Identification of Kv1.3 potassium channel interacting proteins in mammalian cells.

Alzheimer's disease (AD) is a devastating neurodegenerative disease characterized by progressive neuronal loss, accumulation of amyloid-beta(Aβ) plaques, and neuroinflammation. Microglia, the immune cell of the brain, transition to disease associated microglia (DAM) in AD. A subset of DAM, known as proinflammatory DAM, promote neuronal injury and release proinflammatory cytokines. Our lab established that proinflammatory DAM highly express Kv1.3 potassium channel, which consist of a homotetramer of Kv1.3 that interacts with a homotetramer of Kvβ2. Blockade of the Kv1.3 channel reduces microglial immune responses in mouse models. Both Kv1.3 and Kvβ2 contain motifs that interact with immune signaling proteins, however more evidence is needed to support this hypothesis. TurboID is a biotin ligase that biotinylates proteins within a 10nm radius. Split-TurboID consists of two functionally inactive fragments of TurboID that within proximity of each other, fuse and actively biotinylate proteins within proximity. We will identify molecular interactors of Kv1.3 channels in mammalian cells using proximity labeling.We fused TurboID or Split-TurboID to the N- or C-terminal Kv1.3 and Kvβ2 using a 15 amino-acid inert linker and transfected the constructs into Hek293 cells. TurboID not fused to Kv1.3 and sham transfection acted as controls. Flow cytometry and patch-clamp electrophysiology confirmed the presence of functional Kv1.3 channels. Biotinylated proteins were streptavidin bead affinity-enriched from cell lysates and confirmed using western blots.Label free quantitation mass spectrometry (LFQ-MS) identified 2,195 biotinylated proteins of which very few were enriched in negative controls. There were 313 proteins as N-term interactors, 385 as C-term interactors, and 226 proteins common to both N and C term fusions, representing proteins indirectly associated with Kv1.3 channels. we identified 14 unique N-term interactors (including metabolic proteins, eg. Naca, Txnl1, Stub1) and 38 unique C-term interactors (including membrane trafficking proteins, eg. Ank3, Plekha7, Erlec1). Split-TurboID interactions between Kv1.3 and Kvβ2 indicate that these interacting proteins are largely dependent on the fully functional Kv1.3 with the Kvβ2 subunit present. We packaged these plasmids into lentiviruses to transduce into mouse microglial cell lines.Our findings indicate Kv1.3 channels interacting with Kvβ2 are a novel molecular mechanism of immune regulation in mammalian cells.