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Proc Natl Acad Sci U S A. 2018 Jul 31;115(31):E7331-E7340. doi: 10.1073/pnas.1805757115. Epub 2018 Jun 25.

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

Author information

1
Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523.
2
Program in Molecular, Cellular, and Integrative Neurosciences, Colorado State University, Fort Collins, CO 80523.
3
Institute of Ophthalmology, University College London Institute of Ophthalmology, London EC1V 9EL, United Kingdom.
4
Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523; michael.tamkun@colostate.edu.
5
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523.

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 Aplysia 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.

KEYWORDS:

ER/PM junctions; Kv2.1; Kv2.2; VAP; subsurface cisternae

Comment in

PMID:
29941597
PMCID:
PMC6077746
DOI:
10.1073/pnas.1805757115
[Indexed for MEDLINE]
Free PMC Article

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