Kv2 potassium channels form endoplasmic reticulum/plasma membrane junctions via interaction with VAPA and VAPB

Abstract

Kv2.1 exhibits two distinct forms of localization patterns on the neuronal plasma membrane: One population is freely diffusive and regulates electrical activity via voltage-dependent K conductance while a second one localizes to micrometer-sized clusters that contain densely packed, but nonconducting, channels. We have previously established that these clusters represent endoplasmic reticulum/plasma membrane (ER/PM) junctions that function as membrane trafficking hubs and that Kv2.1 plays a structural role in forming these membrane contact sites in both primary neuronal cultures and transfected HEK cells. Clustering and the formation of ER/PM contacts are regulated by phosphorylation within the channel C terminus, offering cells fast, dynamic control over the physical relationship between the cortical ER and PM. The present study addresses the mechanisms by which Kv2.1 and the related Kv2.2 channel interact with the ER membrane. Using proximity-based biotinylation techniques in transfected HEK cells we identified ER VAMP-associated proteins (VAPs) as potential Kv2.1 interactors. Confirmation that Kv2.1 and -2.2 bind VAPA and VAPB employed colocalization/redistribution, siRNA knockdown, and Förster resonance energy transfer (FRET)-based assays. CD4 chimeras containing sequence from the Kv2.1 C terminus were used to identify a noncanonical VAP-binding motif. VAPs were first identified as proteins required for neurotransmitter release in and are now known to be abundant scaffolding proteins involved in membrane contact site formation throughout the ER. The VAP interactome includes AKAPs, kinases, membrane trafficking machinery, and proteins regulating nonvesicular lipid transport from the ER to the PM. Therefore, the Kv2-induced VAP concentration at ER/PM contact sites is predicted to have wide-ranging effects on neuronal cell biology.

Optical measurement of voltage sensing by endogenous ion channels

“…In cultured HEK293 cells, COS-1 cells, and central neurons, 28 clusters mark cortical endoplasmic reticulum junctions with the plasma membrane (Antonucci et 29 al. 2001, Fox et al 2015, Cobb et al 2016, Johnson et al 2018, 30 Kirmiz, Vierra, et al 2018. After incubation with membrane-impermeant GxTX-594 (100 nM 31 for 5 minutes), fluorescence localized to regions with Kv2-GFP signal (Fig 1 A).…”

Mapping Functional Protein Neighborhoods in the Mouse Brain

“…For instance, the original poster-child for a biological transistor, the voltage-gated sodium channel, is now known to reside within large, multi-protein complexes, including structural and signaling elements, as well as other ion channels (Lee et al, 2014;Meadows and Isom, 2005) . Indeed, channel isoforms can serve purely as structural elements, rather than channels (Johnson et al, 2018) . Furthermore, the evolution of neuronal protein interactions is now known to be more plastic than previously appreciated (Liebeskind et al, 2016) , increasing the need for methods such as those we apply here, which are applicable in principle to any organism or cell type.…”

High throughput Characterization ofKCNB1variants Associated with Developmental and Epileptic Encephalopathy

“…Classification of KCNB1 variants into different categories of defects indicates that molecular chaperones to increase cell-surface expression may be a suitable therapeutic strategy for some patients. A subtype-selective activator of KV2.1 may be a viable therapy for others, although this would not address non-conducting functions of KV2.1 31,40 , which may contribute to disease pathophysiology. Alternatively, gene therapy approaches that increase expression of the WT allele may hold promise for treatment of patients with KCNB1 variants.…”