DOI: 10.5061/dryad.63xsj3vfq
title: Functionally-coupled ion channels begin co-assembling at the start of their synthesis

description: Calcium binding to BK channels lowers BK activation threshold,
 substantiating functional coupling with calcium-permeable channels. This
 coupling requires close proximity between different channel types, and the
 formation of BK–CaV1.3 hetero-clusters at nanometer distances exemplifies
 this unique organization. To investigate the structural basis of this
 interaction, we tested the hypothesis that BK and CaV1.3 channels assemble
 before their insertion into the plasma membrane. Our approach incorporated
 four strategies: (1) detecting interactions between BK and CaV1.3 proteins
 inside the cell, (2) identifying membrane compartments where intracellular
 hetero-clusters reside, (3) measuring the proximity of their mRNAs, and
 (4) assessing protein interactions at the plasma membrane during early
 translation. These analyses revealed that a subset of BK and CaV1.3
 transcripts are spatially close in micro-translational complexes, and
 their newly synthesized proteins associate within the endoplasmic
 reticulum (ER) and Golgi. Comparisons with other proteins, transcripts,
 and randomized localization models support the conclusion that BK and
 CaV1.3 hetero-clusters form before their insertion at the plasma membrane.
description: <strong>Cell culture:</strong> We
 used tsA-201 cells to co-express BK and Ca<sub>V</sub>1.3
 channels heterologously. Cells were grown in DMEM (Gibco) supplemented
 with 10% fetal bovine serum and 0.2% penicillin/streptomycin. We used rat
 insulinoma (INS-1) cells to study endogenous levels of transcripts and
 proteins of channels. INS-1 cells were cultured in RPMI high glutamate
 medium (Gibco) with 10% fetal bovine serum, 0.2% penicillin/streptomycin,
 10 mM HEPES (Gibco), 1 mM sodium pyruvate (Gibco), and 50 μM
 2-mercaptoethanol. Both cell types were passaged twice a week and
 incubated in 5% CO<sub>2</sub> at 37°C.
 <strong>Plasmids and transfection:</strong>
 Cells were transfected with 0.1– 0.4 μg DNA per plasmid and
 plated for 24 hours on poly-D-Lysine coated coverslips. Lipofectamine 3000
 (Invitrogen, RRID: L30000) was used for the transfection. DNA clones of
 Ca<sub>V</sub>1.3, BK channels, PH-PLCδ GFP, ER moxGFP,
 pmGFP-Sec16S and Golgi-mGFP were obtained from Addgene (RRID:SCR_002037).
 The Ca<sub>V</sub>1.3 α** subunit construct used in our study
 corresponds to the rat Ca<sub>V</sub>1.3e splice variant
 containing exons 8a, 11, 31b, and 42a, with a deletion of exon 32. The BK
 channel construct corresponds to the VYR splice variant of the mouse BKα
 subunit (KCNMA1). Auxiliary subunits for Ca<sub>V</sub>1.3
 channels, Ca<sub>V</sub>β3 and Ca<sub>V</sub>α2δ1
 (from Diane Lipscombe, Brown University, RI), were transfected as well. No
 BK channel auxiliary subunits were transfected.
 <strong>Antibodies:</strong>
 Ca<sub>V</sub>1.3 channels were immuno-detected with
 a rabbit primary antibody recognizing residues 809 to 825 located at the
 intracellular II-III loop of the channel (DNKVTIDDYQEEAEDKD), kindly
 provided by Drs. William Catterall and Ruth Westenbroek [32]. BK channels
 were detected using the anti-Slo1 mouse monoclonal antibody clone L6/60.
 The goat polyclonal GFP antibody was against the recombinant full-length
 protein corresponding to <em>Aequorea victoria</em> GFP.
 Anti-58K Golgi protein antibody**** was used to mark the Golgi.
 Specificity of antibodies was tested in un-transfected tsA-201 cells
 (Figure 2- Supplementary 1). The secondary antibodies tagged with Alexa
 dyes were Donkey anti-mouse Alexa-647, Donkey anti-rabbit Alexa-555,
 Donkey anti-goat Alexa-488 (Molecular Probes). 
 <strong>Immunostaining:</strong>
 Cells were fixed with freshly prepared 4% paraformaldehyde for 10
 minutes. After washing, aldehydes were reduced with 0.1%
 NaBH<sub>4</sub> for 5 minutes and then washed again.
 Nonspecific binding was blocked with 3% bovine serum albumin (Thermo
 Scientific). Cells were permeabilized with 0.25% v/v Triton X-100 in PBS
 for 1 hour. Primary antibodies were used at 10 μg/ml in blocking solution
 and incubated overnight at 4°C. After washing, secondary antibodies at 2
 μg/ml were incubated for 1 hour at 21˚C. Washing steps indicated in all
 methods include 3 cycles of rinsing and rocking for 5 minutes with PBS at
 21˚C. Cells were imaged using an inverted AiryScan microscope or an ONI
 Nanoimager with super-resolution capabilities, in total internal
 reflection fluorescence (TIRF) mode, and with a Z-resolution of 50
 nm. <strong>Proximity ligation assay
 (PLA):</strong> Cells were fixed with freshly
 prepared 4% paraformaldehyde for 10 minutes. After washing, aldehydes were
 reduced with 50 mM glycine for 15 minutes. After another round of washes,
 PLA was performed according to manufacturer instructions (Duolink® In Situ
 Red Starter Kit). Cells were blocked and permeabilized with Duolink
 blocking solution. Primary antibodies were used at 10 μg/ml in Duolink
 antibody diluent and incubated overnight at 4°C. The Duolink® In Situ PLA®
 probe anti-rabbit PLUS and anti-mouse MINUS were used as secondary
 antibodies, followed by ligation and amplification. For PLA combined with
 immunostaining, PLA was followed by a secondary antibody incubation with
 Alexa Fluor-488 at 2 μg/ml for 1 hour at 21˚C. Since GFP fluorescence
 fades significantly during the PLA protocol, resulting in reduced signal
 intensity and poor image resolution, GFP was labeled using an antibody
 rather than relying on its intrinsic fluorescence. Coverslips were mounted
 using ProLong™ Gold Antifade Mountant with DAPI. Cells were imaged using
 an inverted Zeiss AiryScan microscope.
 <strong>Single-molecule fluorescence in situ hybridization
 (RNAscope<sup>TM</sup>):</strong>
 Manual RNAscope assay was performed using RNAscope Multiplex
 Fluorescent V2 Assay according to the manufacturer's protocol. The
 RNAscope assay consists of target probes and a signal amplification system
 composed of a preamplifier, amplifier, and label probe. A schematic
 RNAscope assay procedure is shown in Figure 5- Supplementary 1. Briefly,
 cells were fixed with 4% paraformaldehyde for 30 minutes, washed,
 dehydrated, and then rehydrated with ethanol, and permeabilized with 0.1%
 Tween-20 in PBS. Next, cells were quenched with
 H<sub>2</sub>O<sub>2</sub> and treated with
 Protease III. Probes were hybridized for 2 hours at 40°C followed by
 RNAscope amplification and then fluorescence detection. Coverslips were
 mounted using ProLong™ Gold Antifade Mountant with DAPI. We used the
 following RNAscope probes: RNAscope 3-plex Positive Control Probes,
 RNAscope 3-plex negative control probes, RNAscope™ Probe- Rn-Ryr,
 RNAscope™ Probe- Rn-Scn9a, RNAscope Probe- Rn-Kcnma1-C3, RNAscope Probe-
 Rn-Cacna1d-C2, and RNAscope Probe- Rn-Gapdh. Cells were imaged on the
 inverted AiryScan microscope. For PLA and RNAscope experiments, we used
 custom-made macros written in ImageJ. Processing of PLA data included
 background subtraction. To assess colocalization, fluorescent signals were
 converted into binary images, and channels were multiplied to identify
 spatial overlap. Specificity of RNAscope probes was tested in
 un-transfected tsA-201 cells (Figure 5- Supplementary 2). For RNAscope
 combined with immunostaining, RNAscope was followed by blocking in PBS
 supplemented with 0.01 % Tween-20 and 3% BSA for 1 h at 21˚C. Samples were
 then probed for BK protein using primary antibody overnight at 4˚C
 followed by secondary antibody incubation with Alexa Fluor-488 at 2 μg/ml
 for 1 hour at 21˚C. Coverslips were mounted using ProLong™ Gold Antifade
 Mountant. Cells were imaged using an inverted Zeiss AiryScan
 microscope. <strong>High resolution
 imaging:</strong> Cells were imaged using an
 inverted AiryScan microscope (Zeiss LSM 880) run by ZEN black v2.3
 software and equipped with a plan apochromat 63X oil immersion objective
 with 1.4 NA. Fluorescent dyes were excited with a 405 nm diode, 458–514 nm
 argon, 561 nm, or 633 nm laser. Emission light was detected using an
 Airyscan 32 GaAsP detector and appropriate emission filter sets. The point
 spread functions were calculated using ZEN black software and 0.1 μm
 fluorescent microspheres. The temperature inside the microscope housing
 was 22°C. Images were analyzed using ImageJ (NIH).
 <strong>Super-resolution imaging:</strong>
 Direct stochastic optical reconstruction microscopy (dSTORM)
 images of BK and Ca<sub>V</sub>1.3 overexpressed in tsA-201
 cells were acquired using an ONI Nanoimager microscope equipped with a
 100X oil immersion objective (1.4 NA), an XYZ closed-loop piezo 736 stage,
 and triple emission channels split at 488, 555, and 640 nm. Samples were
 imaged at 35°C. For single-molecule localization microscopy, fixed and
 stained cells were imaged in GLOX imaging buffer containing 10 mM
 β-mercaptoethylamine (MEA), 0.56 mg/ml glucose oxidase, 34 μg/ml catalase,
 and 10% w/v glucose in Tris-HCl buffer. Single-molecule localizations were
 filtered using NImOS software (v.1.18.3, ONI). Localization maps were
 exported as TIFF images with a pixel size of 5 nm. Maps were further
 processed in ImageJ (NIH) by thresholding and binarization to isolate
 labeled structures. To assess colocalization between the signal from two
 proteins, binary images were multiplied. Particles smaller than 400
 nm<sup>2</sup> were excluded from the analysis to reflect the
 spatial resolution limit of STORM imaging (20 nm) and the average size of
 BK channels. To examine spatial localization preference, binary images of
 BK were progressively dilated to 20 nm, 40 nm, 60 nm, 80 nm, 100 nm, and
 200 nm to expand their spatial representation. These modified images were
 then multiplied with the Ca<sub>V</sub>1.3 channel to quantify
 colocalization and determine BK occupancy at increasing distances from
 Ca<sub>V</sub>1.3. To ensure consistent comparisons across
 distance thresholds, data were normalized using the 200 nm measurement as
 the highest reference value, set to 1.
 <strong>Image scrambling:</strong>
 Images were binarized as TIFF images, and their respective cell
 perimeter coordinates were exported as CSV files by ImageJ. Processed
 binary images were then analyzed by our SpotScrambler (https://github.com/jehuang2/SpotScramble) Python program. SpotScrambler first extracts the areas of fluorescent particles in the binary image. SpotScrambler then redraws the fluorescent particles as circles at randomized coordinates within cell perimeter boundaries. SpotScrambler accurately preserves particle number and particle sizes, averaging less than 1% difference in total area of fluorescent particles between pre-SpotScrambler and post-SpotScrambler images. To ensure reliable randomization for each experiment, results were averaged between 3 trials of SpotScrambler. <strong>Data analysis</strong> Excel (Microsoft), and Prism (GraphPad) were used to analyze data. ImageJ was used to process images. One-way ANOVA and non-parametric statistical test (Mann-Whitney Wilcoxon) were used to test for statistical significance. p-values &lt;0.05 were deemed statistically significant. The number of cells used for each experiment is detailed in each figure legend.
description: # Functionally-coupled ion channels begin co-assembling at the start of
 their synthesis Dataset DOI:
 [10.5061/dryad.63xsj3vfq](https://doi.org/10.5061/dryad.63xsj3vfq) ##
 Description of the data and file structure These datasets are presented in
 our original publication titled *Functionally-Coupled Ion Channels Begin
 Co-assembling at the Start of Their Synthesis*. We have uploaded Excel
 files corresponding to each figure panel, each containing the underlying
 data for the associated graph. Detailed descriptions of the datasets are
 provided below: ## Figure 2: BK and CaV1.3 Hetero-Clusters Are Found
 Inside the Cell Figure_2_panel_D.xlsx **Panel D** This dataset examines
 the use of PLA to detect BK–CaV1.3 hetero-clusters. **Excel columns:** 1.
 PLA puncta density (puncta/μm²) between BK and CaV1.3 2. PLA puncta
 density under negative control (BK primary antibody only)
 Figure_2_panel_F.xlsx **Panel F** This dataset shows BK–CaV1.3
 hetero-clusters localizing both at the plasma membrane and
 intracellularly. **Excel columns:** 1. Percentage of PLA puncta
 colocalized with plasma membrane signal 2. Percentage of PLA puncta found
 inside the cell ## Figure 3: BK and CaV1.3 Hetero-Clusters Localize at ER
 and ER Exit Sites (ERES) Figure_3_panel_C.xlsx **Panel C** ER tubule
 distance measurements in live and fixed cells. **Excel columns:** 1. Live
 tsA201 cells (KDEL-moxGFP) 2. Fixed tsA201 cells (KDEL-moxGFP, anti-GFP)
 3. Live INS-1 cells (KDEL-moxGFP) 4. Fixed INS-1 cells (KDEL-moxGFP,
 anti-GFP) *Units: nm* Figure_3_panel_E.xlsx **Panel E** PLA puncta
 colocalized with ER in two systems. **Excel columns:** 1. tsA201 cells
 (overexpression) 2. INS-1 cells (endogenous) Figure_3_panel_G.xlsx **Panel
 G** PLA puncta colocalized with ERES. **Excel columns:** 1. tsA201 cells
 (overexpression) 2. INS-1 cells (endogenous) ## Figure 4: BK and CaV1.3
 Hetero-Clusters Transit Through the Golgi Figure_4_panel_E.xlsx **Panel
 E** PLA puncta colocalized with Golgi. **Excel columns:** 1. tsA201 cells
 (overexpression) 2. INS-1 cells (endogenous) ## Figure 5: BK and CaV1.3
 mRNA Colocalization (RNAscope) Figure_5_panel_C_.xlsx **Panel C** mRNA
 density in INS-1 cells. **Excel columns:** 1. KCNMA1 2. CACNA1D 3. GAPDH
 Figure_5_panel_D_.xlsx **Panel D** mRNA counts in INS-1 cells. **Excel
 columns:** 1. CACNA1D 2. KCNMA1 Figure_5_panel_E_.xlsx **Panel E** mRNA
 counts in INS-1 cells. **Excel columns:** 1. GAPDH 2. KCNMA1
 Figure_5_panel__F.xlsx **Panel F** KCNMA1 mRNA colocalization (%) in INS-1
 cells. **Excel columns:** 1. With CACNA1D 2. With GAPDH 3. With randomized
 scramble ## Figure 6: BK and RyR2 mRNA Colocalization
 Figure_6_panel_C_.xlsx **Panel C** KCNMA1 mRNA colocalization (%) in INS-1
 cells. **Excel columns:** 1. With RyR2 2. With randomized scramble 3. With
 SCN9A ## Figure 7: BK and CaV1.3 mRNA Colocalize in Micro-Translational
 Complexes Figure_7_panel_C.xlsx **Panel C** RNAscope and BK protein
 immunostaining in INS-1 cells. **Excel columns:** 1. Active translation
 sites 2. Micro-translational complexes ## Figure 8: Formation of BK and
 CaV1.3 Hetero-Clusters in INS-1 Cells \_Figure_8_panel_B.xlsx **Panel B**
 Homo-cluster density (# clusters/μm²). **Excel columns:** 1. BK channels
 2. CaV1.3 \_Figure_8_panel_C.xlsx **Panel C** Cluster size distribution
 and medians. **Excel columns:** 1. Bin size (cluster area, nm²) 2. BK
 cumulative frequency 3. CaV1.3 cumulative frequency 4. Median cluster size
 (BK) 5. Median cluster size (CaV1.3) \_Figure_8_panel_D.xlsx **Panel D**
 Area of BK clusters occupied by CaV1.3 within 40 nm. **Excel columns:** 1.
 Genuine data 2. Randomized scramble ## Figure 9: Early Formation of BK and
 CaV1.3 Hetero-Clusters at the Plasma Membrane \_Figure_9_panel_D.xlsx
 **Panel D** BK cluster size distribution over time in tsA201 cells.
 **Excel columns:** 1. Bin size (cluster area, nm²) 2. BK after 18h
 expression 3. BK after 24h expression 4. BK after 48h expression 5. Median
 cluster size (18h) 6. Median cluster size (24h) 7. Median cluster size
 (48h) \_Figure_9_panel_E.xlsx **Panel E** CaV1.3 cluster size distribution
 over time in tsA201 cells. **Excel columns:** 1. Bin size (cluster area,
 nm²) 2. CaV1.3 after 24h expression 3. CaV1.3 after 48h expression 4.
 Median cluster size (24h) 5. Median cluster size (48h) ## File:
 Figure_S_4-1_.xlsx Description: The Excel file contains two columns both
 represents PLA puncta density observed between first column: CaV1.3 and BK
 and second column with RyR2. Experiment was performed in INS-1 cells
 (endogeounuse system). ## Code/software **Image scrambling:** Images were
 binarized as TIFF images, and their respective cell perimeter coordinates
 were exported as CSV files by ImageJ. Processed binary images were then
 analyzed by our SpotScrambler
 ([https://github.com/jehuang2/SpotScramble](https://github.com/jehuang2/SpotScramble)) Python program. SpotScrambler first extracts the areas of fluorescent particles in the binary image. SpotScrambler then redraws the fluorescent particles as circles at randomized coordinates within cell perimeter boundaries. SpotScrambler accurately preserves particle number and particle sizes, averaging less than 1% difference in total area of fluorescent particles between pre-SpotScrambler and post-SpotScrambler images. To ensure reliable randomization for each experiment, results were averaged between 3 trials of SpotScrambler.