Remodeling neuronal ER-PM junctions is a conserved nonconducting function of Kv2 plasma membrane ion channels - PubMed (original) (raw)

Remodeling neuronal ER-PM junctions is a conserved nonconducting function of Kv2 plasma membrane ion channels

Michael Kirmiz et al. Mol Biol Cell. 2018.

Abstract

The endoplasmic reticulum (ER) and plasma membrane (PM) form junctions crucial to ion and lipid signaling and homeostasis. The Kv2.1 ion channel is localized at ER-PM junctions in brain neurons and is unique among PM proteins in its ability to remodel these specialized membrane contact sites. Here, we show that this function is conserved between Kv2.1 and Kv2.2, which differ in their biophysical properties, modulation, and cellular expression. Kv2.2 ER-PM junctions are present at sites deficient in the actin cytoskeleton, and disruption of the actin cytoskeleton affects their spatial organization. Kv2.2-containing ER-PM junctions overlap with those formed by canonical ER-PM tethers. The ability of Kv2 channels to remodel ER-PM junctions is unchanged by point mutations that eliminate their ion conduction but eliminated by point mutations within the Kv2-specific proximal restriction and clustering (PRC) domain that do not impact their ion channel function. The highly conserved PRC domain is sufficient to transfer the ER-PM junction-remodeling function to another PM protein. Last, brain neurons in Kv2 double-knockout mice have altered ER-PM junctions. Together, these findings demonstrate a conserved in vivo function for Kv2 family members in remodeling neuronal ER-PM junctions that is distinct from their canonical role as ion-conducting channels shaping neuronal excitability.

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Figures

FIGURE 1:

FIGURE 1:

Kv2.2 associates with ER–PM junctions in mammalian brain neurons in situ and in culture and in heterologously expressing HEK293T cells. (A–C) Single z-stack images of multiplex immunofluorescence labeling of adult mouse brain sections showing neocortex (A) and hippocampal CA1 region (B) immunolabeled for Kv2.2 (green) and RyR (magenta), or cultured hippocampal neurons (C) immunolabeled for Kv2.2 (green), Kv2.1 (blue), and RyR (red), as indicated. Scale bar in Kv2.2 neocortex panel is 10 µm and holds for all brain panels. Scale bar in MAP2 CHN panel is 10 µm and holds for all CHN panels in that row. Scale bar in Kv2.2 magnified inset is 2.5 µm and holds for all panels in that row. Panels to the right of each set of images are the corresponding normalized fluorescence intensity values across the individual line scans depicted by the white line in the merged images. Image exposure time for brain sections was optimized for the labeling of each brain region independently. (D) Images of fixed HEK293T cells coexpressing GFP-Kv2.2 (green) and BFP-SEC61β (magenta). The top row shows a single N-SIM optical section taken through the center of a cell. The scale bar is 1.25 µm. The bottom rows show a 2D projection of a 3D reconstruction (middle row), and a single orthogonal slice through the 3D reconstruction (bottom row) of a cell imaged with a Zeiss Airyscan microscope. Scale bar in the GFP-Kv2.2 panel of the 3D reconstruction is 2.5 µm and holds for all panels in the bottom two rows. Panels to the right of each set of rows are the corresponding normalized fluorescence intensity values across the individual line scans depicted by the arrows (top) or white line (bottom) in the merged images.

FIGURE 2:

FIGURE 2:

Exogenous Kv2 expression remodels ER–PM junctions in HEK293T cells and cultured rat hippocampal neurons. (A, B) TIRF images of live HEK293T cells expressing DsRed2-ER5 (magenta) either alone (A) or coexpressed with (in green) GFP-Kv2.2, GFP-Kv2.1, or GFP-Kv1.4, as indicated (B). Scale bar in A is 5 µm and holds for all panels. (C) Graph of mean ER–PM junction (EPJ) size per cell measured from HEK293T cells coexpressing DsRed2-ER5 and GFP-Kv2.2, GFP-Kv2.1, or GFP-Kv1.4 or expressing DsRed2-ER5 alone (control). (D) Graph of percentage of the PM area per cell occupied by cortical ER measured from HEK293T cells coexpressing DsRed2-ER5 and GFP-Kv2.2, GFP-Kv2.1, or GFP-Kv1.4 or expressing DsRed2-ER5 alone (control). (E) Graph of PCC between DsRed2-ER5 and GFP-Kv2.2, GFP-Kv2.1, or GFP-Kv1.4 measured from HEK293T cells coexpressing DsRed2-ER5 and GFP-Kv constructs. The dashed line denotes a PCC of 0.5. Bars on all graphs are mean ± SD. See Supplemental Tables 1–3 for values and statistical analyses for C–E. (F) TIRF image of a live CHN expressing DsRed2-ER5 (magenta) alone. (G) TIRF image of a live CHN coexpressing DsRed2-ER5 (magenta) and GFP-Kv2.2 (green). Scale bar in DsRed2-ER5 panel is 10 µm and holds for all panels in that row. Magnified images are shown in bottom row. Scale bar in DsRed2-ER5 magnified inset panel is 2.5 µm and holds for all panels in that row. (H) Scatterplot shows sizes of Kv2.2 clusters and associated ER–PM junctions (EPJs, as indicated by DsRed2-ER5 in TIRF) in HEK293T cells (red points) and CHNs (black points). n = 3 cells each.

FIGURE 3:

FIGURE 3:

ER–PM junction-localized Kv2.2 channels are expressed on the cell surface. (A) TIRF images of a live HEK293T cell expressing GFP-Kv2.2 (green) and DsRed2-ER5 (red) and surface-labeled for Kv2 channels with GxTX-633 (blue). Heat map shows overlap of GFP-Kv2.2 and GxTX-633 pixels. Scale bar is 5 µm. (B) Fluorescence intensity values across the individual line scan depicted by the white line in the merged image. (C) Graph of PCC between GxTX and Kv2.2 or DsRed2-ER5 measured from live HEK293T cells surface labeled with GxTX-633 and coexpressing GFP-tagged Kv2.2 and DsRed2-ER5. Bars are mean ± SD. See Supplemental Table 5 for values and statistical analyses for C.

FIGURE 4:

FIGURE 4:

Kv2-mediated ER–PM junctions are located at sites depleted in components of the cortical actin cytoskeleton. (A) Mouse brain section immunolabeled for Kv2.2 (green), Kv2.1 (red), and ankG (blue). Scale bar for large image is 20 µm and for Kv2.2 inset is 3 µm and holds for all inset panels. (B) Projected z-stack of optical sections taken from a CHN immunolabeled for Kv2.2 (green), Kv2.1 (red), and ankG (blue). Scale bar for large image is 20 µm and for Kv2.2 inset is 3 µm and holds for all inset panels. (C) Single optical section taken from the cell body of a CHN immunolabeled for Kv2.2 (green) and Kv2.1 (red) and labeled for F-actin with phalloidin (blue). Scale bar for merged panel is 10 µm and holds for all panels in set. Panels below each set of images show the corresponding normalized fluorescence intensity values across the line scans indicated in the merged images in that column. (D) TIRF images of live HEK293T cells coexpressing GFP-Kv2.2 (green) and BFP-SEC61β (blue) in conjunction with (in red) mCherry-actin (top row) or ankG-mCherry (bottom row). Scale bar for GFP-Kv2.2 panel in top row is 5 µm and holds for all panels in set. Panels to the right of each row show the corresponding normalized fluorescence intensity values across the line scan depicted by the white line in the merged images.

FIGURE 5:

FIGURE 5:

Disrupting the actin cytoskeleton impacts spatial organization of Kv2.2-mediated ER–PM junctions. (A, B) TIRF images of a live HEK293T cell coexpressing GFP-Kv2.2 (green) and DsRed2-ER5 (magenta), prior to (Rest) and 15 min after latrunculin A (After LatA) treatment. Scale bar in GFP-Kv2.2 Rest panel is 5 µm and holds for all panels. Graphs show values measured from cells before (Rest) and after a 15-min treatment (After LatA) with 10 µM LatA. (C) Mean Kv2.2 cluster size per cell. (D) Mean ER–PM junction (EPJ) size per cell. (E) Number of ER–PM junctions per cell. (F) PCCs between Kv2.2 and DsRed2-ER5. See Supplemental Tables 6–9 for values and statistical analyses for C–F.

FIGURE 6:

FIGURE 6:

Kv2.2-containing ER–PM junctions colocalize with multiple components of mammalian ER–PM junctions. (A) TIRF images of live HEK293T cells coexpressing DsRed- or GFP-tagged Kv2.2 and various members of the E-Syt, JP, and STIM families of ER-localized PM tethers. Scale bar in top left GFP-Kv2.2 panel is 10 µm and holds for all panels. Heat maps show pixel overlap of Kv2.2 and ER–PM tether signals. STIM-containing cells were treated with 2 µM thapsigargin for 5 min prior to imaging. (B) Graph of PCC between Kv2.2 and ER–PM tethers. Bars are mean ± SD. (C) Graph of MOC values between Kv2.2 and ER–PM tether signals. Bars are mean ± SD. See Supplemental Table 10 for values and statistical analyses for B and C. (D) TIRF images of a live HEK293T cell coexpressing BFP-STIM1 (blue), DsRed-Kv2.2 (green), and GFP-Orai1 (red) prior to (Rest) and immediately after 5 min of treatment with 2 µM thapsigargin (+Thap). (E) TIRF images of a live HEK293T cell coexpressing DsRed-Kv2.2 (green) and GFP-Orai1 (magenta) prior to (Rest) and immediately after 5 min of treatment with 2 µM thapsigargin (+Thap). Scale bar in top left DsRed-Kv2.2 panel is 5 µm and holds for panels in D, E. (F) Graph of PCC between Orai1 and Kv2.2 (black) or STIM1 (red) measured from cells with BFP-STIM1 coexpression before (Rest) and after (+Thap) thapsigargin treatment. (G) Graph of PCC between Orai1 and Kv2.2 measured from cells without BFP-STIM1 coexpression before (Rest) and after (+Thap) thapsigargin treatment. Bars on all graphs are mean ± SD. See Supplemental Table 11 for values and statistical analyses for F and G.

FIGURE 7:

FIGURE 7:

Acutely formed ER–PM junctions are distinct from Kv2.2-containing ER–PM junctions. (A) TIRF images of CFP fluorescence in a live HEK293T cell coexpressing CFP-CB5-FKBP and Lyn11-FRB before (Rest) and immediately after treatment with 5 µM rapamycin (+Rap). Scale bar is 5 µm and holds for both panels. Graph to right of panels shows fluorescence intensity of CFP-CB5-FKBP across the individual line scan depicted by the white lines before (Rest) and immediately following treatment with 5 µM rapamycin (+Rap). (B) TIRF images of a live HEK293T cell coexpressing DsRed-Kv2.2 (green), CFP-CB5-FKBP (magenta), and Lyn11-FRB. Top row: Prior to rapamycin treatment (Rest). Middle row: Same cell immediately following 5 µM rapamycin treatment (+Rap). Bottom row: Same cell after subsequent 15-min treatment with 10 µM LatA (+LatA). Panels to the right of each row are the corresponding normalized fluorescence intensity values across the individual line scans depicted by the white line in the merged images. Scale bar is 10 µm and holds for all panels. (C) Graph of PCC between DsRed-Kv2.2 and CFP-CB5-FKBP. Bars are mean ± SD. See Supplemental Table 12 for values and statistical analysis.

FIGURE 8:

FIGURE 8:

Mutations that eliminate K+ conductance do not impact Kv2.2 channel clustering. (A) Exemplar whole-cell voltage clamp recordings (left) and corresponding graphs of current levels vs. command voltage (right) of HEK293T cells expressing GFP (control), GFP-Kv2.2, GFP-Kv2.2 P412W, GFP-Kv2.1, or GFP-Kv2.1 P404W. Recordings shown are representative responses to 100-ms steps from –100 mV to –40, 0, and +40 mV. Note the lack of outward currents in control, GFP-Kv2.2 P412W, and GFP-Kv2.1 P404W recordings. (B, C) Summary graphs showing whole cell current at +40 mV for cells expressing Kv2.2 (B) or Kv2.1 (C) isoforms. See Supplemental Table 13 for values and statistical analyses for B and C. (D) Deconvolved widefield image of a live CHN expressing GFP-Kv2.2 P412W or GFP-Kv2.1 P404W. Scale bar is 5 µm and holds for both panels. (E) TIRF images of live HEK293T cells expressing GFP-Kv2.2 P412W or GFP-Kv2.1 P404W and surface labeled with GxTX-633. Scale bar in the Kv2.2 P412W panel is 5 µm and hold for all panels. (F, G) Graphs of mean cluster size per cell measured from CHNs expressing GFP-Kv2.2 or GFP-Kv2.2 P412W (F) or GFP-Kv2.1 or GFP-Kv2.1 P404W (G). Bars are mean ± SD. See Supplemental Table 14 for values and statistical analyses for F and G. (H, I) Graph of PCC between Kv2 and GxTX from HEK293T cells expressing GFP-Kv2.2 or GFP-Kv2.2 P412W (H) or GFP-Kv2.1 or GFP-Kv2.1 P404W (I). Bars are mean ± SD. See Supplemental Table 15 for values and statistical analyses for H and I.

FIGURE 9:

FIGURE 9:

Separation of function point mutations shows that clustering, but not conduction, is necessary for Kv2-mediated remodeling of ER–PM junctions. (A) TIRF images of live HEK293T cells expressing GFP-tagged Kv2.2 mutants (nonconducting P412W and nonclustering S605A in green) and DsRed2-ER5 (magenta). (B) TIRF images of live HEK293T cells expressing GFP-tagged Kv2.1 mutants (nonconducting P404W and nonclustering S586A in green) and DsRed2-ER5 (magenta). Scale bar in top left panels in A and B is 5 μm and holds for all panels in set. (C–E) Comparisons of cells expressing wild-type and mutant Kv2 isoforms (Kv2.2 P412W, Kv2.2 S605A, Kv2.1 P404W, or Kv2.1 S586A); control refers to cells expressing DsRed2-ER5 alone. (C) Mean ER–PM junction (EPJ) size per cell. (D) Percent PM per cell occupied by cortical ER. (E) PCC between DsRed2-ER5 and wild-type and mutant Kv2 isoforms. Bars on all graphs are mean ± SD. See Supplemental Tables 16–18 for values and statistical analyses. (F, G) Exemplar whole-cell voltage clamp recordings (left) and graphs of the corresponding normalized conductance–voltage relationship from HEK293T cells expressing GFP-Kv2.2 or GFP-Kv2.2 S605A (F) and GFP-Kv2.1 or GFP-Kv2.1 S586A (G). Different colors represent data from distinct cells. Recordings shown are representative responses to 200-ms steps from –100 mV to –40, 0, and +40 mV. Note the lack of a significant impact of the declustering point mutation on the properties of the whole cell currents. See Table 1 for values and statistical analyses for whole-cell current density and midpoint of voltage activation, respectively.

FIGURE 10:

FIGURE 10:

The PRC domain can act autonomously to transfer the ER–PM junction remodeling function of Kv2 channels to another PM protein. TIRF images of fixed and immunolabeled HEK293T cells coexpressing BFP-SEC61β (magenta) and in green either Kv2.2 (A), Kv2.1 (B), Kv1.5 (C), Kv1.5N-Kv2.1C (D), or Kv1.5N-Kv2.1PRC (E). Scale bar in A is 10 µm and holds for all panels.

FIGURE 11:

FIGURE 11:

Genetic ablation of Kv2.2 and Kv2.1 alters RyR localization in mouse brain neurons. (A–D) Projected z-stack images of CA1 hippocampus from mouse brain sections from wild-type (A, WT), Kv2.1 knockout (B, Kv2.1KO), Kv2.2 knockout (C, Kv2.2KO), or Kv2.1 and Kv2.2 double-knockout (D, Kv2 dKO) mice immunolabeled for RyR (red), Kv2.2 (green), and Kv2.1 (blue). (A) RyR, Kv2.2, and Kv2.1 immunolabeling from WT mouse. (B) RyR and Kv2.2 immunolabeling from Kv2.1 KO mouse. (C) RyR and Kv2.1 immunolabeling from Kv2.2 KO mouse. (D) RyR immunolabeling from Kv2.1/Kv2.2 dKO mouse. Scale bar in A is 10 µm and holds for all panels in set. Panels to the right of each row are the corresponding normalized fluorescence intensity values across the individual line scans depicted by the white line in the merged images. (E) Enlarged selections of RyR-labeling of WT and Kv2.1/Kv2.2 dKO images as indicated by boxes in panels A and D, respectively. Scale bar in WT RyR inset panel is 1.25 µm and holds for both panels. (F) Graph of mean RyR cluster size measured from each genotype. Bars are mean ± SEM. See Supplemental Table 20 for values and statistical analyses.

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