GAT-1 VARIANTS IN EPILEPSY: MOLECULAR & RESCUE MECHANISMS

The human -aminobutyric acid (GABA) transporter-1 (hGAT-1) belongs to the solute carrier 6 (SLC6) gene family (member SLC6A1). Numerous point mutations in the hGAT-1 gene have been linked to epilepsy. The molecular mechanisms underlying this disease are currently unclear. Many mutations in other SLC6 family members are known to impair protein folding, causing their retention in the endoplasmic reticulum (ER) and precluding their delivery to the cell surface. Such misfolding events subsequently give rise to severe pathological conditions, e.g. folding-deficient variants of the dopamine transporter (DAT, SLC6A3) and the creatine transporter-1 (CRT-1, SLC6A8) trigger infantile/juvenile parkinsonism-dystonia and severe X- linked mental retardation, respectively. In some cases, folding defects can be corrected by treatment with pharmacological and/or chemical chaperones. Our preliminary data indicate that all of the epilepsy-linked mutations in hGAT-1 greatly reduce or completely abolish GABA uptake activity, and that a large proportion of these are absent from the cell surface due to GAT-1 protein folding defects. Some mutants appear to be properly trafficked to the cell surface, but nonetheless render the protein incapable of transport. Our aim is to elucidate the molecular basis of epilepsy triggered by GAT-1 variants and, in addition, assess the efficacy of diverse small molecules in their ability to rescue their cell surface expression and function. We plan to systematically examine and categorise the variants, using state-of the-art computer simulation models to study the structural effects of mutations at the atomic level, and use these discernments as a complimentary approach to guide for in-depth biochemical and pharmacological characterisation in vitro, in HEK293 cells, as well as in primary neuronal cultures. We are also the very first to examine these GAT-1 variants using Drosophila melanogaster as an animal model, to explore the effects of mutations on animal behaviour and molecular features of this debilitating disease. We will utilise the pharmacochaperoning approach to assess the efficacy of diverse small molecules in restoring the activity of misfolded variants, both in vitro (i.e. in HEK293 cells) and in vivo (i.e. in living flies). Taken together, this fundamental work will provide crucial new insights into (i) the molecular and structural basis of epilepsy in patients harbouring mutations in hGAT-1 and (ii) a proof-of principle that misfolded variants associated with epilepsy are amenable to rescue by pharmacochaperoning. This grants innovative therapeutic prospects for the treatment of the affected patients and justifies a rational search for additional compounds to remedy the epilepsy symptoms induced by (at least some) pathological mutations in the hGAT-1 gene.