List of mechanisms described for the interaction between channel subunits, channels of the same type (clusters), or ensembles of channel families permeating different ions.

Representation of molecular complex, cluster, and ensemble.

BK and CaV1.3 ensembles are found inside the cell.

A. Diagram of the hypothesis: Ensembles 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 ensembles. Proximity ligation assay is used to detect the ensembles. C. Confocal images of fluorescent puncta from 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 ensembles of BK and CaV1.3 to the negative control. Data points are from n = 12 cells from BK and CaV1.3 clusters and from n = 14 cells from 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 and CaV1.3 channels and GFP. The plane of the PM was set as zero in the Z axis. PLA puncta are shown in magenta, and the plasma membrane in green. Distance from the PM plane is labeled with the Z values on top of each image. Enlargements of the same region in Z are shown in the insets. F. Scatter dot plot comparing BK and CaV1.3 cluster abundance at PM and inside the cell. Data points are from n = 12 cells. Scale bar is 10 μm and 1 μm in the insets.

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.

BK and CaV1.3 ensembles localize at ER and ER exit sites (ERES).

A. Diagram of the hypothesis: Ensembles 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. Enlargement of a selected region 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. Confocal images of PLA puncta (magenta) and ER (green) in tsA-201 (left) and INS-1 (right) cells. Cells were transfected with BK, CaV1.3, and KDEL-moxGFP. PLA puncta reveal ensembles of BK and CaV1.3. ER is shown in green. E. Comparison of BK and CaV1.3 ensembles found at the ER and relative to all PLA puncta in the cell. Values are given in percentages. F. Representative image of ER exit sites labeled with sec16-GFP. Magnification is shown in the inset. G. Thresholded images of PLA puncta and ER exit sites. Left and right are the same as in D, but cells were transfected with Sec16-GFP instead of KDEL. H. Percentage of BK and CaV1.3 ensembles found at ER exit sites relative to all PLA puncta. Grey bars show the percentage of ensembles found in the ER. 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; n = 23 INS-1 cells for ER exit sites. Scale bar is 10 μm and 2 μm in the magnifications.

BK and CaV1.3 ensembles go through the Golgi.

A. Diagram of the hypothesis: PLA puncta detecting ensembles between BK (magenta) and CaV1.3 (cyan) channels can be found at the Golgi membrane. B. Representative confocal 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 (left) in green and 58K-Golgi (middle) in red. Overlay (right). D. Representative confocal 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 percentage of BK and CaV1.3 ensembles 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. 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.

Diagram illustrating our interpretation of the percentage of BK-CaV1.3 ensembles found in the cell.

This illustration is based on experiments shown in figures 1 to 4, but percentages were modified to represent overlap of fluorescent signals and limited resolution. We also show that the ensembles found in the ER exit sites (ERES) are also accounted in the ER.

BK mRNA (KCNMA1), CaV1.3 mRNA (CACNA1D) co-localize.

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 RNA scope experiments showing KCNMA1 mRNA in magenta CACNA1D mRNA in cyan, and GAPDH mRNA in green. Right, magnification of three ROI. 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 bar is 10 μm and 1 μm in the magnifications.

Illustration of RNA scope 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.

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 Receptors type 2 (RyR2), and (F) NaV1.7 channels. Scale bar is 10 μm.

BK (KCNMA1) and RyR-2 (RyR2) mRNA co-localize.

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 bar is 10 μm and 1 μm in the magnifications.

Formation of BK and CaV1.3 ensembles in INS-1 cells.

(A) Representative localization map of antibodies against BK (magenta) or CaV (cyan) channels. Magnifications are shown in the insets on the right. (B) Scatter dot plot of channel density of BK and CaV1.3 channels in INS-1 cells. (C) Cumulative frequency distribution of the size of BK and CaV1.3 channels. Inset compares median size of BK and CaV channels. (D) Comparison of colocalization between BK and CaV1.3 and BK and scrambled BK images. Data points are from n = 13 cells. Scale bar is 5 μm and 300 nm in the magnifications.

Ensembles 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 CaV channels (A) 18 hours, (B) 24 hours, or (C) 48 hours after DNA transfection into the cell. Enlargements are shown in insets. (D) Size distribution of BK clusters at 18, 24, and 48 hours. Inset compares median BK cluster areas. (E) Size distribution of CaV clusters at 24 and 48-hour time points. Inset compares median CaV cluster areas. CaV1.3 clusters are not present at the 18-hour time point. (F) Comparison of colocalization plots between BK and CaV1.3 channels at 24 and 48-hour time points. Data points are from n = 10 cells. Scale bar is 10 μm and 300 nm in enlargements.