Identification of Novel Kv1.3 Channel-Interacting Proteins Using Proximity Labelling in T-Cells

Potassium channels regulate membrane potential, calcium flux, cellular activation and effector functions of adaptive and innate immune cells. The voltage-activated Kv1.3 channel is an important regulator of T cell-mediated autoimmunity and microglia-mediated neuroinflammation. Kv1.3 channels, via protein-protein interactions, are localized with key immune proteins and pathways, enabling functional coupling between K+ efflux and immune mechanisms.

To gain insights into proteins and pathways that interact with Kv1.3 channels, we applied a proximity-labeling proteomics approach to characterize protein interactors of the Kv1.3 channel in activated T-cells. Biotin ligase TurboID was fused to either N or C termini of Kv1.3, stably expressed in Jurkat T cells, and biotinylated proteins in proximity to Kv1.3 were enriched and quantified by mass spectrometry.

We identified over 1,800 Kv1.3 interactors including known interactors (beta-integrins, Stat1), although the majority were novel. We found that the N-terminus of Kv1.3 preferentially interacts with protein synthesis and protein trafficking machinery, while the C-terminus interacts with immune signaling and cell junction proteins. T-cell Kv1.3 interactors we found consisted of 335 cell surface proteins, including T-cell receptor complex, mitochondrial, calcium and cytokine-mediated signaling pathway, and lymphocyte migration proteins. 178 Kv1.3 interactors in T-cells also represent genetic risk factors for T cell-mediated autoimmunity, including STIM1, which was further validated using co-immunoprecipitation.

Our studies revealed novel proteins and molecular pathways that interact with Kv1.3 channels in adaptive (T-cell) and innate (microglia) immune cells, providing a foundation for understanding how Kv1.3 channels may regulate immune mechanisms in autoimmune.