organic cations [48]. Within the hMATE1 homology model based on the N or M structure, the four glutamate residues localize to the hydrophilic cleft, which would likely represent the path for substrate translocation [63]. Whether all four of these glutamates are directly involved in substrate binding or contribute to overall secondary structure stability is unclear but nonetheless impact hMATE1 transporter activity.
Using the crystal structure of AtDTX14, it has been proposed that eukaryotic MATE proteins rely upon conformation changes in the 7th transmembrane domain. The 7th transmembrane domain is predicted to undergo protonation at a conserved acidic residue in the C‐lobe that leads to electrostatic repulsion and a bent conformation. Bending of this domain is anticipated to collapse the cavity of the C‐lobe leading to hydrogen‐binding and release of substrate. While in the straight conformation, the proton is absent and the 7th transmembrane domain would be capable of binding positively charged substrates [65]. As the 7th transmembrane domain moves from the straight to the bent confirmation, the proteins are expected to undergo “rocking” between the inward‐open and outward‐open configurations.
3.5 EXPRESSION AND REGULATION
3.5.1 Transcriptional Regulation
Analysis of the upstream regions of hMATE1 and rMate1 have identified critical regions responsible for basal promoter activity (SLC47A1: −65 to −25 and Slc47a1: −146 to −38) [68]. These regions lack canonical TATA and CCAAT boxes but instead contain two GC‐rich sites [68]. Basal promoter activity is increased with overexpression of the Sp1 transcription factor and blocked by mithramycin A, which is known to bind GS boxes and prevent Sp1 binding and activity [68]. Within the basal promoter region of hMATE1, there are two sites for Sp1 binding, both of which have been demonstrated to be functional based on mutagenesis studies [68]. Within the human population, there was a single‐nucleotide polymorphism (SNP) at position −32 (G/A) that reduces luciferase activity when expressed in vitro, likely due to impaired Sp1 activity [68].
Hepatocyte nuclear factor (HNF) 4 alpha has emerged as a novel transcriptional regulator of mMate1 [69]. ChIP‐Seq has revealed binding of HNF4 alpha within the region of Slc47a1 and Slc47a2. Inhibition of HNF4 alpha in embryonic rat kidney cultures markedly reduced rMate1 mRNA expression [69]. By comparison, over‐expression of HNF4 alpha in mouse embryonic fibroblasts increased mMate1 mRNA. Recognizing that HNF4 alpha is a master regulator of xenobiotic processing genes, it is possible the upregulation of mMate1 occurs directly or indirectly by activating upstream signaling paths.
The transcriptional regulator of stress responses, nuclear factor e2‐related factor 2 (NRF2), also influences MATE expression. Downregulation of NRF2 in human proximal tubule cells reduced hMATE2‐K mRNA [70]. Likewise, pharmacological activation of NRF2 or genetic disruption of its negative regulator KEAP1 induced the expression of hMATE2‐K mRNA. It has been presumed that stimulation of NRF2 signaling may be the mechanism by which hMATE2‐K levels are enhanced in response to shear stress when cultured in a microfluidic system [70]. The ability of NRF2 to similarly regulate MATE1 expression in human proximal tubule epithelial cells has been demonstrated after treatment with the pharmacological activator, bardoxolone‐methyl [71].
Two nuclear receptors have also emerged as potential regulators of Mate transporters, namely, the peroxisome proliferator‐activated receptor (PPAR) alpha and the farnesoid × receptor (FXR). Daily treatment of mice with metformin for 30 days increased mMate1 mRNA in the kidneys of wild‐type mice, with no change observed in mice lacking PPAR alpha [72]. This observation was further supported by the ability of the PPAR alpha agonist, gemfibrozil, to elevate mMate1 expression in mouse kidney tubular cells [72]. Likewise, treatment with obeticholic acid, a nonsteroidal agonist of FXR, increased the mRNA expression of Mate1 in the livers of rats [73].
3.5.2 Short‐Term Regulation
Short‐term regulation of transporter activity can be accomplished through numerous cellular signaling pathways involving phosphorylation and dephosphorylation. In HEK293 cells expressing hMATE1 or hMATE2‐K, pharmacological inhibition of casein kinase II can stimulate transporter activity [74]. Blockade of another kinase, p56lck tyrosine kinase, also greatly enhanced hMATE2‐K activity; however, this upregulation of function was absent under more acidic conditions. Conversely, a modest reduction in hMATE1 activity could be observed with inhibition of p56lck tyrosine kinase, as well as calcium/calmodulin [74]. Activation of protein kinases A and C attenuated hMATE2‐K function but had no effect on hMATE1. Clearly, the short‐term regulation of hMATE1 and hMATE2‐K activity is dynamic with multiple phosphorylation events able to turn on/off activity within minutes.
3.5.3 Sex and Age
Expression of Mate transporters can vary by sex and age. Within the kidneys of rats, rMate1