KCC2, a potassium-chloride co-transporter, is the major chloride exporter in neurons and is responsible for maintaining the low neuronal resting chloride concentration. It is critical for synaptic transmission of inhibitory signals by neurotransmitters. Mutations in SLC12A5 (the gene encoding KCC2) have been reported in individuals with neurodevelopmental and psychiatric disorders, including autism spectrum disorder (ASD), epilepsy and schizophrenia. Further, results from KCC2 mutant mice have highlighted the importance of this protein in proper neuronal function.
Despite its significant role in both neural function and dysfunction, biophysical characterizations of KCC2 are currently lacking, making it hard to assess how mutations in KCC2 may lead to altered function. Challenges in generating a high-resolution structure of KCC2 include its large size and membrane localization, which makes it difficult to produce recombinantly and maintain in solution.
Charles Craik and his colleagues at the University of California, San Francisco, have expertise in studying similarly difficult transporters1,2. In the current project, they plan to use this experience to perform a detailed biophysical characterization of KCC2. They will accomplish this goal by producing reagent quality and quantity recombinant KCC2 for fragment antigen-binding (Fab) or nanobody-stabilized negative-stain electron microscopy (EM). In preliminary studies, funded by an earlier SFARI Explorer Award, they have succeeded in optimizing the production and purification protocol for KCC2 orthologues expressed in insect cells. They now plan to validate KCC2 quality by an activity assay followed by the development and preparation of Fab- or nanobody-bound KCC2 for EM. Craik and his team also plan to study disease-related mutations by recombinant production of the mutants and functional characterization using the KCC2 activity assay.
Taken together, findings from these studies will provide an initial biophysical characterization of KCC2. This will provide valuable information for future rational drug design efforts to regulate KCC2 activity, which may be used to treat a variety of disorders, including ASD.