hMATE1 was recently identified as an important transporter in the kidney and liver for structurally diverse cationic xenobiotics including therapeutic drugs and chemical toxins.1, 2, 3
It is abundantly expressed in the adrenal gland, suggesting that it also functions in the disposition of endogenous substrates that may be synthesized and secreted by the gland. Thus, genetic variation in hMATE1 could potentially contribute to variation in drug disposition and response as well as to physiologic and pathophysiologic variation among people. This study represents the first comprehensive screen of the coding region of hMATE1 for genetic and functional variation in an ethnically diverse sample population. We identified six nonsynonymous single nucleotide polymorphisms (SNPs) in hMATE1. Among these, three variants were singletons and three variants had allele frequency of >1% in the total population. Two common variants had allele frequencies of 5% in African American or Mexican American samples. No nonsynonymous variants were identified in samples from individuals with European ancestry. The overall nonsynonymous variation of hMATE1 is low compared to OCT1 and OCTN1 (see ), but comparable to OCT2, another transporter in the kidney in the SLC superfamily, which is believed to work in concert with MATE1 in renal secretion of xenobiotics.12, 13
These data are consistent with purifying selection of MATE1 and are consistent with an important physiologic function for the transporter.
For our functional studies, we selected four model compounds: paraquat, metformin, TEA and the anticancer drug, oxaliplatin. TEA represents a classical model organic cation used to study the function of renal organic cation transporters. Paraquat is a toxic herbicide that has been banned from many countries. Nephrotoxicity, hepatoxicity and pulmonary toxicity, which occur after accidental or suicidal ingestion, are the major toxicities of this herbicide. hMATE1 is expressed in abundance in lung, kidney and liver; therefore, genetic variants in MATE1 may have a function in risk for toxicity after ingestion. Metformin is an important anti-diabetic drug, which is cleared almost exclusively in the kidney. hMATE1 appears to be an important protein in its secretion from tubule cell to tubule lumen. Genetic variation in hMATE1 may contribute to variation in renal clearance of metformin. Finally, oxaliplatin is an anticancer drug transported by hMATE1, which could potentially affect the tissue distribution and tubular secretion of this agent. Genetic variants in hMATE1 may have a function in anticancer effects and/or adverse effects of oxaliplatin.
Our cellular assays indicated that the six nonsynonymous variants were functionally heterogeneous. Such functional heterogeneity has been reported previously for SLC22A
transporters such as OAT3 and OCT1.14, 15
Two of the hMATE1 variants (G64D and V480M) produced a complete loss of function for all the substrates tested and appeared as singletons only in our Asian population sample. From the sequence alignment of hMATE1 with its species orthologs, the G64 residue occurs in an evolutionarily conserved amino acid residue identical among human, mouse and rat orthologs of hMATE1. A change in an evolutionarily conserved amino acid residue would be expected to cause a functional change. Further, substitution of a chemically different amino acid at a functionally critical position could also cause a loss of function. For example, Otsuka et al.1
showed that amino acid replacement from glutamate to glutamine at position 273 (E273Q), the substitution of an essential amino acid residue found in the bacterial NorM protein, led to complete loss of function for TEA uptake. G64D, a substitution of a negatively charged aspartic acid for a neutral amino acid, glycine, in the first extracellular loop of MATE1, represents a large chemical change, which could have contributed to the loss of function. Furthermore, we found that hMATE1-G64D tagged with GFP is expressed mainly in the intracellular space and less on the plasma membrane () compared to hMATE1 reference, which may explain its functional loss.
Because V480M is not an evolutionarily conserved residue and the substitution of methionine for valine is a conservative chemical change, the loss of function in this singleton variant was not expected. In further studies with a GFP chimera, we observed that the substitution appeared to change the intracellular localization of the transporter. That is, we found that hMATE1-V480M tagged with GFP was poorly localized to the plasma membrane (), which rendered it functionally inert. Though further studies are needed, it is possible that valine at position 480 is critical for trafficking of hMATE1 to the plasma membrane.
We also identified two variants (L125F and V338I) that significantly reduced the transport function in a substrate-dependent manner. hMATE1-L125F had a reduced ability to transport all three model substrates, paraquat, metformin and TEA, but maintained the transport ability for oxaliplatin. hMATE1-V338I had reduced function with respect to metformin and TEA but not with respect to paraquat and oxaliplatin. Similarly, hMATE1-C497S exhibited a decreased transport of metformin and TEA; however it seemed to have an enhanced ability to transport paraquat and oxaliplatin. Chemically, paraquat has two positive charges and oxaliplatin after aquation will form monoaqua and diaqua complexes (two positive charges), whereas metformin and TEA carry a single positive charge. Thus, it is possible that different amino acid residues affect the substrate recognition and translocation of divalent organic cations by hMATE1 in comparison to monovalent cations.
Previous studies have demonstrated that hMATE1 expression enhances the cytotoxicity of oxaliplatin.16
In this study we examined the effect of genetic variants of hMATE1 on the cytotoxicity of oxaliplatin, and observed a 2.3- to 3.5-fold enhanced cytotoxicity in cell lines expressing the reference hMATE1 and four of the variant transporters. In contrast, cell lines expressing the hMATE1 variants, hMATE1-G64D and hMATE1-V480M, did not exhibit enhanced cytotoxicity or uptake of oxaliplatin. Because both of these variants are rare, their effects at the population level would be minimal; however, the studies suggest that rare variants of hMATE1 in the coding region could result in a modulation of the disposition and pharmacologic action of oxaliplatin.
The kinetics studies performed on the relatively common variants hMATE1-L125F and hMATE1-V338I did not clearly explain why these variants had reduced transport function for paraquat. The Km, Vmax and Vmax/Km values for hMATE1-L125F were not significantly different from the reference hMATE1 (see ). However, because paraquat transport was only slightly reduced in the hMATE1-L125F variant (see ), small differences in Km and Vmax values would be difficult to detect in kinetic studies. For metformin uptake, in which larger differences in uptake between the hMATE1 reference and the two polymorphic variants (hMATE1-L125F and hMATE1-V338I) were observed, the Vmax/Km ratios exhibited trends toward significantly lower values in cells expressing the variant transporters in comparison to the reference transporter (P=0.051). Confocal microscopy showed that hMATE1-L125F and hMATE1-V338I variants were located on both plasma membrane and in the intracellular spaces, in contrast to the reference hMATE1, which was predominately distributed to the plasma membrane (). These data are consistent with the reduced function of hMATE1-L125F and hMATE1-V338I in comparison to the reference transporter.
In summary, we identified two rare variants of hMATE1 with no function and four variants with altered function. Three of the four variants were polymorphic in a particular ethnic population with allele frequencies equal to or greater than 2%. Because hMATE1 is expressed on the apical membrane of renal tubule cells and the canalicular membrane of hepatocytes, these genetic variants may contribute to variation in renal or biliary elimination of hMATE1 substrates. Clinical studies are clearly needed to determine whether these findings have implications to drug disposition and response.