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.

Representation of molecular complex, homo-cluster, and hetero-cluster.

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 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 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 hetero-clusters localize at 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 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 is 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 ER exit sites. 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 ER exit sites 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 ER exit sites. Scale bar is 10 μm and 2 μm in the magnifications.

BK and CaV1.3 hetero-clusters go through the Golgi.

A. Diagram of the hypothesis: 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 1-4. 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.

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 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 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 bar is 10 μm and 1 μm in the magnifications.

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 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 bar is 10 μm and 1 μm in the magnifications.

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 bar is 5 μm and 300 nm in the magnifications.

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 hours, (B) 24 hours, or (C) 48 hours after DNA transfection into cells. Enlargements are shown in insets. D. Cumulative frequency distributions of BK homo-cluster size at 18, 24, and 48 hours. Inset compares median BK homo-cluster areas. E. Cumulative frequency distributions of CaV1.3 homo-clusters at 24 and 48 hours. Inset compares median CaV1.3 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.