Kv2.1 mediates spatial and functional coupling of L-type calcium channels and ryanodine receptors in mammalian neurons
- School of Medicine, University of California, Davis, United States
- University of California, Davis, United States
Figures
Figure 1
Kv2.1 reversibly associates with Cav1.2 and RyRs in cultured hippocampal neurons.
(A) Single optical section image of a rat CHN immunolabeled for PSD-95, Cav1.2, and MAP2 (scale bar: 20 μm). Note large population of somatic Cav1.2 channels distinct from excitatory synapses …
Figure 2 with 1 supplement
Kv2.1 spatially associates with Cav1.2 and RyRs in brain neurons.
(A) Panels show exemplar images of the hippocampus acquired from a brain section from an adult rat immunolabeled for Kv2.1 (red), Cav1.2, (green) and RyRs (blue), and the merged image (scale bar: …
Figure 2—figure supplement 1
Cav1.2 spatially associates with Kv2.2 in brain neurons.
(A) Confocal optical section obtained from the dentate gyrus of a rat brain section immunolabeled for Kv2.2 (red) and Cav1.2 (green). Nuclei are shown in blue (scale bar: 10 μm). (B) Confocal …
Figure 3 with 2 supplements
LTCCs are recruited to Kv2-induced EPJs.
(A) TIRF images of a HEK293T cell cotransfected with DsRed (red), GFP-Cav1.2 (green), BFP-SEC61β (blue) and LTCC auxiliary subunits Cavβ3 and Cavα2δ1 (not shown) and labeled with GxTX-633 (scale …
Figure 3—figure supplement 1
Kv2.1 increases clustering of surface Cav1.2 channels.
(A) Optical sections of HEK293T cells transfected with and immunolabeled for cell surface Cav1.2-HA and Kv1.5 (upper panels) or Kv2.1 (lower panels) (scale bar: 10 μm and holds for all panels). (B) …
Figure 3—figure supplement 2
Cav1.3s is recruited to Kv2-induced EPJs.
(A) TIRF images of a HEK293T cell cotransfected with the short isoform of Cav1.3 (GFP-Cav1.3 (green), BFP-SEC61β (blue) and auxiliary subunits Cavβ3 and Cavα2δ (not shown). Scalebar is 10 μm and …
Figure 4 with 2 supplements
Spontaneous Ca2+ signals are generated at Kv2.1-associated EPJs.
(A) Widefield image of a rat CHN transfected with GCaMP3-Kv2.1 (also see Video 1). Arrows indicate selected Kv2.1 clusters whose fluorescent intensity profiles are plotted in panel B (scale bar: 10 …
Figure 4—figure supplement 1
GCaMP3-Kv2.1WT and GCaMP3-Kv2.1P404W show comparable PM expression in HEK293T cells.
(A) TIRF images of a HEK293T cell transfected with GCaMP3-Kv2.1WT. GxTX-633 labeling is shown in red and GCaMP3 fluorescence is shown in green in the merge panel (scale bar: 10 µm). (B) As in A but …
Figure 4—figure supplement 2
Relationship of Ca2+ sparks to global Ca2+ spikes.
(A) Neuron expressing GCaMP3-Kv2.1 and exhibiting Ca2+ sparks at the cluster indicated by ROI 1 (note that Ca2+ sparks were undetectable at the adjacent cluster indicated by ROI 2) (scale bar: 10 …
Figure 5
Spontaneous Ca2+ signals at Kv2.1-associated EPJs are produced by RyR- and LTCC-mediated CICR.
(A) Representative GCaMP3-Kv2.1 fluorescence traces from CHNs treated with pharmacological probes of CICR. Different colors indicate spatially distinct ROIs within the same neuron. Dashed lines …
Figure 6 with 1 supplement
Kv2.1 expression increases the frequency of LTCC- and RyR-mediated sparks reconstituted in HEK293T cells.
(A) TIRF images of a HEK293T cell cotransfected with DsRed-Kv2.1 (red), Cav1.2 (green), YFP-RyR2 (blue), and auxiliary subunits Cavβ3, Cavα2δ1, and STAC1 (not shown) (scale bar: 10 μm). (B) TIRF …
Figure 6—figure supplement 1
Kv2.1 increases the frequency of Cav1.3s and RyR-mediated sparks reconstituted in HEK293T cells.
(A) TIRF image of HEK293T cell expressing the short isoform of Cav1.3 (Cav1.3s), RyR2, STAC1, and the LTCC auxiliary subunits β3 and α2δ1, and loaded with Cal-590 AM (scale bar: 10 µm and holds for …
Figure 7 with 1 supplement
Cav1.2 channel activity is increased by coexpression with Kv2.1P404W.
(A) Representative Ca2+ current trace families recorded from HEK293T cells transfected with Cav1.2-GFP and auxiliary subunits Cavβ3 and Cavα2δ1, without (+ pcDNA3 empty vector) with cotransfection …
Figure 7—figure supplement 1
Cav1.2 channel activity is increased in cells coexpressing STAC1 upon coexpression with Kv2.1P404W.
(A-D) Data recorded from HEK293T cells transfected with Cav1.2-GFP and auxiliary subunits Cavβ3, Cavα2δ1, and STAC1, without (+pCDNA3, in black) or with Kv2.1P404W (in red). (A) Representative Ca2+ …
Figure 8
Kv2.1P404W increases Cav1.2 single channel activity.
(A, B) Maximum z-projections of TIRF images of Cav1.2-mediated Ca2+ sparklets in a representative HEK293T cell transfected with Cav1.2 and auxiliary subunits and loaded with Fluo-5F via the patch …
Figure 9
LTCC activity is reduced in Kv2.1 KO hippocampal neurons.
(A) Representative Ba2+ current traces recorded from WT (left) and Kv2.1 KO CHNs (right) recorded at +10 mV in vehicle or in the presence of the LTCC inhibitor nimodipine (10 µM). (B) Representative …
Figure 10
Increased immunolabeling for Cav1.2 in Kv2.1 KO brain sections.
(A) Column shows exemplar images of the hippocampus acquired from brain sections of adult WT mice immunolabeled for Kv2.2 (red), Cav1.2 (green) and Kv4.2 (blue) (scale bar: 200 μm). (B) As in A but …
Figure 11
Reduced association of Cav1.2 and RyRs and decreased spark frequency in Kv2.1 KO CHNs.
(A) A single optical section image of a WT mouse CHN immunolabeled for Kv2.1, Cav1.2, and RyRs (scale bar: 10 μm). (B) As in D but acquired from a Kv2.1 KO mouse CHN. (C–F) Morphology and spatial …
Videos
Video 1
Spontaneous somatic Ca2+ signals detected at GCaMP3-Kv2.1 clusters in rat CHNs.
Stack of widefield images of a rat CHN transfected with GCaMP3-Kv2.1 and imaged at 10 Hz.
Video 2
Spontaneous somatic Ca2+ signals detected by TIRF microscopy in rat CHNs loaded with Cal-590 AM.
Stack of TIRF images of rat CHNs loaded with Cal-590 AM and imaged at 30 Hz. Regular wave-like signals are a TIRF imaging artifact. Images have been normalized to the first image without detectable …
Video 3
Caffeine increases the frequency of somatic Ca2+ sparks in CHNs.
Images of a rat CHN transfected with GCaMP3-Kv2.1 acquired at 5 Hz. Neuron is treated with 5 mM caffeine at t = 84 s; the increased Ca2+ spark frequency is apparent from t = 87 s-101s. Images have …
Video 4
Bay K8644 increases the frequency of somatic Ca2+ sparks in CHNs.
Rat CHN transfected with GCaMP3-Kv2.1 and imaged in the presence of 500 nM Bay K8644. Images were acquired at 11.3 Hz and have been normalized to the first image without detectable Ca2+ signals (i.e.…
Video 5
Video depicting increase in Ca2+ spark frequency upon addition of Bay K8644.
Rat CHN transfected with GCaMP3-Kv2.1 and imaged at 20 Hz. 500 nM Bay K 8644 is added starting at approximately t = 25 s. Non-normalized GCaMP3-Kv2.1 images are shown on the left, images normalized …
Video 6
Tetracaine blocks Ca2+ sparks reconstituted in HEK293T cells.
Stack of TIRF images acquired at 20 Hz of a single HEK293T cell transfected with RyR2, Cav1.2, and auxiliary subunits and loaded with Cal-590 AM. 100 µM tetracaine was added at t = 7000 ms. Regular …
Video 7
Ca2+ sparklets in a control HEK293T cell expressing Cav1.2.
Stack of TIRF images acquired at approximately 100 Hz of a single HEK293T cell transfected with Cav1.2, PKCα, and auxiliary subunits and loaded with Fluo-5F via the patch pipette, before (left) and …
Video 8
Ca2+ sparklets in a HEK293T cell coexpressing Cav1.2 and Kv2.1.
Stack of TIRF images acquired at approximately 33 Hz of a single HEK293T cell transfected with Cav1.2, DsRed-Kv2.1P404W, PKCα, and auxiliary subunits and loaded with Fluo-5F via the patch pipette, …
Tables
Table 1
LTCC subunits and other Ca2+ signaling proteins specifically copurifying with Kv2.1
https://doi.org/10.7554/eLife.49953.005| Protein | Rank | Mean | SEM (n = 3) |
|---|---|---|---|
| Kv2.1 | 1 | 100.000 | NA |
| Kv2.2 | 3 | 31.638 | 0.518 |
| VAPA | 5 | 25.344 | 1.733 |
| RyR3 | 10 | 12.477 | 0.881 |
| Cavβ4 | 12 | 11.133 | 1.411 |
| VAPB | 15 | 7.600 | 1.393 |
| Cavβ2 | 18 | 5.623 | 0.79 |
| Cav1.3 | 19 | 5.730 | 1.652 |
| Cavβ3 | 23 | 5.070 | 1.033 |
| Hippocalcin | 24 | 4.583 | 0.831 |
| Neurocalcin-delta | 25 | 4.590 | 0.856 |
| SR/ER calcium ATPase 2 | 28 | 4.226 | 2.4 |
| Hippocalcin-like protein 1 | 29 | 4.360 | 0.288 |
| Cavβ1 | 33 | 3.800 | 0.697 |
| Calcineurin catalytic subunit γ | 35 | 3.583 | 0.718 |
| RyR2 | 36 | 3.140 | 0.903 |
| Calcineurin subunit B | 37 | 3.197 | 0.469 |
| Calcium-transporting ATPase | 39 | 2.873 | 0.447 |
| SR/ER calcium ATPase 1 | 40 | 2.530 | 1.21 |
| Cav1.2 | 43 | 2.427 | 0.766 |
-
Values in table are spectral counts normalized to Kv2.1 over three independent experiments.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Human) | HEK293T | ATCC Cat # CRL-3216 | RRID:CVCL_0063 | |
| Strain (R. norvegicus) | Sprague Dawley | Charles River | ||
| Strain (M. musculus) | C57/BL6J mice | The Jackson Laboratory | RRID:IMSR_JAX:000664 | |
| Strain (M. musculus) | Kcnb1-/- mice | PMID: 17767909; PMID: 24494598 | RRID:MGI:3806050 | maintained on the C57BL/6J background |
| Antibody | numerous | See Table 2 | ||
| Recombinant DNA reagent | GCaMP3-Kv2.1 | This paper | ||
| Recombinant DNA reagent | GCaMP3-Kv2.1P404W | This paper | ||
| Recombinant DNA reagent | DsRed-Kv2.1 | PMID: 30012696 | ||
| Recombinant DNA reagent | DsRed-Kv2.1P404W | PMID: 30012696 | ||
| Recombinant DNA reagent | DsRed-Kv2.2 | PMID: 30012696 | ||
| Recombinant DNA reagent | Kv2.1S586A | PMID: 10719893 | ||
| Recombinant DNA reagent | Kv1.5 | PMID: 8636142 | ||
| Recombinant DNA reagent | BFP-Sec61β | Addgene | Plasmid #49154 | |
| Recombinant DNA reagent | Cav1.2-eGFP | PMID: 25714924 | ||
| Recombinant DNA reagent | Cav1.2-tagRFP | PMID: 25714924 | ||
| Recombinant DNA reagent | Cav1.3S-GFP | PMID: 27187148 | ||
| Recombinant DNA reagent | Cav1.2 | Addgene | Plasmid #26572 | |
| Recombinant DNA reagent | Cav1.2-HA | PMID: 15090038 | Gift from Dr. Valentina Di Biase | |
| Recombinant DNA reagent | Cavα2δ1 | Addgene | Plasmid #26575 | |
| Recombinant DNA reagent | Cavβ3 | Addgene | Plasmid #26574 | |
| Recombinant DNA reagent | YFP-RyR2 | PMID: 17452324 PMID: 20427316 | Gift from Dr. S.R. Wayne Chen | |
| Recombinant DNA reagent | STAC1 | DNASU | Plasmid # HsCD00445396 | |
| Chemical compound | Cal-590 AM | AAT Bioquest | Cat# 20510 | |
| Chemical compound | Rhod-2 | AAT Bioquest | Cat# 21068 | |
| Chemical compound | Fluo-4 AM | Invitrogen | Cat# F14201 | |
| Chemical compound | Fluo-5F | Invitrogen | Cat# F14221 | |
| Chemical compound | Caffeine | Sigma | Cat# C0750 | |
| Chemical compound | Thapsigargin | Millipore | Cat# 586005 | |
| Chemical compound | Nimodipine | Alomone | Cat# N-150 | |
| Chemical compound | Nitrendipine | Alomone | Cat# N-155 | |
| Chemical compound | Bay K8644 | Alomone | Cat# B-350 | |
| Chemical compound | Tetracaine | Sigma | Cat# T7508 | |
| Chemical compound | Tetrodotoxin | Alomone | Cat #T-550 | |
| Chemical compound | Amphotericin B | Millipore | Cat# 171375 | |
| Software | Photoshop | Adobe Systems | RRID:SCR_014199 | |
| Software | Axiovision | Carl Zeiss MicroImaging | RRID:SCR_002677 | |
| Software | pClamp | Molecular Devices | RRID:SCR_011323 | |
| Software | TILLvisION | TILL Photonics | ||
| Software | Fiji | PMID: 22743772 | RRID:SCR_002285 | |
| Software | Prism | GraphPad Software | RRID:SCR_002798 |
Table 2
Antibody information.
https://doi.org/10.7554/eLife.49953.029| Antigen and antibody name | Immunogen | Manufacturer information | Concentration used | Figures |
|---|---|---|---|---|
| PSD-95 (K28/43) | Fusion protein aa77–299 of human PSD-95 | Mouse IgG2a mAb, NeuroMab, RRID:AB_10807979 | Tissue culture supernatant (1:5) | Figure 1 |
| Cav1.2 (N263/31) | Fusion protein aa 808–874 of rat Cav1.2 | Mouse IgG2b mAb, NeuroMab, RRID:AB_11001554 | Tissue culture supernatant (1:5) | Figure 1, Figure 2, Figure 2—figure supplement 1, Figure 6, Figure 10, Figure 11 |
| Cav1.2 (L57/23) | Fusion protein aa1507–1733 of rabbit Cav1.2 | Mouse IgG2a mAb, In-house (Trimmer Laboratory) RRID:AB_2802123 | Tissue culture supernatant, neat | Figure 1, Figure 3 |
| Cav1.3 (ACC-005) | Synthetic peptide aa 859–875 of rat Cav1.3 | Rabbit pAb, Alomone catalog # ACC-005, RRID:AB_2039775 | Affinity purified, 10 μg/mL | Figure 1 |
| Kv2.1 (KC) | Synthetic peptide aa 837–853 of rat Kv2.1 | Rabbit pAb, In-house (Trimmer Laboratory), RRID:AB_2315767 | Affinity purified, 1:100 | Table 1 (immunopurifications) |
| Kv2.1 (K89/34R) | Synthetic peptide aa 837–853 of rat Kv2.1 | Recombinant mouse IgG2a mAb, In-house (Trimmer Laboratory), RRID:AB_2750677 | Tissue culture supernatant (1:5) | Figure 1, Figure 2, Figure 3, Figure 3—figure supplement 2, Figure 4, Figure 11 |
| Kv2.1 (K39/25R) | Synthetic peptide aa 211–229 of human Kv2.1 | Recombinant mouse IgG2a mAb, In-house (Trimmer Laboratory), RRID:AB_2750663 | Tissue culture supernatant (1:5) | Figure 3—figure supplement 1 |
| MAP2 (AB5622-I) | KLH-conjugated three peptides from N-and C-terminal regions of rat MAP2 | Rabbit pAb, Millipore catalog # AB5622-I, RRID: AB_2800501 | Affinity purified, 1:1000 | Figure 1, Figure 4, Figure 5 |
| RyRs (34C) | Partially purified chicken pectoral muscle ryanodine receptor | Mouse IgG1 mAb, Developmental Studies Hybridoma RRID:AB_528457 | Concentrated tissue culture supernatant, 3 μg/ml | Figure 1, Figure 2, Figure 4, Figure 5, Figure 6, Figure 11 |
| Cav3.1 (N178A/9) | Fusion protein aa 2052–2172 of mouse Cav3.1 | Mouse IgG1 mAb, NeuroMab, RRID:AB_10673097 | Tissue culture supernatant (1:5) | Figure 3—figure supplement 2 |
| Kv1.5 (Kv1.5e) | Synthetic peptide aa 271–284 of rat Kv1.5 | Rabbit pAb, in-house (Trimmer Laboratory), RRID:AB_2722698 | Affinity purified, 5 μg /ml | Figure 3—figure supplement 1 |
| Kv2.2 (Kv2.2C) | Fusion protein aa 717–907 of rat Kv2.2 | Rabbit pAb, in-house (Trimmer Laboratory), RRID:AB_2801484 | Affinity purified, 1:100 | Figure 2—figure supplement 1, Figure 10 |
| Kv4.2 (K57/41) | Synthetic peptide aa 209–225 of human Kv4.2 | Mouse IgG1 mAb, In-house (Trimmer Laboratory), RRID:AB_2802124 | Affinity purified, 10 μg /ml | Figure 1, Figure 10 |
| Anti-HA (12CA5) | Amino acids 98–106 of the human influenza virus hemagglutinin protein | Mouse IgG2b mAb, In-house (Trimmer Laboratory) RRID: AB_2532070 | Pure, 5 μg/mL | Figure 3 |
| Anti-HA (2–2.2.14-647) | HA peptide YPYDVPDYA | Mouse IgG1 mAb, Thermo Fisher Scientific catalog # 26183-A647, RRID: AB_2610626 | Affinity purified, 1 μg /ml | Figure 3—figure supplement 1 |
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.49953.030
