Large-scale experimental assessment of variant effects on the structure and function of the citrate transporter SLC13A5

Citrate is an essential metabolite playing critical roles in metabolism and as a neuromodulator during cellular differentiation and development. Mutations in the human citrate transporter SLC13A5, highly expressed in neurons, have been associated with a debilitating form of epileptic encephalopathy. In this study, we used deep mutational scanning to reveal the effect of 90% of all possible single missense variants on the structure and function of SLC13A5. Computational analyses and a detailed experimental validation of 38 variants revealed an accuracy of 86% and provided mechanistic interpretations for deleterious mutations, including the effect on protein stability, trafficking, and citrate transport. Analyses of blood citrate concentration from individuals enrolled in the UK Biobank study supported our analyses. The results illustrate an unbiased mutational landscape of the citrate transporter, illuminate mechanisms of pathogenicity, and offer a platform for the analysis of specific variants as well as opportunities for the future development of intervention strategies.