A number of studies have lead to the suggestion that disruptions to chloride homeostasis play a role in a variety of neurological and neurodevelopmental disorders. The neuron-specific potassium chloride co-transporter, KCC2, is the major chloride exporter in neuronal cells, and mutations in SLC12A5 (the gene encoding KCC2) have been reported in individuals with some neurodevelopmental disorders, such as autism spectrum disorder (ASD), epilepsy and schizophrenia. Further, results from KCC2 knockout and knockdown mice highlight the importance of this protein in proper neuronal function.
KCC2 functions to establish a chloride ion concentration gradient across neuronal membranes. This allows inhibitory neurotransmitters to propagate their signals by triggering chloride influx at postsynaptic receptors. While past studies have suggested a role for KCC2 in neural function and dysfunction, biophysical characterizations of KCC2 are lacking, making it hard to assess how mutations in KCC2 may lead to altered function.
Charles Craik’s laboratory plans to perform a detailed biophysical characterization of KCC2. To do so, they plan to first develop a reproducible protocol for the production of milligram quantities of biochemically validated, functional KCC2. Biophysical characterization of KCC2 will be conducted by antibody-fragment-stabilized electron microscopy. The laboratory has previously used fragments of antibodies (Fabs) and nanobodies for the characterization of other transporters and enzymes1,2,3. Craik and colleagues find that Fabs can stabilize proteins, increase protein monodispersion required for electron microscopy (EM) and significantly improve EM results by providing a high-quality fiducial marker for class averaging.
There is currently only very limited understanding of the biophysical properties of KCC2. This study is expected to help identify the oligomeric state of KCC2 and help to evaluate the hypothesis that oligomerization of KCC2 is required for its activity. Furthermore, this isolation protocol can also facilitate crystallization through Fab-mediated crystal contacts, providing high-quality material for future atomic-level structural determination3. Craik’s team anticipates that their KCC2 biophysical data will be valuable in designing new drugs that modulate the function of KCC2 and which may be used to treat a variety of neurodevelopmental disorders, including ASD.