Genetic polymorphisms in organic cation transporter 1 (OCT1) in Chinese and Japanese populations exhibit altered function

Abstract

Organic cation transporter 1 (OCT1; SLC22A1) seems to play a role in the efficacy and disposition of the widely used antidiabetic drug metformin. Genetic variants in OCT1 have been identified largely in European populations. Metformin is increasingly being used in Asian populations where the incidence of type 2 diabetes (T2D) is on the rise. The goal of this study is to identify genetic variants of OCT1 in Chinese and Japanese populations, which may potentially modulate response to metformin. We used recent data from the 1000 Genomes Project (Chinese and Japanese) and direct sequencing of selected amplicons of OCT1 in 66 DNA samples from Japanese patients with T2D. A total of six nonsynonymous variants were identified. Three of them (Q97K, P117L, and R206C) had not been functionally characterized previously and had allele frequencies of 0.017, 0.023 and 0.008, respectively. The uptake of metformin in cells expressing Q97K, P117L, and R206C was significantly reduced relative to the OCT1 reference (62 ± 4.3, 55 ± 6.8, and 22 ± 1.5% for Q97K, P117L, and R206C, respectively). Kinetic studies indicated that P117L and R206C exhibited a reduced V(max), whereas Q97K showed an increased K(m). The green fluorescent protein (GFP)-tagged Q97K and P117L variants localized to the plasma membrane, whereas the GFP-tagged R206C was retained mainly in the endoplasmic reticulum. Replacement of the highly conserved R206 with different amino acids modulated the subcellular localization and function of the transporter. This study suggests that nonsynonymous variants of OCT1 in Chinese and Japanese populations may affect the differential response to metformin.

Fig. 1.

Functional analyses of three nonsynonymous variants of OCT1. Variants of OCT1 were expressed in stably transfected HEK293 cells and assayed for activity by measurement of uptake of radiolabeled probe substrate (10 μM [¹⁴C]metformin) at 5 min. A, uptake activity of two variants compared with OCT1-ref (control). Uptake activity was calculated as [(OCT1or Variant-Mock)/OCT1-Mock] × 100. Values are expressed as mean ± S.D. ***, P < 0.001 versus OCT1-ref. B, kinetics of metformin transport by reference OCT1 and variants P117L and R206C. HEK293 cells stably expressing either OCT1-ref, OCT1-P117L, OCT1-R206C, or Mock were incubated in the presence of 10 μM [¹⁴C]metformin and varying concentrations of unlabeled metformin for 5 min. C, metformin-stimulated AMPK phosphorylation in cell lines stably transfected mock, OCT1, and OCT1's three variants. Immunoblots were performed against phospho-AMPKα (Thr172), AMPKα, and β-actin.

Fig. 2.

Subcellular localization and quantification of surface expression of OCT1 and three genetic variants. A, OCT1-variant GFP fusion constructs were transiently expressed in HEK293 cells and visualized by fluorescence microscopy. The nucleus, which was stained with 4′,6-diamidino-2-phenylindole, is shown in blue. GFP-OCT1 proteins are shown in green. In GFP-Mock cells, colocalization of GFP-derived signal and that from nucleus stain is shown in cyan. The red arrow in R206C indicates that this variant changed the OCT1 protein subcellular localization. B, Colocalization of GFP-R206C and ER. Orange indicates the GFP-R206C was well colocalized with ER. C, biotinylation of the plasma membrane of GFP-tagged OCT1 and its variants. Q97K and P117L had similar cell surface expression levels to the OCT1-ref. R206C showed dramatically reduced cell surface expression compared with OCT1-ref.

Fig. 3.

Subcellular localization of different substitutions of R206. A, OCT1-variant GFP fusion constructs were transiently or stably expressed in HEK293 cells and visualized by confocal microscopy. Red arrows indicate the expression of OCT1 protein localized to the plasma membrane, and the white arrow shows the protein localized in the membrane transporting apparatus ER. B, colocalization of GFP-R206 substitutions on the plasma membrane. The plasma membrane was stained with Alexa Fluor 594-labeled WGA, shown in red. GFP-R206 substitutions are shown in green. Colocalization of GFP-derived signal and that from the plasma membrane stain is shown in orange.

Fig. 4.

Fluorescent intensity quantification and uptake assay of GFP-tagged OCT1 and its mutations. A, quantification of fluorescent intensity of GFP-tagged OCT1, its variants, or various substitutions of R206 with flow cytometry. All of the mutations (red peaks) were compared with GFP-OCT1 (gray peaks). B, uptake assays of metformin in cells expressing GFP-OCT1 and its mutations. Uptake activity was calculated as [(GFP-OCT1 or GFP-variant − Mock)/OCT1 − Mock] × 100. ***, P < 0.001 versus GFP-OCT1. Data represent the mean ± S.D. from triplicate wells in a representative experiment.

Fig. 5.

Sequence alignment of the proximal region of R206 of OCT1 with its homologs. A, species are listed as representative species from invertebrate (Drosophila) to primates (Homo sapiens). The genes are identified or predicted cation transporters. Arginine 206 is not only highly conserved in all of the cation transporters of all of the species but it is also highly conserved in human organic anion transporters, OAT1–OAT3. B, topology of OCT1 revealed that Q97K and P117 were located at the extra-large loop and R206 was between the third and fourth transmembrane domains. Three variants are highlighted in the topology figure as red. C, cysteine mutation of highly conserved homologous arginine in OCT3 and OAT2 showed similar intracellular retention to the R206C in OCT1.