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 and rMate2‐K mRNA begin to be expressed at postnatal days 21 and 1, respectively, and peak at 6 months of age before declining with advanced age [75]. Interestingly, older rats (~18 months) have heightened susceptibility to the nephrotoxicity of the Mate1 substrate, cisplatin [76]. This increased susceptibility is likely due to the reduced expression of Mate1 protein as expression of other cisplatin transporters were unchanged in 18‐month‐old rats [76].
Sex differences in Mate transporters have been observed in mice, but not in humans, and may reflect differential regulation by hormones across species. Expression of mMate1 mRNA is slightly higher in the livers of female mice compared with males. However, within female kidneys, mMate1 mRNA levels are lower than that observed in male mice [14]. Interestingly, expression of mMate2 mRNA in the kidneys can be reduced by removal of the ovaries in female mice [77]; supplementation with 17ß‐estradiol restores mMate2 expression. By comparison, there is no change in mMate1 mRNA in ovariectomized mice. Treatment of mice with testosterone for 7 days increases the protein expression of mMate1 and mMate2 [78]. These findings provide initial insights into the hormonal regulation of Mate transporters in mice.
3.5.4 Pregnancy
Pregnancy is accompanied by a number of physiologic changes, including increased cardiac output, as well as hepatic and renal blood flow. There has been much interest into adaptive changes in expression of drug transporters within the mother, placenta, and fetus. Pregnant mice exhibit reduced expression of renal mOct1 and 2, as well as mMate1 mRNA and protein [79, 80]. Within the livers of pregnant mice, there is a downregulation of Oct1 with no change in mMate1 levels compared with virgin mice [79]. These data stand in contrast to a study performed in pregnant women prescribed metformin. Notably, the renal clearance of metformin, as well as the endogenous Mate substrate NMN, was markedly increased during mid‐ and late‐pregnancy compared with early pregnancy or the postpartum period [81]. While a significant increase in glomerular filtration rate contributes to enhanced metformin and NMN elimination from the kidneys, the data also suggest enhanced tubular secretion. The mechanism(s) responsible for divergent regulation of the organic cation transport systems during pregnancy (reduced in mice, increased in humans) are unclear and warrant consideration.
3.5.5 Disease Models
3.5.5.1 Kidney Disease and Injury
Acute and chronic renal disease is a significant cause of morbidity and mortality and have been associated with the altered clearance of drugs and toxicants. In rodents, acute and chronic kidney disease can be recapitulated experimentally using surgical nephrectomy, ischemia‐reperfusion injury, and toxicant‐induced damage. Nephrectomy, or 5/6 removal of kidney tissue, mirrors clinical features of
