Proximity Labeling Proteomics Reveals Kv1.3 Potassium Channel Immune Interactors in Microglia.

Area of Science:

• Neuroscience
• Immunology
• Cell Biology

Background:

• Microglia are brain immune cells regulating inflammation.
• Disease-associated microglia (DAM) show increased pro-inflammatory markers and altered metabolism.
• Kv1.3 channel inhibition reduces DAM pro-inflammatory signature, but its interaction mechanisms are unclear.

Purpose of the Study:

• To identify proteins interacting with Kv1.3 during the transition to DAM.
• To investigate the role of Kv1.3 domains in protein interactions during inflammation.
• To understand Kv1.3's function beyond ion conduction in neuroinflammation.

Main Methods:

• Utilized TurboID proximity labeling fused to Kv1.3 in BV-2 microglia activated by TLR4.
• Employed mass spectrometry to identify interacting proteins.
• Used electrophysiology, Western blotting, and flow cytometry to assess Kv1.3 channel activity and biotinylation.
• Investigated Kv1.3 domain-specific interactions and functional coupling with STAT1.

Main Results:

• Kv1.3 N-terminus mediates trafficking to cell surface and mitochondria (e.g., NUDC, TIMM50).
• Kv1.3 C-terminus interacts with immune signaling proteins (e.g., STAT1, TLR2, C3) during inflammation.
• 70 proteins, including ND3, Snx3, and Sun1, interact with Kv1.3 via its C-terminal PDZ-binding domain.
• Kv1.3 blockade confirmed functional coupling with interferon-mediated STAT1 activation.

Conclusions:

• Kv1.3 channel has roles beyond potassium transport in inflammatory contexts.
• Kv1.3 modulates key immune signaling proteins like STAT1 and C3 in microglia.
• Understanding Kv1.3 interactions provides insights into neuroinflammation and potential therapeutic targets.