Regulation of signaling and function of the voltage-gated sodium channel complex by protein:protein interactions
As fundamental determinants of neuronal function, voltage-gated Na+ (Nav) channels are important targets for therapeutic development against a wide range of health conditions. Dysfunction of Nav channels in the CNS is associated with disorders ranging from neurological (i.e., epilepsy, neurodegeneration) to psychiatric (i.e., major depression disorder, schizophrenia). Unfortunately, commercially available drugs targeting Nav channels are directed against highly conserved domains across Nav isoforms, giving rise to severe side effects such as cardiotoxicity and movement disorders. Thus, there is an unmet need for discovering new probes and pathways that regulate Nav channels that could potentially help designing new medications. Recent evidence suggests that protein:protein interactions (PPI) between Nav channels and their accessory proteins play a key role in regulating neuronal firing, and that minimal disturbances to these tightly controlled PPI can lead to persistent maladaptive plasticity. These PPI interfaces are highly specific and provide ideal targets for drug development, especially in the CNS where selectivity and specificity are vital for limiting side effects. However, for the most part how these protein:channel interactions are regulated in the cell is still poorly understood, and methods for assessing these interactions are lacking. Therefore, the goal of the present study was to develop robust assays to reconstitute the Nav channel complex in cells and identify cellular pathways and small molecules regulating PPI interfaces with the Nav channel complex. Specifically, we focused on the PPI between Nav1.6 and its regulatory protein, fibroblast growth factor 14 (FGF14). Using a newly developed assay we screen cellular pathways followed by biophysical validation, we discovered a mechanism by which the JAK2 tyrosine kinase might directly influence neuronal firing through phosphorylation of FGF14. Furthermore, we conducted a high-throughput screening of ~45,000 small molecules and identified potent modulators of the FGF14:Nav1.6 complex that are functionally active and predicted to be permeable to the blood-brain barrier. While providing a robust in-cell screening platform that can be adapted to search for any channelopathy-associated regulatory protein, these results lay the potential groundwork for a new class of drugs targeting Nav channels with a broad range of applicability for CNS disorders.