Identification of the substrate and characterization of transport properties for the cataract associated monocarboxylate transporter MCT12

Abstract

Purpose: Mutations in the gene SLC16A12, encoding the monocarboxylate transporter MCT12, have been implicated in congenital cataract with microcornea and glucosuria as well as in age related cataracts. We hypothesized that disturbed solute homeostasis is part of the underlying pathomechanism. To better understand the function of the orphan transporter MCT12, we aimed to identify its substrate.

Methods: The heterologous expression system of Xenopus laevis oocytes and a metabolomics approach were combined. Human reference and mutant SLC16A12 cRNA were injected into oocytes and transport activity was recorded. Urine of wild type and Slc16a12 knock-out rats was analyzed. Expression pattern of SLC16A12 and SLC6A8 from various human tissues were investigated.

Results: This novel combination of approaches proved successful as it allowed us to identify creatine as the substrate that is specifically transported by MCT12. This facilitated trans-membrane transport followed Michaelis Menten kinetics, was insensitive to sodium and chloride ions and to its phosophorylated form, was not affected by the presence of its biosynthetic precursors but showed pH sensitivity. Creatine transport by MCT12 was further shown by genetic and physiological experiments: a non-synonymous mutation that was identified in a patient with age-related cataract showed a significant reduction of creatine transport in the oocyte system and the urine of Slc16a12 knock-out rats had significantly increased levels of creatine. Previously, another creatine transporter CRT1, encoded by SLC6A8, had been reported which, when mutated lead to mental retardation but no eye phenotypes. In support, relative expression of SLC16A12 and SLC6A8 showed complementary tissue-specificity.

Conclusions: Creatine is a highly important cellular energy buffer and its disturbed transport can have serious consequences either in cataract and glucosuria or mental retardation, depending on the effective transporter. Partial, inefficient transport due to mutations in SLC16A12 may result in the age-related form of cataracts. Overall, the heterologous expression system in combination with metabolomics is a powerful tool that could find numerous applications.

Keywords: 445 cataract • 592 metabolism • 533 gene/expression