Identification of VAPA and VAPB as Kv2 channel-interacting proteins defining endoplasmic reticulum–plasma membrane junctions in mammalian brain neurons

Michael Kirmiz, Nicholas C. Vierra, Stephanie Palacio, James Trimmer

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Membrane contacts between endoplasmic reticulum (ER) and plasma membrane (PM), or ER-PM junctions, are ubiquitous in eukaryotic cells and are platforms for lipid and calcium signaling and homeostasis. Recent studies have revealed proteins crucial to the formation and function of ER-PM junctions in non-neuronal cells, but little is known of the ER-PM junctions prominent in aspiny regions of mammalian brain neurons. The Kv2.1 voltage-gated potassium channel is abundantly clustered at ER-PM junctions in brain neurons and is the first PM protein that functions to organize ER-PM junctions. However, the molecular mechanism whereby Kv2.1 localizes to and remodels these junctions is unknown. We used affinity immunopurification and mass spectrometry-based proteomics on brain samples from male and female WT and Kv2.1 KO mice and identified the resident ER vesicle-associated membrane protein-associated proteins isoforms A and B (VAPA and VAPB) as prominent Kv2.1-associated proteins. Coexpression with Kv2.1 or its paralog Kv2.2 was sufficient to recruit VAPs to ER-PM junctions. Multiplex immunolabeling revealed colocalization of Kv2.1 and Kv2.2 with endogenous VAPs at ER-PM junctions in brain neurons from male and female mice in situ and in cultured rat hippocampal neurons, and KO of VAPA in mammalian cells reduces Kv2.1 clustering. The association of VAPA with Kv2.1 relies on a “two phenylalanines in an acidic tract” (FFAT) binding domain on VAPA and a noncanonical phosphorylation-dependent FFAT motif comprising the Kv2-specific clustering or PRC motif. These results suggest that Kv2.1 localizes to and organizes neuronal ER-PM junctions through an interaction with VAPs.

Original languageEnglish (US)
Pages (from-to)7562-7584
Number of pages23
JournalJournal of Neuroscience
Volume38
Issue number35
DOIs
StatePublished - Aug 29 2018

Fingerprint

Endoplasmic Reticulum
Cell Membrane
Neurons
Membranes
Brain
Proteins
Shab Potassium Channels
Cluster Analysis
R-SNARE Proteins
Voltage-Gated Potassium Channels
Calcium Signaling
Eukaryotic Cells
Phenylalanine
Proteomics
Blood Proteins
Mass Spectrometry
Protein Isoforms
Membrane Proteins
Homeostasis
Phosphorylation

Keywords

  • Ion channel
  • Membrane contact sites
  • Neuron
  • Subcellular

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

Identification of VAPA and VAPB as Kv2 channel-interacting proteins defining endoplasmic reticulum–plasma membrane junctions in mammalian brain neurons. / Kirmiz, Michael; Vierra, Nicholas C.; Palacio, Stephanie; Trimmer, James.

In: Journal of Neuroscience, Vol. 38, No. 35, 29.08.2018, p. 7562-7584.

Research output: Contribution to journalArticle

Kirmiz, Michael ; Vierra, Nicholas C. ; Palacio, Stephanie ; Trimmer, James. / Identification of VAPA and VAPB as Kv2 channel-interacting proteins defining endoplasmic reticulum–plasma membrane junctions in mammalian brain neurons. In: Journal of Neuroscience. 2018 ; Vol. 38, No. 35. pp. 7562-7584.
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