Novel single nucleotide polymorphisms of organic cation transporter 1 (SLC22A1) affecting transport functions

doi:10.1016/j.bbrc.2003.11.175

Biochemical and Biophysical Research Communications

Volume 313, Issue 3, 16 January 2004, Pages 789-793

https://doi.org/10.1016/j.bbrc.2003.11.175 Get rights and content

Abstract

Organic cation transporter OCT1 (SLC22A1) plays an essential role in absorption, distribution, and excretion of various xenobiotics including therapeutically important drugs. In the present study, we analyzed the functional properties of the single nucleotide polymorphisms (SNPs) in SLC22A1 gene found in Japanese control individuals. Four mutations resulting in the amino acid changes (F160L, P283L, R287G, and P341L) were functionally characterized in Xenopus oocyte expression system. Two new SNPs, identified in Japanese population, P283L and R287G exhibited no uptake of both [¹⁴C]TEA and [³H]MPP⁺, although their protein expressions were detected in the plasma membrane of the oocytes injected with their cRNAs. Uptake of [¹⁴C]TEA by P341L was reduced to 65.1% compared to wild type, whereas F160L showed no significant change in its transport activity. This study suggests that the newly found OCT1 variants will contribute to inter-individual variations leading to the differences in cationic drug disposition and perhaps certain disease processes.

Section snippets

Materials and methods

Materials. [¹⁴C]TEA (2.05 GBq/mmol) and [³H]MPP⁺ (2.96 TBq/mmol) were purchased from American Radiolabeled Chemicals (USA). All other chemicals and reagents were of analytical grade. Rabbit polyclonal antibodies were raised against a synthetic carboxyl-terminal peptide of human OCT1, ENLGRKAKPKENC, as reported previously [14].

Isolation of human OCT1 cDNA. Specific PCR primers were designed based on the nucleotide sequence for human OCT1 (GenBank Accession Nos. U77086 and NM_003057) [9]: forward

Polymorphisms in OCT1 in Japanese population

In JSNP database, we found four nonsynonymous nucleotide polymorphisms of OCT1: F160L (JSNP ID: ssj0008476), P283L (ssj0005319), R287G (ssj0005320), and P341L (ssj0008480). SNPs F160L and P341L were same as those reported previously for American population [11], [12], while SNPs P283L and R287G are only found in Japanese population. As shown in Fig. 1, F160 is located in the middle of the second transmembrane domain (TMD): P283 and R287 are at the 5^′-end of long intracellular loop between 6th

Acknowledgements

The anti-hOCT1 polyclonal antibody was supplied by Transgenic Inc. Kumamoto, Japan. This work was supported in part by grants from the Ministry of Education Science, Sports, Culture and Technology of Japan, and from the Japanese Society for the Promotion of Science (JSPS). This work was performed in collaboration with PharmaSNP consortium.

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Consistent with our findings, G401S and G465R mutations were significantly associated with functional changes in hOCT1 activity as well as drug response which may alter the pharmacokinetics and pharmacodynamics of numerous drug such a metformin, tropisetron, ondansetron, morphine, and tramadol [9,75,79,80]. Alternatively, as deduce by the study of Sakata collaborators, P283L and R287G substitutions were associated significantly associated with impaired hOCT1 transport function which may influence pharmacokinetics of hOCT1 substrates [19]. We believe that further Molecular dynamics (MD) simulation and in vitro functional studies are required to directly determine the effect of these mutant hOCT1 proteins on Imatinib response.
Non-synonymous single nucleotide polymorphisms (nsSNPs) in hOCT1 (encoded by SLC22A1 gene) are expected to affect Imatinib uptake in chronic myeloid leukemia (CML). In this study, sequence homology-based genetic analysis of a set of 270 coding SNPs identified 18 nsSNPs to be putatively damaging/deleterious using eight different algorithms. Subsequently, based on conservation of amino acid residues, stability analysis, posttranscriptional modifications, and solvent accessibility analysis, the possible structural-functional relationship was established for high-confidence nsSNPs. Furthermore, based on the modeling results, some dissimilarities of mutant type amino acids from wild-type amino acids such as size, charge, interaction and hydrophobicity were revealed. Three highly deleterious mutations consisting of P283L, G401S and R402G in SLC22A1 gene that may alter the protein structure, function and stability were identified. These results provide a filtered data to explore the effect of uncharacterized nsSNP and find their association with Imatinib resistance in CML.
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The most studied transporter in relation to metformin genetics and disposition is OCT1, which is essential for the hepatic uptake of metformin. OCT1 is encoded by the gene SLC22A1 which is highly polymorphic and a number of coding missense SNPs affect its activity (Kerb et al., 2002; Leabman et al., 2002; Sakata et al., 2004; Shu et al., 2003). In a study of 20 healthy volunteers, it was demonstrated that the presence of at least one of four reduced function variants (R61C/rs12208357, G401S/rs34130495, 420del/rs72552763, and/or G465R/rs34059508) attenuates the effects of a short course of metformin on glucose tolerance tests (Shu et al., 2007).
Pharmacogenetic studies of antidiabetic drugs have so far focused largely on response to metformin, which is the first-line therapy for treatment of type 2 diabetes (T2D). The first studies of metformin pharmacogenetics were focused on candidate genes that were implicated in metformin pharmacokinetics and transport. Since 2011, genome-wide association studies have been conducted in large cohorts of individuals with T2D identifying genes that are associated with glycemic response to metformin. There have been fewer pharmacogenetic studies of other antidiabetic drugs, and those have been largely limited to candidate gene studies with small sample sizes. Understanding the pharmacogenetics of antidiabetes medications is important for the integration of genetic screening into therapeutic decision making, and to achieve the goal of “precision medicine” for patients with T2D. In this chapter, we provide a review of the pharmacogenetics investigations of metformin and other antidiabetes medications. In addition, we highlight the importance of collaborative efforts with large sample size and representation from multiple ethnic groups in pharmacogenetics studies.
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Membrane transporters are recognized to be an important class of proteins for regulating cellular and physiologic solute and fluid balance. Transporter proteins can be generally separated into two major classes—uptake and efflux transporters. Not only are transporter proteins critical to human physiology in the maintenance of normal homeostasis via transport of endogenous substrates, but they are also, in many cases, important to the disposition of numerous xenobiotic compounds such as environmental toxins, dietary constituents, drugs, and their metabolites. In particular, over the past two decades, members of the solute carrier (SLC) families, including the organic anion transporting polypeptide (SLCO) family, the organic cation/anion/zwitterion transporter (SLC22) family, and the sodium bile salt cotransport (SLC10) family, have been determined to have important physiological, pathological, and therapeutic implications. In this article, we focus on uptake transporter proteins and provide a comprehensive overview of the significant roles these proteins play in normal physiology and the drug disposition process.
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Biochemical and Biophysical Research Communications

Abstract

Section snippets

Materials and methods

Polymorphisms in OCT1 in Japanese population

Acknowledgements

References (23)

Polymorphic human cytochrome P450 enzymes: an opportunity for individualized drug treatment

Trends Pharmacol. Sci.

Polymorphisms in OATP-C. Identification of multiple allelic variants associated with altered transport activity among European- and African-Americans

J. Biol. Chem.

Molecular cloning and characterization of a new multispecific organic anion transporter from rat brain

J. Biol. Chem.

Pharmacogenomics: translating functional genomics into rational therapeutics

Science

Molecular mechanisms of genetic polymorphisms of drug metabolism

Annu. Rev. Pharmacol. Toxicol.

Functional polymorphisms of the human multidrug-resistance gene: multiple sequence variations and correlation of one allele with P-glycoprotein expression and activity in vivo

Proc. Natl. Acad. Sci.

Identification of functional variant MDR1 alleles among European American and African American

Clin. Pharmacol. Ther.

Genetic polymorphisms of human organic anion transporters OATP-C (SLC21A6) and OATP-B (SLC21A9): allele frequencies in the Japanese population and functional analysis

J. Phamacol. Exp. Ther.

Organic cation transporters in intestine, kidney, liver, and brain

Annu. Rev. Physiol.

Cloning and functional expression of a human liver organic cation transporter

Mol. Pharmacol.

Cloning and characterization of two human polyspecific organic cation transporters

DNA Cell Biol.

Cited by (96)

Comprehensive in-silico analysis of damage associated SNPs in hOCT1 affecting Imatinib response in chronic myeloid leukemia

Pharmacogenetics of Antidiabetic Drugs

Uptake Transporters

Absorption characteristics of the total alkaloids from Mahonia bealei in an in situ single-pass intestinal perfusion assay

Structural insights into human organic cation transporter 1 transport and inhibition

Association of Met420del Variant of Metformin Transporter Gene SLC22A1 with Metformin Treatment Response in Ethiopian Patients with Type 2 Diabetes