Kv2 channels create endoplasmic reticulum / plasma membrane junctions: a brief history of Kv2 channel subcellular localization

Abstract

The potassium channels Kv2.1 and Kv2.2 are widely expressed throughout the mammalian brain. Kv2.1 provides the majority of delayed rectifying current in rat hippocampus while both channels are differentially expressed in cortex. Particularly unusual is their neuronal surface localization pattern: while half the channel population is freely-diffusive on the plasma membrane as expected from the generalized Singer & Nicolson fluid mosaic model, the other half localizes into micron-sized clusters on the soma, dendrites, and axon initial segment. These clusters contain hundreds of channels, which for Kv2.1, are largely non-conducting. Competing theories of the mechanism underlying Kv2.1 clustering have included static tethering to being corralled by an actin fence. Now, recent work has demonstrated channel clustering is due to formation of endoplasmic reticulum/plasma membrane (ER/PM) junctions through interaction with ER-resident VAMP-associated proteins (VAPs). Interaction between surface Kv2 channels and ER VAPs groups channels together in clusters. ER/PM junctions play important roles in inter-organelle communication: they regulate ion flux, are involved in lipid transfer, and are sites of endo- and exocytosis. Kv2-induced ER/PM junctions are regulated through phosphorylation of the channel C-terminus which in turn regulates VAP binding, providing a rapid means to create or dismantle these microdomains. In addition, insults such as hypoxia or ischemia disrupt this interaction resulting in ER/PM junction disassembly. Kv2 channels are the only known plasma membrane protein to form regulated, injury sensitive junctions in this manner. Furthermore, it is likely that concentrated VAPs at these microdomains sequester additional interactors whose functions are not yet fully understood.

Targeted disruption of Kv2.1-VAPA association provides neuroprotection against ischemic stroke in mice by declustering Kv2.1 channels

“…With this information, we hypothesized that the region encompassing amino acids 602 to 608 within Kv2.2 CT was most likely to be responsible for disrupting Kv2.1 clusters, likely by outcompeting a “cluster targeting” sequence on existing channels that recruited them to the cluster microdomain. Kv2.1 and Kv2.2 target somatodendritic clusters via noncanonical two phenylalanine in an acidic tract (FFAT) motif sequences, namely SFISCAT and SFTSCAT ( 17 – 19 ), which overlap substantially with our proposed domain (fig. S1B).…”

“…S1B). These sequences interact with the ER linker VAPs ( 17 , 19 ). As these sequences omit one critical residue strongly implicated in cluster formation (S602 in Kv2.2 and S587 in Kv2.1), we opted to evaluate a sequence that includes all four critical proclustering residues (fig.…”

“…Kv2.1 and Kv2.2 channels are recruited to ER-PM junctions via CT PRC-mediated interaction with VAPA/B ( 17 – 19 ). As indicated earlier, this process occurs at noncanonical FFAT binding motifs within the PRC domains of Kv2.1 and Kv2.2 (fig.…”

“…Conversely, mutation of these same residues to aspartic acid restores CD4-linked PRC-VAPA interaction ( 19 ). Although these phosphorylation events had been proposed to render the PRC domain as containing noncanonical FFAT motifs ( 17 – 19 ), we were also unable to observe demonstrable binding to VAPA with these amino acids sequences in the peptide array. The pseudo-phosphorylated mutant DP-2 peptide sequences lacked VAPA binding capacity in our peptide spot array assay (fig.…”

“…Following oxidative or nitrosative injury, free zinc displacement from metallothionein by reactive oxygen intermediate species (ROS) triggers dual phosphorylation of Kv2.1 channels by Src and p38, resulting in a calcium/calmodulin-dependent protein kinase II–dependent interaction between syntaxin and the proximal CT domain of the channel ( 3 , 8 , 10 , 25 , 32 – 34 ). This process results in exocytotic insertion of proapoptotic Kv2.1 channels into the PM, likely at specialized Kv2.1 cluster domains that form ER-PM junctions ( 18 , 19 ) and act as scaffolding sites for up to 85% of Kv2.1 channels that are trafficked to the membrane ( 15 ). We know that this population of proapoptotic Kv2.1 is distinct from the existing, freely dispersed population of physiologically active Kv2.1 that regulates neuronal excitability and neuronal firing rates in normal brain tissue, as these channels are de novo inserted in a delayed manner in response to injury ( 3 , 8 , 10 , 25 , 32 – 34 ).…”