Molecular mechanisms regulating Kv2.1-induction of endoplasmic reticulum / plasma membrane contact sites
Date
2019
Authors
Johnson, Ben, author
Tamkun, Michael, advisor
Amberg, Gregory, committee member
Di Pietro, Santiago, committee member
Prenni, Jessica, committee member
Tsunoda, Susan, committee member
Journal Title
Journal ISSN
Volume Title
Abstract
Kv2 voltage gated potassium channels localize to 'clusters' on the soma, axon initial segment, and dendritic arbor of hippocampal neurons. For decades the molecular mechanism behind this localization pattern was unknown. In 2015 our lab determined that this behavior was due to the channels interacting with an unknown endoplasmic reticulum resident protein and thereby forming endoplasmic reticulum / plasma membrane (ER/PM) junctions. The channel clusters covering the surface of cells represented those domains. The work in this dissertation examines in increased detail the mechanism, regulation, and possible functions associated with these sites. ER/PM junctions are domains with a variety of roles. They regulate both calcium and lipid homeostasis, they are involved in vesicular trafficking, and they oversee a host of cell signaling pathways. Junctions represent 12% of the neuronal soma surface and are also present in both the axon and the dendritic arbor. These are sites that exhibit a high degree of dynamic flux, both in composition and in structure. Residency of junction proteins is governed by the calcium concentration of the ER, the calcium concentration of the cytosol, the activity of the excitable cell, and the lipid composition of the PM. In turn these residents influence the nature of the junction, determining the function and nanoarchitecture of these domains. In this work we use a proximity-based biotinylation approach to identify VAMP-associated proteins (VAPs) as the Kv2 channel interactor responsible for the formation of ER/PM junctions. We characterize the amino acid motif necessary to generate interaction between the two proteins, finding an unconventional FFAT motif located in the channel C-terminus. We examine the protein composition of these novel junctions by investigating their relationship with other known ER/PM tethers such as Nir2, STIM1 and the junctophilins. We use super resolution imaging techniques to observe ER membrane behavior at these locations and study how that behavior changes during the concentration of additional protein residents. Lastly, we investigate the mechanisms underlying Kv2-VAP junction disassembly during neuronal activity and insult. We find that Kv2.1-VAP unbinding during glutamate stimulation is mediated by serine residues downstream of the Kv2.1 FFAT motif. This dispersal of Kv2-VAP ER/PM junctions during calcium influx is mirrored by junctophilin-induced junction disassembly, suggesting a common mechanism regulating ER/PM junctions throughout the hippocampus. This dissertation examines a novel microdomain formed by Kv2 channels and presents data describing how this domain is created and regulated on a molecular level. It represents the first in-depth study of this topic.
Description
Rights Access
Subject
ER/PM junction
plasma membrane
VAP
Kv2
endoplasmic reticulum
subsurface cisternae