Using Bimolecular Fluorescence Complementation to Visualize Protein-Protein Interactions of Potassium Channels in Live Cells

Bimolecular fluorescence complementation (BiFC) is a technique that allows for the direct visualization of protein-protein interactions in their cellular environment. In this study, a fluorescence emission is observed in BiFC when the proteins tagged with two complimentary non-fluorescent fragments interact to facilitate fusion of the split fluorophore. Here, I used BiFC to show that the potassium leak channel subunits hK2P1 and hK2P2 readily interact to form heterodimers. This is significant given that there are opposing views presented by research groups about the association of these subunits. Total internal reflection fluorescence (TIRF) microscopy verified that these heterodimers are in the plasma membrane, where functional K2P channels should be located. Electrophysiology via whole-cell patch clamp, however, gave results consistent with that of homomeric hK2P2 channels, and not heteromeric channels demonstrating hK2P1 function. The addition of SENP1 to detect whether the observed channels were SUMOylated gave electrophysiology data supporting that only hK2P1 homodimers are SUMOylated, consistent with literature. Coupling BiFC with Förster resonance energy transfer (FRET) microscopy, I investigated the simultaneous interaction between three proteins, the observed split K2P channels and SUMO2. Only the FRET time constant for homomeric hK2P1 in the presence of SUMO was deemed significant when compared to channels in the absence of SUMO. While the use of BiFC advocates that the heterodimers are indeed formed, it seems that functionality needs to be evaluated further.