Ryanodine receptors are large (~2.2 MDa) homotetrameric Ca²⁺-activated Ca²⁺ ion channels located in the endoplasmic and sarcoplasmic reticulum of virtually every cell (Lanner et al., 2010; Van Petegem, 2015; Meissner, 2017). They are distributed in discrete clusters whose various shapes and sizes contribute to the spatial and temporal characteristics of Ca²⁺ release. Of the three highly homologous isoforms, ventricular myocytes only express the type II ryanodine receptor (RyR2) and its intracellular location is restricted to the junctional sarcoplasmic reticulum (jSR) membrane in close apposition to the sarcolemma (Carl et al., 1995; Sun et al., 1995). The small distance between the two membranes,~15 nm, provides a space of restricted diffusion where a small influx of Ca²⁺ through sarcolemmal L-type Ca²⁺ channels (Ca_v1.2) can open the RyR2 via Ca²⁺-induced Ca²⁺ release (CICR) (Franzini-Armstrong, 2018). This architecture is critical for normal cardiac function (Louch et al., 2006; Song et al., 2006).

In diastole, and in the absence of Ca²⁺ influx through Ca_v1.2, RyR2 open stochastically but with a very low probability (Satoh et al., 1997). A single tetramer can either open without further effect, or it can activate other RyR2 through inter-RyR2 CICR producing a Ca²⁺ spark (Cheng et al., 1993; Santiago et al., 2010; Sobie et al., 2006). This diastolic SR Ca²⁺ release, with and without sparks, is normal and asymptomatic in a healthy individual, but it is elevated in both acquired and inherited cardiac diseases such as hypertrophy, heart failure (Song et al., 2005) and catecholaminergic polymorphic ventricular tachycardia (CPVT) (Uchinoumi et al., 2010), leading to increased morbidity and mortality. Less Ca²⁺ in the SR results in a negative inotropic effect, and the elevated release reduces the rate of decline of the Ca²⁺ transient producing a negative lusitropic effect. Removing the excess Ca²⁺ from the myoplasm is energetically costly since it utilizes ATP. If the leak is sufficiently large, the Na⁺/Ca²⁺ exchanger can depolarize the cell and seed a potentially lethal arrhythmia, a common cause of death in cardiac disease (Bers, 2014). For these reasons, elevated SR Ca²⁺ release is a significant driver behind current efforts to understand how RyR2 are regulated and how they function.

Abnormal RyR2 activity and Ca²⁺ release has also been linked to many other disorders, including atrial fibrillation (Dobrev et al., 2011), cognitive dysfunction (Liu et al., 2012), Alzheimer’s disease (Stutzmann et al., 2006), and diabetes mellitus (Santulli et al., 2015). All of these, in addition to heart failure and arrhythmia, have been suggested to originate, at least in part, through RyR2 phosphorylation and a decrease in its affinity for the immunophilin FKBP12.6 (calstabin 2) (Kushnir et al., 2018; Marx et al., 2000). While this mechanism remains contentious (Camors and Valdivia, 2014; Xiao et al., 2004), as do the actions of the FKBPs even in the absence of disease (Gonano and Jones, 2017), the adverse effects of pathologic Ca²⁺ release through abnormally functioning RyR2 has been well established.

Recently, pathologically induced changes in the RyR2 cluster sizes was identified as the likely cause of cerebral artery smooth muscle tone dysfunction in the mdx mouse (Pritchard et al., 2018) and in rat ventricle the probability of a Ca²⁺ spark was found to be correlated to the size of the cluster (Galice et al., 2018). These results suggest that tetramer movement and cluster size might be variables that contribute to regulating RyR2 function in both health and disease. Intuitively, the receptors’ relative positions should also be of importance in regulating the positive feedback of inter-RyR2 CICR and could therefore be modulated, but mathematical models have produced conflicting results (Cannell et al., 2013; Tanskanen et al., 2007; Iaparov et al., 2019). Empirical studies are few, but phosphorylation and Mg²⁺ were seen to reorient the tetramers and their relative positions were correlated with the Ca²⁺ spark frequency (Asghari et al., 2014). We therefore set out to test the impact of FKBP binding, hypothesizing that it would alter distribution and spark frequency, and to examine the effect on RyR2 cluster sizes.

We used correlative microscopy to examine the position and orientation of RyR2 tetramers relative to each other, and Ca²⁺ sparks from within the same cells, and found that exposing the tetramers to a saturating concentration of either FKBP12 or FKBP12.6 resulted in a reduced SR Ca²⁺ release and a more densely packed side-by-side tetramer array. Exposing similarly treated myocytes to cell-wide phosphorylation produced the opposite result; increased SR Ca²⁺ release and more widely spaced tetramers. dSTORM microscopy observed widespread changes in RyR2 cluster sizes and in the number of tetramers per cluster that supported and extended those obtained from correlative microscopy.

We conclude that RyR2 are not distributed randomly or independently of each other on the jSR membrane. Rearranging the tetramer array and changing the cluster sizes is part of the normal operation of the mammalian ventricle and are likely mechanisms that alter the probability of Ca²⁺ spark formation.

RyRs can have from zero to 4 FKBPs associated with them. The amount and the proportion of each isoform in the native cell is uncertain, and the level of saturation of the binding sites is estimated to be between 45–100% (Guo et al., 2010; Jeyakumar et al., 2001; Zissimopoulos et al., 2012). To isolate the effects of each isoform, and to ensure saturation of the RyR2 tetramers, we delivered 10 μmol/L of purified FKBP12 or FKBP12.6 to the permeabilized myocytes. The results thus obtained illuminate the inherent ability to cluster and move for those RyR2 channels with four FKBPs bound.

The images in Figure 1A are representative line scans of Ca²⁺ sparks recorded from saponin permeabilized myocytes at room temperature in a solution containing 100 nmol/L Ca²⁺ and 1 mmol/L Mg²⁺, conditions comparable to those in a resting myocyte. The images have been placed into an array with columns designating the treatment (buffer [Control]; 10 µmol/L FKBP12; 10 µmol/L FKBP12.6), and rows indicating whether the cells were (bottom) or were not (top) exposed to the phosphorylation cocktail.

Figure 1

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Figure 1—source data 1

Statistical analysis of calcium spark data.

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Figure 1—source data 2

Number of sparks, cells and rats examined.

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The distributions of most of the Ca²⁺ spark parameters were not normal, so they were displayed as box plots (Figure 1B). For each spark parameter in the six experimental groups there are fifteen different comparisons, and for this reason we used a color code to indicate statistical differences. Data that were not significantly different from each other share the same color, while those that are significantly different, do not. Some box plots contain two or more colors; for example, the spark mass plot in Figure 1B, phosphorylation is bi-colored because it was not significantly different from control (white) or FKBP12.6 plus phosphorylation (blue), but the latter two were significantly different from each other. The probabilities for the comparisons for each parameter and whether they have statistical significance is shown in Figure 1—source data 1; an explanation of the table columns is given in the Materials and methods section.

The immunophilins significantly reduced the spark frequency and most of the Ca²⁺ spark parameters relative to control. Comparing control, FKBP12 and FKBP12.6 treated cells to their phosphorylated counterparts demonstrated that phosphorylation significantly increased many of the Ca²⁺ spark parameters, although the degree depended on the particular immunophilin. FKBP12 plus phosphorylation produced far fewer sparks than FKBP12.6 plus phosphorylation (p<10⁻¹²), possibly reflecting their differing effects on RyR2 gating (Gonano and Jones, 2017). The spark frequency of phosphorylation alone lies between the two and is significantly different from both (p<2×10⁻⁴). The number of sparks analyzed, cells examined, and animals used are in Figure 1—source data 2.

Given these results, and those from our previous study, we hypothesized that changes in the tetramers’ distribution would accompany the functional changes and used correlation microscopy to examine dyads in the same cells from which we had recorded the Ca²⁺ sparks.

RyR2 is a homotetramer resembling a square-capped mushroom about 27 nm wide and 12 nm deep (Peng et al., 2016). Its size, shape and position on the jSR membrane makes it one of the few proteins that can be identified in an electron micrograph without the aid of additional markers (Franzini-Armstrong, 1994), a significant advantage of this technique. We examined the distribution of RyR2 tetramers in junctions using the Multi Planner Viewer (Amira software), simultaneously viewing three orthogonal sections of a selected junction enabling a 360-degree view of the dual aligned 3D data sets, and of every RyR2 (Figure 2). The 3D view is essential because the dyadic cleft has a complex geometry. It is usually uneven and often curved such that a single plane cannot view all of the tetramers at once. Multiple planes and multiple points of view are required.

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Using Amira, a single plane from a dual-tilt tomogram is displayed in Figure 2A (control) showing the jSR (single arrow) adjacent to a t-tubule (double arrow). Viewpoints within the tomogram’s volume are determined by orthogonal planes: XY; red (Figure 2A,C), YZ; green (Figure 2B) and XZ; blue (Figure 2D). A single XZ plane that bisects the cleft enabling all of the tetramers’ positions and orientations to be visualized and mapped is usually impossible because of the uneven and undulating surface of the jSR membrane. We therefore positioned a XZ plane as parallel to the jSR surface as possible and, progressing in 0.5 nm increments, moved through the cleft to identify a plane where a given tetramer was clearly seen; the tomogram was then tilted in X and Y until its four corners were clear, at which point a red box of the appropriate size (27 nm on a side) was manually positioned over the tetramer and the image was captured. This was done for each tetramer in the tomogram. These images were then used to inform the positioning of the boxes in the RyR_fit program. For displaying all of the RyR2 in the tomogram, we used RyR_fit to manually insert all of the red boxes onto a single XZ plane with a tilt of 0°. Not all of the tetramers are clearly visible in a single image plane, so some of the boxes are atop what appear to be oddly shaped blobs. For clarity, we redisplay the boxes on a black background. Of the 28 XZ planes that spanned this cleft (Figure 2D), the selected tetramer (arrows) was clearly seen in planes 2–6, and plane 4 (outlined in blue) was tilted in X and Y to visualize its corners (Figure 2E); in this instance rotations of ~5° in X and 0° in Y gave a clear result (Figure 2Fi). All of the red boxes, identifying the tetramers’ positions and orientations, were displayed on a single XZ plane (Figure 2Gi) and then on a black background (Figure 2Gii); the tetramer fitted in Figure 2D and E is highlighted with an asterisk. Mapping the position and orientation of the tetramers labelled a, b and c (Figure 2Fii) is shown in Figure 2—figure supplement 1. A 3D model of this tomogram’s RyR2, and its jSR and t-tubule membranes, can be seen in the accompanying movie, Video 1.

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Measurement of the tetramers’ centre-to-centre nearest neighbor distances (NND), also derived from RyR_fit, is displayed in Figure 2Hi, and a histogram of all the results obtained from the control group (five tomograms, 98 tetramers) is in Figure 2Hii. The histogram has a bimodal distribution with modes at 28 and 34 nm, comparable to our previous results (Asghari et al., 2014). As can be seen in Figure 2Hi, the first mode corresponds to tetramers that are side-by-side, while the second shows tetramers that are mostly in a checkerboard-like arrangement. Using our classification criteria, 50.0% of the tetramers had neighbors that were only in a checkerboard configuration and 27.6% had neighbors that were only side-by-side. 8.2% had neighbors in both side-by-side and checkerboard configurations while 14.3% were isolated (Table 1).

Tetramer arrangements recorded in each of the indicated experimental conditions using the RyR_fit program which is available in Table 1, Source code 1.

Comparable images of a myocyte treated with the phosphorylation cocktail are shown in Figure 3 and a model of the complete tomogram is in the movie Video 2. The images in Figure 3 demonstrate that the tetramers adopted a more ordered and less densely packed configuration when phosphorylated, with 75.0% of them in a checkerboard and only 6.3% that were side-by-side (64 tetramers, four tomograms; Table 1). These changes resulted in a unimodal histogram with a single broad peak centred at 34 nm.

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Permeabilized myocytes treated with saturating concentrations of either FKBP12 (Figure 4A) or FKBP12.6 (Figure 4C) produced dramatic effects on the tetramer distribution, orienting them into a more densely packed side-by-side configuration; 57.1% for FKBP12 (119 tetramers, five tomograms) and 82.8% for FKBP12.6 (122 tetramers examined; Table 1). Both proteins shifted the histograms of the centre-to-centre NND to a single mode with a peak at 28 nm (Figure 4Aiii and 4Ciii), reflecting the preponderance of the side-by-side configuration when the tetramers were completely saturated with either of these proteins. Models of these complete tomograms are in the movies Video 3 (FKBP12) and Video 4 (FKBP12.6).

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Video 3

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Exposing immunophilin-treated tetramers to cell-wide phosphorylation partially reversed the proteins’ effects (Figure 4B and D), but seemed more effective in separating tetramers treated with FKBP12.6 than with FKBP12. As summarized in Table 1, FKBP12.6 plus phosphorylation increased the percentage of tetramers in a checkerboard to 71.3%, with side-by-side tetramers reduced to only 5.2% (115 tetramers examined in eight tomograms). The comparable values for FKBP12 plus phosphorylation were 60.5% checkerboard and 13.2% side-by-side (129 tetramers examined in seven tomograms). Models of these tomograms are in Video 5 (FKBP12 + phosphorylation cocktail) and Video 6 (FKBP12.6 + phosphorylation).

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To determine which, if any, of the tetramer distributions could have been derived from the same population, the histograms were expressed as cumulative distribution functions (CDF; Figure 5) to enable statistical analysis using the k-sample Anderson-Darling test. In all cases, phosphorylation produced a highly significant rightward shift in the distribution, showing that phosphorylation overwhelms the effect of the immunophilins. In particular, FKBP12.6 shifted from being the leftmost curve to being the rightmost. For FKBP12, the shift was less dramatic and it was noticeable that the effects on spark frequency were significantly less than those for FKBP12.6 (Figure 1B).

Figure 5

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Statistical Analysis of the Cumulative Distribution Functions.

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Tomograms currently provide the highest possible resolution with which to view the tetramers, but to determine their position and orientation the images must be limited to a small area within a single dyadic cleft. To determine whether the results were truly representative of wide-spread changes in the cells we imaged the distribution of RyR2 near the myocyte surface using our custom-built superresolution microscope in near-TIRF mode (Tafteh et al., 2016). The results are displayed in Figure 6A.

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Statistical Analysis of the Cumulative Distribution Functions for the Alpha Shape Areas.

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Statistical Analysis of the Cumulative Distribution Functions for the Tetramers per Cluster.

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Images of tetramer clusters near the cell surface were placed into an array with columns designating the treatment (buffer [Control]; 10 µmol/L FKBP12; 10 µmol/L FKBP12.6), and rows indicating whether the cells were (bottom) or were not (top) exposed to the phosphorylation cocktail. The images demonstrate dramatic, widespread, changes in the appearance and shape of the clusters. The immunophilins appeared to have fractured the clusters into smaller pieces, while all of the phosphorylated cells appeared to have larger clusters than their non-phosphorylated counterparts.

As demonstrated in Figures 2H, 3F and 4, the centre-to-centre NND between tetramers rarely exceeded 50 nm. We therefore defined a cluster in the dSTORM images as a group of lit pixels that had no pixel separated from its nearest neighbor by more than 50 nm. Each cluster had an alpha shape drawn around it enabling calculation of the enclosed area. The clusters were irregularly shaped and sometimes included subgroups (arrows, Figure 6B). We then pooled all of the areas from a given experimental group and plotted them as a CDF (Figure 6C). Regardless of the experimental treatment the areas varied in size by two orders of magnitude. The median cluster area and the number of clusters analyzed in each experimental group are listed in Table 2. The k-sample Anderson Darling test indicated that the six distributions were not all drawn from the same population (p<0.01). The clusters produced by saturating concentrations of FKBP12 and 12.6 were not significantly different from each other, but were significantly smaller compared to control (p<0.01). Cells exposed to the phosphorylation cocktail, both with and without immunophilins, had significantly larger clusters than the control cells (p<0.01). Importantly, these results show that hyperphosphorylation takes precedence over excess FKBP12 or 12.6.

To quantify the dSTORM images, we used the higher resolution tomographic images to guide our analysis. We first drew alpha shapes around the tetramer arrays in the tomograms (Figure 6D) to calculate the median tetramer coverage/cluster for each of the experimental groups giving the percentage of the area covered by the tetramers; the values were different for the different experimental groups (Table 3). This is a measure of the group behavior of the array and is not comparable to the NND. The phosphorylated value was slightly larger than the control because ordering the array reduced the area of the alpha shape so that the tetramers occupied slightly more of the area defined. In the immunophilin treated cells the receptors clustered together reducing the alpha shape area and increasing the proportion covered by the tetramers. Phosphorylation of the immunophilin treated cells had the opposite effect – separating the tetramers, increasing the alpha shape area and decreasing the proportion covered. For each of the dSTORM images we multiplied the cluster area by coverage/cluster giving the area covered by the tetramers. When this was divided by the area occupied by a single tetramer, 729 nm (Van Petegem, 2015), the result was the number of tetramers per cluster in the dSTORM images (Table 3). The pooled values for each experimental group were plotted as a CDF, Figure 6E, where the steps in the curves reflected rounding to the nearest integer. The values shown, from two to ~100, are broadly consistent with published estimates (Baddeley et al., 2009; Hayashi et al., 2009). The k-sample Anderson Darling test showed that each curve was sampled from a different population (p<0.01). These quantitative results support the visual impression; the immunophilins fractured the clusters into smaller units with fewer members. While phosphorylation, either with or without the immunophilins present, produced larger clusters with more members.

Median mean cluster areas and number of clusters examined. The alpha shapes and areas were generated using CGAL.

Median tetramer coverage per cluster recorded from the indicated number of tomograms using the RyR_fit program (Source code 2). Mean and median number of tetramers per cluster.

We have provided compelling evidence that inhibitory factors, such as the immunophilins, are associated with the RyR2 tetramers moving into a largely side-by-side configuration and decreasing the cluster size. In direct contrast, excitation due to phosphorylation, even in the presence of the inhibitory immunophilins, is associated with the tetramers moving apart into a checkerboard-like configuration and increasing the cluster size. These findings show that our understanding of RyR2 function has been incomplete and a full understanding should incorporate these dynamic effects. The results confirm and extend our earlier findings, and demonstrate that normal operation of the mammalian ventricular dyad involves movement and rearrangement of the tetramers and changes in the cluster size.

Phosphorylation

Phosphorylation of RyR2 has been reported to reduce the affinity for FKBPs resulting in their dissociation and an increase in the tetramers’ open probability and spark frequency; a phenomenon allegedly responsible for the increased diastolic Ca²⁺ leak in heart failure (Marx et al., 2000). The finding is controversial as many laboratories have been unable to reproduce the results, and predictions from the hypothesis have failed to materialize (Xiao et al., 2004; Guo et al., 2010; Alvarado et al., 2017; Xiao et al., 2007).

Multiple lines of evidence support the conclusion that the immunophilins did not dissociate from RyR2. If they had, then results obtained from cells treated with the phosphorylation cocktail and cells treated with either FKBP12 or FKBP12.6 plus the phosphorylation cocktail would be equivalent. This was not the case in any of our experiments. The Ca²⁺ sparks had significant differences in their frequency, FDHM, FWHM and mass (Figure 1 and Figure 1—source data 1). The nearest neighbour distances between the tetramers were not equivalent (Figure 5 and Figure 5—source data 1). In our dSTORM images there were significant differences in the areas of the clusters (Figure 6C, Figure 6—source data 1 and Table 2) as well as in the number of tetramers per cluster (Figure 6E, Figure 6—source data 2 and Table 3). Western blots showed an insignificant change in the intensity of the immunophilin bands following phosphorylation relative to their non-phosphorylated counterparts (Figure 7 and Figure 7—source data 1).

Figure 7

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Figure 7—source data 1

Statistical Analysis of the Western Blots.

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These results are consistent with many others who have concluded that phosphorylation does not dissociate the immunophilins from RyR2. Co-immunoprecipitation experiments have demonstrated the continued binding of immunophilins to RyR2 despite phosphorylation by either CaMKII (Kohlhaas et al., 2006) or PKA (Xiao et al., 2004). Experiments with fluorescent FKBP12 and FKBP12.6 found no change in the K_d, B_max or binding kinetics of the immunophilins in response to PKA activation (Guo et al., 2010). Finally, Western blots found no significant decrease in the intensity of the immunophilin bands following phosphorylation (Jiang et al., 2002; Stange et al., 2003; Xiao et al., 2005; Xiao et al., 2006).

Exposing immunophilin-treated tetramers to cell-wide phosphorylation largely reversed the immunophilins’ effects, despite their remaining bound. The effects of phosphorylation plus FKBPs were much closer to phosphorylation alone than to FKBPs alone, making it likely that phosphorylation predominates over the FKBPs in determining the tetramers’ function and that the FKBPs, phosphorylation and the combination of the two result in different conformations of RyR2. It is notable that phosphorylation was significantly less effective in reversing FKBP12’s effects on spark frequency, FDHM and spark mass compared to FKBP12.6, despite its significantly lower affinity for RyR2 (Figure 1B). Ca²⁺ sparks in cells treated with FKBP12.6 plus the phosphorylation cocktail were significantly more frequent than in any other treatment group (Figure 1A and B) and when combined with the increase in spark mass, these cells would have the largest diastolic Ca²⁺ release.

The experiments used ventricular myocytes from adult rats. Animal handling was done in accordance with the guidelines of the Canadian Council on Animal Care and approved by the animal research committee of the University of British Columbia (UBC). All chemicals were purchased from Sigma-Aldrich (Oakville, ON) unless otherwise stated.

Correlation microscopy

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Isolation of rat ventricular myocytes was performed as previously described (Rodrigues and Severson, 1997). Isolated, permeabilized myocytes were exposed to one of (10 min exposure each): a) buffer (as control), b) phosphorylation cocktail, c) 10 µmol/L FKBP12.6, d) 10 µmol/L FKBP12.6, then the phosphorylation cocktail, e) 10 µmol/L FKBP12, f) 10 µmol/L FKBP12, then the phosphorylation cocktail. The phosphorylation cocktail was composed of (µmol/L): 10 c-AMP, 10 IBMX, 10 Okadaic acid and 0.5 Calycullin A (pH7.2; adjusted with KOH) (Asghari et al., 2014). Ca²⁺ sparks were recorded on a Zeiss LSM 700 confocal microscope in line scan mode (63x objective) using 30 µmol/L Fluo-4 (Invitrogen). Sparks were analyzed using SparkMaster (Picht et al., 2007). Ca²⁺ spark frequency was corrected for SR Ca²⁺ content to isolate effects on the array from those due to changes in SR filling (Li et al., 2002). Spark duration and spread were recorded as full duration half maximum (FDHM; ms) and full width half maximum (FWHM; μm). Cells were then fixed, in place, and prepared for electron microscopy. A gridded coverslip enabled tomograms to be acquired from cells whose Ca²⁺ sparks had been recorded. Groups of cells prepared and treated identically were exposed to 20 mmol/L caffeine to assess SR Ca²⁺ content. The experimental pipeline for this novel correlative light and electron microscopy technique is detailed in Figure 8.

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Measurements of FKBP12 and FKBP12.6 concentration in rat myocytes have produced wildly different results: (i) 1.8 FKBP12.6/RyR2 (45% occupancy) and no FKBP12 (Zissimopoulos et al., 2012); (ii) At most 20% of the subunits occupied by FKBP 12.6 and many of the rest by FKBP12 (Guo et al., 2010); (iii) FKBP12/RyR2 = 1.75 and FKBP12.6/RyR2 = 3.5 (Jeyakumar et al., 2001). Given these uncertainties we used high concentrations, 10 μmol/L of FKBP12 and FKBP12.6, to displace the endogenous proteins and to ensure that all four binding sites on each RyR2 were occupied with the desired isoform. We avoided rapamycin because recent experiments demonstrated that it may not displace FKBP12, only partially displace FKBP12.6, and may have direct effects on RyR2 by depressing both the Ca²⁺ spark frequency and spread (Gonano and Jones, 2017; Guo et al., 2010; Richardson et al., 2017).

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided where required.

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Author details

1. Parisa Asghari

   Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, Canada

   Contribution

   Conceptualization, Investigation, Formal analysis, Supervision, Validation, Visualization, Methodology, Project administration

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2285-3242

2. David RL Scriven

   Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, Canada

   Contribution

   Data curation, Conceptualization, Software, Formal analysis, Visualization, Methodology, Investigation, Validation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1828-1405

3. Myles Ng

   Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, Canada

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

4. Pankaj Panwar

   Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada

   Contribution

   Resources

   Competing interests

   No competing interests declared

5. Keng C Chou

   Department of Chemistry, University of British Columbia, Vancouver, Canada

   Contribution

   Resources, Software

   Competing interests

   No competing interests declared

6. Filip van Petegem

   Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada

   Contribution

   Resources

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2728-8537

7. Edwin DW Moore

   Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, Canada

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Validation, Project administration

   For correspondence

   edwin.moore@ubc.ca

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7519-5592

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