Neuronal ER–plasma membrane junctions organized by Kv2–VAP pairing recruit Nir proteins and affect phosphoinositide homeostasis

Michael Kirmiz,Taryn E Gillies,Eamonn J Dickson,James S Trimmer

doi:10.1074/jbc.ra119.007635

Michael Kirmiz, Taryn E Gillies + Show 2 more

Open Access

https://doi.org/10.1074/jbc.ra119.007635

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Abstract

The association of plasma membrane (PM)-localized voltage-gated potassium (Kv2) channels with endoplasmic reticulum (ER)-localized vesicle-associated membrane protein-associated proteins VAPA and VAPB defines ER-PM junctions in mammalian brain neurons. Here, we used proteomics to identify proteins associated with Kv2/VAP-containing ER-PM junctions. We found that the VAP-interacting membrane-associated phosphatidylinositol (PtdIns) transfer proteins PYK2 N-terminal domain-interacting receptor 2 (Nir2) and Nir3 specifically associate with Kv2.1 complexes. When coexpressed with Kv2.1 and VAPA in HEK293T cells, Nir2 colocalized with cell-surface-conducting and -nonconducting Kv2.1 isoforms. This was enhanced by muscarinic-mediated PtdIns(4,5)P2 hydrolysis, leading to dynamic recruitment of Nir2 to Kv2.1 clusters. In cultured rat hippocampal neurons, exogenously expressed Nir2 did not strongly colocalize with Kv2.1, unless exogenous VAPA was also expressed, supporting the notion that VAPA mediates the spatial association of Kv2.1 and Nir2. Immunolabeling signals of endogenous Kv2.1, Nir2, and VAP puncta were spatially correlated in cultured neurons. Fluorescence-recovery-after-photobleaching experiments revealed that Kv2.1, VAPA, and Nir2 have comparable turnover rates at ER-PM junctions, suggesting that they form complexes at these sites. Exogenous Kv2.1 expression in HEK293T cells resulted in significant differences in the kinetics of PtdIns(4,5)P2 recovery following repetitive muscarinic stimulation, with no apparent impact on resting PtdIns(4,5)P2 or PtdIns(4)P levels. Finally, the brains of Kv2.1-knockout mice had altered composition of PtdIns lipids, suggesting a crucial role for native Kv2.1-containing ER-PM junctions in regulating PtdIns lipid metabolism in brain neurons. These results suggest that ER-PM junctions formed by Kv2 channel-VAP pairing regulate PtdIns lipid homeostasis via VAP-associated PtdIns transfer proteins.

Highlights

The association of plasma membrane (PM)-localized voltagegated potassium (Kv2) channels with endoplasmic reticulum (ER)-localized vesicle-associated membrane protein-associated proteins VAPA and VAPB defines ER–PM junctions in mammalian brain neurons
We include here data for the Kv2.1 auxiliary subunit AMIGO-1, which exhibits an extensive association and colocalization with Kv2.1 in brain [38, 51], yet for which peptide spectra are present at relatively low abundance in the Kv2.1 IP samples relative to Kv2.2, VAPA, VAPBB, and the Nir proteins, and for TMEM24/C2CD2L, recently reported to colocalize with Kv2.1 at neuronal ER–PM junctions [52], and which we recovered in Kv2.1 IPs from WT brain samples (Fig. 1A)
We found that mCherry–N-terminal domain-interacting receptor 2 (Nir2) was not recruited to ER–PM junctions mediated by CB5/lyn11 to the same extent as those mediated by Kv2.1, such that Pearson’s correlation coefficient (PCC) measurements of colocalization of mCherry–Nir2 and CB5 were significantly lower than those between mCherry–Nir2 and CFP–Kv2.1 in these experiments (Fig. 7J)

Summary

Introduction

The association of plasma membrane (PM)-localized voltagegated potassium (Kv2) channels with endoplasmic reticulum (ER)-localized vesicle-associated membrane protein-associated proteins VAPA and VAPB defines ER–PM junctions in mammalian brain neurons. The brains of Kv2.1-knockout mice had altered composition of PtdIns lipids, suggesting a crucial role for native Kv2.1-containing ER–PM junctions in regulating PtdIns lipid metabolism in brain neurons These results suggest that ER–PM junctions formed by Kv2 channel–VAP pairing regulate PtdIns lipid homeostasis via VAP-associated PtdIns transfer proteins. Junctions between the plasma membrane (PM) and endoplasmic reticulum (ER) or ER–PM junctions serve as a vital platform for information transduction between these two membranes [1,2,3,4] These MCS were initially discovered in EM studies in mammalian muscle cells [5] and soon after in mammalian brain neurons (6 –8), and they can engage Ͼ10% of the somatic neuronal PM [9]. Relatively little is known at the molecular level of the components underlying lipid trafficking and homeostasis at the ER–PM junctions that are prominent in mammalian brain neurons

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