Kv2.1-Induced Er/Pm Junctions Modify The Cell Surface Diffusion Landscape

The Kv2.1 voltage-gated K+ channel is widely expressed in the nervous system. In addition to its classical function as a delayed rectifier K+ channel, Kv2.1 has a structural role where it interacts with the cortical endoplasmic reticulum (ER) to form ER-plasma membrane (ER/PM) junctions in a variety of cell types. Within these micron-sized domains the ER membrane is held within 20 nm of the PM. Our previous work has shown that ER/PM junctions serve as membrane trafficking hubs where endo/exocytosis events are localized. We also showed that large membrane proteins such as voltage-gated Na+ channels are excluded from the Kv2.1-induced ER/PM contact sites. In order to test the possibility that the Kv2.1-induced ER/PM junctions influence the overall cell surface diffusion landscape we used TIRF-based single molecule tracking to examine the diffusion of CD4 and beta adrenergic receptors, two very different membrane proteins. Single-molecule tracking was performed while simultaneously imaging ER/PM junctions induced either by Kv2.1, Junctophilin-4 (JP4), or an artificial membrane bridge based on rapamycin ligands. The analysis of local mean square displacement (MSD) shows that the diffusion of these molecules is typically anomalous and the diffusive state displays a marked correlation to the proximity to ER/PM contact sites. This analysis indicates that Kv2.1 ER/PM junctions not only alter protein diffusion in the vicinity of these membrane microdomains, but also concentrate PM proteins directly over the ER. Most interestingly, differences between Kv2.1-induced junctions vs. junctions formed with Junctophilin or the rapamycin bridge indicate altered diffusion is specific to the presence of Kv2.1. These results suggest the dynamics of membrane proteins are regulated to enhance the collision with signaling molecules and recruitment to clathrin coated pits in the vicinity of the Kv2.1-induced ER/PM junctions.