The cataract and glucosuria associated monocarboxylate transporter MCT12 is a new creatine transporter

Abstract

Creatine transport has been assigned to creatine transporter 1 (CRT1), encoded by mental retardation associated SLC6A8. Here, we identified a second creatine transporter (CRT2) known as monocarboxylate transporter 12 (MCT12), encoded by the cataract and glucosuria associated gene SLC16A12. A non-synonymous alteration in MCT12 (p.G407S) found in a patient with age-related cataract (ARC) leads to a significant reduction of creatine transport. Furthermore, Slc16a12 knockout (KO) rats have elevated creatine levels in urine. Transport activity and expression characteristics of the two creatine transporters are distinct. CRT2 (MCT12)-mediated uptake of creatine was not sensitive to sodium and chloride ions or creatine biosynthesis precursors, breakdown product creatinine or creatine phosphate. Increasing pH correlated with increased creatine uptake. Michaelis-Menten kinetics yielded a Vmax of 838.8 pmol/h/oocyte and a Km of 567.4 µm. Relative expression in various human tissues supports the distinct mutation-associated phenotypes of the two transporters. SLC6A8 was predominantly found in brain, heart and muscle, while SLC16A12 was more abundant in kidney and retina. In the lens, the two transcripts were found at comparable levels. We discuss the distinct, but possibly synergistic functions of the two creatine transporters. Our findings infer potential preventive power of creatine supplementation against the most prominent age-related vision impaired condition.

Figure 1.

Immunofluorescence images. Membrane localization of human MCT12 (hMCT12) in Xenopus laevis oocytes injected with SLC16A12 cRNA. Noninjected oocytes and those injected with the chaperone CD147 alone served as controls (top row). Membrane localized signals (arrow) were detected only in oocytes injected with MCT12, irrespective of the presence of chaperone CD147.

Figure 2.

Creatine is transported by MCT12. (A) Significantly lower creatine levels (6.3-fold) were detected in oocytes coinjected with SLC16A12 and its chaperone CD147 compared with oocytes injected only with CD147. (B) Creatine efflux in noninjected oocytes (NI), and oocytes expressing CD147 or CD147 + hMCT12. Content of ¹⁴C creatine in the medium was recorded as disintegrations per minute at different time points (0, 15 and 60 min). (C) Creatine uptake. Measurements were taken 10 min after addition of ¹⁴C-creatine to the medium of oocytes (NI/CD147/CD147 + hMCT12). Uptake is shown as pmol/h/oocyte. Bars indicate SEM.

Figure 3.

Characterization of creatine uptake. (A) Michaelis–Menten kinetics of creatine uptake. V_max = 838.8 pmol/h/oocyte and K_m = 567.4 µM. (B) Ion dependency of creatine uptake. Experiments were performed either in the presence of both sodium and chloride (NaCl) or in sodium (Na⁺) or chloride (Cl⁻)-free medium. (C) pH dependency of creatine uptake. pH of 5.5, 6.5, 7.4 and 8.0 were tested. (D) Effect of potential competitors on creatine uptake. Creatine uptake alone (creatine) is shown as 100%. Bars indicate SEM.

Figure 4.

Effect of mutations on creatine transport and expression studies. (A) SLC16A12 mutation screen. Electropherograms of an unaffected individual and of a patient with ARC showing the heterozygous mutation (arrow) SLC16A12 c.1219G>A, p.G407S. (B) ¹⁴C creatine uptake of oocytes expressing the mutant hMCT12 p.G407S compared with the reference hMCT12. The mutation causes a significant reduction in creatine uptake by 43% (P = 0.0004). Uptake was recorded as pmol/h/oocyte. Bars indicate SEM. (C) Creatine levels in urine of rats. Male Slc16a12 KO or heterozygous (Het) rats and female KO and WT. Displayed are the percentages relative to the unaffected (not KO) males and females at 100%. Bars indicate SEM. (D) Expression of creatine transporter transcripts in human tissues. RT–PCR using primers specific for SLC16A12 (a) and SLC6A8 (b). Amplicon sizes are given in base pairs. Non-template control is water.