Functionally coupled ion channels begin co-assembling at the start of their synthesis
- Department of Pharmacology, University of Washington, United States
- Department of Neurobiology and Biophysics, University of Washington, United States
- Howard Hughes Medical Institute, United States
Figures
Figure 1
Representation of molecular complex, homo-cluster, and hetero-cluster.
Figure 2 with 1 supplement
BK and CaV1.3 hetero-clusters are found inside the cell.
(A) Diagram of the hypothesis: hetero-clusters of BK (magenta) and CaV1.3 (cyan) are on intracellular membranes and on the plasma membrane. (B) Illustration of the technique to detect BK and CaV1.3 hetero-clusters. Proximity ligation assay is used to detect the hetero-clusters. (C) Confocal images of fluorescent puncta from proximity ligation assay (PLA) experiments in tsA-201 cells. Left: Cells were transfected and probed for BK and CaV1.3 channels. Right: negative control. Cells were transfected and probed only for BK channels. Enlargement of a selected region is shown in the inset. (D) Scatter dot plot comparing puncta density of BK and CaV1.3 hetero-clusters to the negative control. Data points are from n=12 cells for BK and CaV1.3 hetero-clusters and from n=14 cells for negative control. p-Values are shown at the top of the graphs. (E) Confocal images of fluorescent PLA puncta at different focal planes co-labeled against GFP at the plasma membrane. Cells were transfected with BK, CaV1.3, and PH-PLCδ-GFP and probed for BK channels, CaV1.3 channels, and GFP. PLA puncta are shown in magenta, and the plasma membrane is shown in green. Enlargements of the representative regions of PM and intercellular hetero-clusters are shown in the insets. (F) Scatter dot plot comparing BK and CaV1.3 hetero-cluster abundance at PM and inside the cell. Data points are from n=12 cells. Scale bars are 10 μm and 1 μm in the insets. For statistical analysis, unpaired t-tests were applied in panel D and paired t-tests in panel F to evaluate significance.
Figure 2—figure supplement 1
Validation of antibodies against BK, CaV1.3, and GFP.
Representative confocal images of tsA-201 cells immuno-tested for BK channels (left), CaV1.3 channels (middle), and GFP proteins (right). Cells were not transfected. Nuclei were stained with DAPI and pseudo-colored in gray. Scale bar is 10 μm for all images.
Figure 3
BK and CaV1.3 hetero-clusters localize at endoplasmic reticulum (ER) and ER exit sites (ERES).
(A) Diagram of the hypothesis: hetero-clusters of BK (magenta) and CaV1.3 (cyan) can be found at the ER membrane. (B) Representative image of the ER labeled with exogenous GFP in INS-1 cells. Cells were transfected with KDEL-moxGFP. Magnification is shown in the inset. (C) Comparison of the ER tubule distance in live and fixed tsA-201 and INS-1 cells. Data points are from n=23 tsA-201 cells, n=27 INS-1 cells. (D) Representative images of proximity ligation assay (PLA) puncta and ER. Left: tsA-201 cells were transfected with BK, CaV1.3, and KDEL-moxGFP. Right: INS-1 cells were transfected only with KDEL-moxGFP. Fixed cells were probed for BK-CaV1.3 hetero-clusters (PLA puncta) and GFP. PLA puncta are shown in magenta. ER is shown in green. (E) Comparison of BK-CaV1.3 hetero-clusters found at the ER and relative to all PLA puncta in the cell. Values are given in percentages. (F) Representative images of PLA puncta and ERES. Left and right are the same as in D, but cells were transfected with Sec16-GFP instead of KDEL. (G) Comparison of BK-CaV1.3 hetero-clusters found at ERES relative to all PLA puncta in the cell. Values are given in percentages. Data points are from n=45 tsA-201 cells for ER, n=21 tsA-201 cells for ERES, n=23 INS-1 cells for ER, and n=23 INS-1 cells for ERES. Scale bars are 10 μm and 2 μm in the magnifications.
Figure 4 with 1 supplement
BK and CaV1.3 hetero-clusters go through the Golgi.
(A) Diagram of the hypothesis: proximity ligation assay (PLA) puncta detecting hetero-clusters between BK (magenta) and CaV1.3 (cyan) channels can be found at the Golgi membrane. (B) Representative image of the Golgi structure with exogenous GFP in INS-1 cells. Cells were transfected with Gal-T-mEGFP. Enlargement is shown in the inset. (C) Representative images of fixed cells co-stained with antibodies against Gal-T-mEGFP in green and 58K-Golgi in red. (D) Representative images of PLA puncta and Golgi. tsA-201 cells were transfected with BK, CaV1.3, and Gal-T-mEGFP (left), and INS-1 cells were transfected only with Gal-T-mEGFP (right). PLA puncta are shown in magenta. Golgi is shown in green. (E) Scatter dot plot of percentages of BK-CaV1.3 hetero-clusters found at the Golgi relative to all PLA puncta in tsA-201 and INS1 cells. Data points are from n=22 tsA-201 cells and n=19 INS-1 cells. (F) Representative image of PLA puncta and Golgi. tsA-201 cells were transfected with BK, CaV1.3, and Gal-T-mEGFP. Left: PLA was done against BK and 58K Golgi (magenta), and Golgi is shown in green. Right: PLA was done against CaV1.3 and 58K Golgi (magenta), and Golgi is shown in green. (G) Diagram illustrating our interpretation of percentages of BK-CaV1.3 hetero-clusters found in the cell. This illustration is based on results shown in Figures 1—3 and Figure 4. Percentages were modified to represent overlap of fluorescent signals and limited resolution. We also show that hetero-clusters found in the ER exit sites (ERES) are also accounted in the ER. Scale bars: 10 μm and 2 μm in panels B and C; 10 μm and 1 μm in panel D; 2 μm in panel F.
Figure 4—figure supplement 1
CaV1.3 channels do not exhibit proximity interactions with ryanodine receptor type 2 (RyR2).
(A) Representative images of proximity ligation assay (PLA) experiments in INS-1 cells labeling against CaV1.3 and BK channels (left) or CaV1.3 and RyR2 (right). (B) Comparison of PLA puncta density for the two conditions. Data points are from n = 16 cells for the interaction between CaV1.3 and BK and n = 12 cells for the interaction between CaV1.3 and RyR2. Statistical significance was assessed using an unpaired t-test.
Figure 5 with 2 supplements
BK mRNA (KCNMA1) and CaV1.3 mRNA (CACNA1D) colocalize.
(A) Diagram of the hypothesis: KCNMA1 and CACNA1D mRNAs are found in close proximity to be translated in the same neighborhood. (B) Images of fluorescent puncta from RNAscope experiments showing KCNMA1 mRNA in magenta, CACNA1D mRNA in cyan, and GAPDH mRNA in green. Right, magnification of three ROIs. (C) Comparison of mRNA density of KCNMA1, CACNA1D, and GAPDH. (D) Correlation plot of mRNA abundance of KCNMA1 and CACNA1D per cell. (E) Correlation plot of mRNA abundance of KCNMA1 and GAPDH per cell. (F) Comparison of colocalization between KCNMA1 mRNA and mRNA from CACNA1D, GAPDH, and scrambled images of CACNA1D. Data points are from n=67 cells. Scale bars are 10 μm and 1 μm in the magnifications. Data were analyzed using ordinary one-way ANOVA with Dunnett’s multiple comparisons test.
Figure 5—figure supplement 1
Illustration of RNAscope methodology.
Steps to detect an mRNA sequence (red zipper) consisting of (1) hybridization, (2) pre-amplification, (3) and labeling. Double ZZ probes are shown in green. Dye labeling the mRNA is shown in magenta.
Figure 5—figure supplement 2
RNA probe validation.
(A) Representative images of INS-1 cells subjected to mRNA probes against a bacterial gene (DapB from Bacillus subtilis). (B) Representative images of INS-1 cells subjected to mRNA probes against three constitutive mammalian genes (ubiquitin C, cyclophilin B, and a polymerase II subunit). (C–F) show tsA-201 cells that were naive to exogenous DNA but treated with mRNA probes against the genes for (C) BK channels, (D) CaV1.3 channels, (E) Ryanodine receptor type 2 (RyR2), and (F) NaV1.7 channels. Scale bar is 10 μm.
Figure 6
BK mRNA (KCNMA1) and RyR-2 mRNA (RyR2) colocalize.
(A) Representative confocal images of KCNMA1 and NaV1.7 (SCN9A) mRNA. (B) Representative images of KCNMA1 and RyR2 mRNA. (C) Comparison of the colocalization between KCNMA1 mRNA and mRNA from RyR2, SCN9A, and scrambled images of KCNMA1. Data points are from n=67 cells. One-way ANOVA. Scale bars are 10 μm and 1 μm in the magnifications. Data were analyzed using ordinary one-way ANOVA with Dunnett’s multiple comparisons test.
Figure 7
BK mRNA (KCNMA1) and CaV1.3 mRNA (CACNA1D) colocalize in micro-translational complexes.
(A) Diagram of the hypothesis: KCNMA1 mRNAs are found in micro-translational complexes. (B) Representative images of KCNMA1 mRNA in magenta, CACNA1D mRNA in cyan, and BK protein in green. (C) Comparison of the frequency of colocalization of KCNMA1 mRNA in active translation and in micro-translational complexes. Data points are from n=57 cells. One-way ANOVA was used as statistical analysis. Scale bars are 10 μm and 1 μm in the magnifications.
Figure 8 with 1 supplement
Formation of BK and CaV1.3 hetero-clusters in INS-1 cells.
(A) Representative localization map of antibodies against BK (magenta) and CaV1.3 (cyan) channels. Magnifications are shown in the insets on the right. (B) Scatter dot plot of homo-cluster densities of BK and CaV1.3 channels in INS-1 cells. (C) Cumulative frequency distributions of homo-cluster sizes of BK and CaV1.3 channels. Inset compares median size of BK and CaV1.3 homo-clusters. (D) Comparison of colocalization between BK and CaV1.3 and between scrambled BK and CaV1.3. Data points are from n=13 cells. Scale bars are 5 μm and 300 nm in the magnifications. Data was analyzed using paired t-tests to evaluate significance.
Figure 8—figure supplement 1
Organization of BK-CaV1.3 hetero-clusters in INS-1 cells.
(A) Proportion of channels in clusters or outside clusters for BK and CaV1.3. Clustered channels were defined as being at a distance of 200 nm or less from the other channel type. Channels found at 200 nm or more were considered non-clustered. Data from 7 cells. (B) Distribution of the nearest neighbor distance from BK channels to CaV1.3 channels in INS-1 cells (magenta, n=1037 BK channels, from N=5 cells). Distribution of the nearest neighbor distance from BK channels to randomized channels (gray, n=363 BK channels). The shift in the distribution from INS-1 cells to randomized images suggests a preferential localization of endogenous BK-CaV1.3 hetero-clusters.
Figure 9
Hetero-clusters of BK and CaV1.3 channels are detected at the plasma membrane soon after their expression begins.
(A–C) Representative localization maps of antibodies against BK and CaV1.3 channels. (A) 18 hr, (B) 24 hr, or (C) 48 hr after DNA transfection into cells. Enlargements are shown in insets. (D) Cumulative frequency distributions of BK homo-cluster size at 18, 24, and 48 hr. Inset compares median BK homo-cluster areas. (E) Cumulative frequency distributions of CaV1.3 homo-clusters at 24 and 48 hr. The inset compares median CaV1.3 cluster areas. CaV1.3 clusters are not present at the 18 hr time point. (F) Comparison of colocalization plots between BK and CaV1.3 channels at 24 and 48 hr time points. Data points are from n=10 cells. Scale bars are 10 μm and 300 nm in enlargements. Statistical significance was assessed using ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test and unpaired t-test in panels D and E, respectively.
Author response image 1
Author response image 2
Tables
Table 1
List of mechanisms described for the interaction between channel subunits, channels of the same type (homo-clusters), or hetero-clusters of channel families permeating different ions.
| Mechanism | Description | Source |
|---|---|---|
| Co-translation of heteromeric channel subunits | mRNA transcripts and nascent proteins of hERG heteromeric subunits form molecular complexes during protein translation | Liu et al., 2016 |
| Co-translation of channels permeating different ions | Potassium channel hERG and sodium channel SCN5A form complexes of mRNA transcripts and nascent proteins during protein translation | Eichel et al., 2019 |
| Membrane curvature sensing | Clusters of Piezo1 channels enriched in membrane invaginations | Yang et al., 2022 |
| ER membrane protein complex | ER membrane complex acts as a chaperone for heteromeric channel assembly | Chen et al., 2023b |
| Scaffolding proteins | Scaffolding protein AKAP150 is required for abnormal gating of CaV1.2-LQT8 channels | Cheng et al., 2011 |
| Random insertion | Clusters of CaV1.2, CaV1.3, BK, and TRPV4 are proposed to be randomly formed into the plasma membranes of smooth muscle, cardiac muscle, hippocampal neurons, and tsA-201 cells | Sato et al., 2019 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (human) | tsA-201 | Sigma | RRID:CVCL_2737 | Authentication: STR profiling; Mycoplasma: negative (see Materials and methods) |
| Cell line (rat) | INS-1 | Sigma | RRID:CVCL_0352 | Authentication: STR profiling/DNA barcoding; Mycoplasma: negative (see Materials and methods) |
| Recombinant DNA reagent | CaV1.3 | Addgene | RRID:Addgene_49333; Addgene:49333 | Plasmid expressing CACNA1D (rat) |
| Recombinant DNA reagent | Slo1 (BK) | Addgene | RRID:Addgene_113566; Addgene:113566 | Mouse BK channel α-subunit expression vector |
| Recombinant DNA reagent | KDEL-mox-GFP (ERmoxGFP) | Addgene | RRID:Addgene_68072; Addgene:68072 | ER-localized moxGFP with KDEL retention sequence |
| Recombinant DNA reagent | pmGFP-Sec16 | Addgene | RRID:Addgene_15775; Addgene:15775 | pmGFP-Sec16S mammalian expression plasmid |
| Recombinant DNA reagent | Golgi-mEGFP | Addgene | RRID:Addgene_182877; Addgene:182877 | Golgi-targeted mEGFP |
| Recombinant DNA reagent | pPH-PLC-δ-GFP (PH-PLCD1-GFP) | Addgene | RRID:Addgene_51407; Addgene:51407 | Biosensor for PI(4,5)P2 |
| Recombinant DNA reagent | CaVβ3 | Diane Lipscombe, Brown University | other | Auxiliary subunit for CaV1.3; repository ID not available; contact depositor |
| Recombinant DNA reagent | CaVα2δ1 | Diane Lipscombe, Brown University | Other | Auxiliary subunit for CaV1.3; repository ID not available; contact depositor |
| Sequence-based reagent (rat) | RNAscope 3-plex Positive Control Probes | Advanced Cell Diagnostics | Cat. No. 320871 | Species-specific housekeeping targets for RNAscope; 3-plex positive control |
| Sequence-based reagent (rat) | RNAscope 3-plex Negative Control Probe (dapB) | Advanced Cell Diagnostics | Cat. No. 320871 | Negative control probe targeting bacterial dapB |
| Sequence-based reagent (rat) | RNAscope Probe—Rn-Ryr | Advanced Cell Diagnostics | Cat. No. 2560931 | Custom target probe |
| Sequence-based reagent (rat) | RNAscope Probe—Rn-Scn9a | Advanced Cell Diagnostics | Cat. No. 317851 | Custom target probe |
| Sequence-based reagent (rat) | RNAscope Probe—Rn-Kcnma1-C3 | Advanced Cell Diagnostics | Cat. No. 1108261-C3 | Channel C3 probe |
| Sequence-based reagent (rat) | RNAscope Probe—Rn-Cacna1d-C2 | Advanced Cell Diagnostics | Cat. No. 409361-C2 | Channel C2 probe |
| Sequence-based reagent (rat) | RNAscope Probe—Rn-Gapdh | Advanced Cell Diagnostics | Cat. No. 409821 | Housekeeping gene control |
| Antibody | Anti-CaV1.3 (Rabbit polyclonal) | Drs. William Catterall and Ruth Westenbroek | Rabbit polyclonal primary recognizing residues 809–825 (II–III loop), (1:100), (1 μl) | |
| Antibody | Anti-Slo1 (clone L6/60) (Mouse monoclonal) | Millipore Sigma | RRID:AB_10805948; Cat. No. MABN70 | Mouse monoclonal, (1:100), (1 μl) |
| Antibody | Anti-GFP (Goat polyclonal) | Abcam | RRID:AB_305643; Cat. No. ab6673 | Rabbit polyclonal to full-length Aequorea victoria GFP, (1:100), (1 μl) |
| Antibody | Anti-58K Golgi (Mouse monoclonal) | Abcam | RRID:AB_2107005; Cat. No. ab27043 | Mouse monoclonal recognizing Golgi protein (1:100), (1 μl) |
| Antibody | Donkey anti-mouse Alexa 647 (Donkey polyclonal) | Invitrogen (Molecular Probes) | RRID:AB_2536183; Cat. No. A-31573 | Secondary antibody donkey polyclonal, (1:1000), (1 μl) |
| Antibody | Donkey anti-rabbit Alexa 555 (Donkey polyclonal) | Invitrogen (Molecular Probes) | RRID:AB_2534017; Cat. No. A10042 | Secondary antibody, donkey polyclonal, (1:1000), (1 μl) |
| Antibody | Donkey anti-goat Alexa 488 (Donkey polyclonal) | Invitrogen (Molecular Probes) | RRID:AB_162542; Cat. No. A-31571 | Secondary antibody, donkey polyclonal, (1:1000), (1 μl) |
| Antibody | Duolink In Situ PLA probe anti-rabbit PLUS (Donkey polyclonal) | Sigma | RRID:AB_2810940 | Proximity ligation assay probe, donkey polyclonal, (1:20), (2 μl) |
| Antibody | Duolink In Situ PLA probe anti-mouse PLUS (Donkey polyclonal) | Sigma | RRID:AB_2810939 | Proximity ligation assay probe, donkey polyclonal, (1:20), (2 μl) |
| Commercial assay or kit | Duolink In Situ Red Starter Kit | Sigma | Cat. No. DUO92008 | PLA detection kit |
| Chemical compound, drug | Lipofectamine 3000 | Invitrogen | Cat. No. L3000001/008/015 (series) | Transfection reagent; see Thermo Fisher catalog for sizes |
| Chemical compound, drug | ProLong Gold Antifade Mountant with DAPI | Invitrogen | Cat. No. P36931 | Mounting medium with DAPI |
| Commercial assay or kit | RNAscope Multiplex Fluorescent v2 Assay | Advanced Cell Diagnostics (Bio-Techne) | Cat. No. 323270 (with TSA Vivid dyes)/323100 (reagent kit v2) | Manual multiplex fluorescent RNA ISH assay |
| Software, algorithm | Prism | GraphPad | RRID:SCR_002798 | Version 10 |
| Software, algorithm | Excel | Microsoft | RRID:SCR_016137 | Microsoft 365 build |
| Software, algorithm | ImageJ | NIH | RRID:SCR_003070 | ImageJ2/Fiji build |
| Software, algorithm | NImOS | ONI | Version: v1.18.3 | |
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/106791/elife-106791-mdarchecklist1-v1.docx
-
Source code 1
Text code of the analysis program SpotScramble in PDF.
- https://cdn.elifesciences.org/articles/106791/elife-106791-code1-v1.zip
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Functionally coupled ion channels begin co-assembling at the start of their synthesis
eLife 14:RP106791.
https://doi.org/10.7554/eLife.106791.4
