Biochemical and Molecular Characterization of the Most-Common SLC13A5-Epilepsy Causing Missense-Mutations

The sodium-coupled citrate transporter (NaCT) is a plasma membrane transporter, which is energized by an inwardly directed electrochemical sodium gradient. It mediates the symport of sodium and the carboxylate citrate into cells. NaCT is expressed in the liver, testis, brain, bone, and teeth, where citrate plays key roles in the synthesis of neurotransmitters, cholesterol, and fatty acids, the generation of energy, and teeth/bone mineralization. In humans, loss-of-function mutations in SLC13A5, the NaCT gene, cause early infantile epileptic encephalopathy type-25 (EIEE25, SLC13A5-Epilepsy), which leads to epilepsy, impaired speech, limited motor skills, developmental delay, and tooth defects. Currently, there is no treatment for EIEE25. Recently, the cryo-electron microscopy structure of the human NaCT was solved in an inward-facing conformation. This was an important advancement in the NaCT field, paving the way for a better understanding of the structure-function relationships for this clinically important transporter. We classified 22 NaCT missense disease-causing mutations based on their localizations in the 3D structure. Class I mutations interfere with the transport function by blocking the elevator-type mechanism for substrate translocation. Class II cause defects in protein folding and protein trafficking to the cell surface, which may be corrected by small molecule therapeutics. As there are no NaCT-specific antibodies, we expressed WT and the mutants with specific epitopes to facilitate detection, which didn’t interfere with the presentation of the mutant phenotypes. The Class I mutations C50R, T142M, and T227M displayed protein and surface expression levels similar to WT. Class II mutants G219R, S427L, and L488P showed significantly decreased protein expression and no plasma membrane expression. Both classes displayed diminished transport activity. These experiments have brought us one step closer to understanding the defects of disease-causing mutations at the molecular level, allowing us to begin dissecting NaCT trafficking pathway(s).