Abstract
The plasma membrane is a well-organized structure of lipids and proteins, segmented into lipid compartments under 200 nm in size. This specific spatial patterning is crucial for the function of proteins and necessitates super-resolution imaging for its elucidation. Here, we establish that the genetically encoded enhanced green fluorescent protein (EGFP), when combined with direct optical reconstruction microscopy (dSTORM), tracks shear- and cholesterol-induced nanoscopic patterning of potassium channels overexpressed in HEK293T cells. Leveraging EGFP in dSTORM (EGFP-STORM), our findings indicate that cholesterol directs the C-terminus of TWIK-related potassium channel (TREK-1) to ceramide-enriched lipid ganglioside (GM1) clusters. In the absence of the C-terminus, the channel associates with phosphatidylinositol 4,5-bisphosphate (PIP2) cluster. Similarly, cholesterol derived from astrocytes repositions EGFP-tagged inward-rectifying potassium (Kir) channels into GM1 clusters. Without cholesterol, the channel aligns with PIP2 lipids. We deduce that cholesterol's interaction with Kir sequesters the channel, separating it from its activating lipid PIP2. Fundamentally, a genetically encoded EGFP tag should make any protein amenable to dSTORM analysis.