Role of FK506-binding protein in Ca2+ spark regulation

Science Bulletin xxx (2017) xxx–xxx

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Article

Role of FK506-binding protein in Ca2+ spark regulation Yan-Ting Zhao a,1, Yun-Bo Guo a,1, Xue-Xin Fan a,1, Hua-Qian Yang a, Peng Zhou a, Zheng Chen b, Qi Yuan b, Haihong Ye c, Guang-Ju Ji b, Shi-Qiang Wang a,⇑ a

State Key Laboratory of Membrane Biology, College of Life Sciences, Peking University, Beijing 100871, China National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China c School of Basic Medical Sciences, Beijing Institute for Brain Disorders Center of Schizophrenia, Capital Medical University, Beijing 100069, China b

a r t i c l e

i n f o

Article history: Available online xxxx Keywords: Ca2+ spark FK506-binding protein Ryanodine receptor Intracellular calcium Excitation-contraction coupling

a b s t r a c t The elementary Ca2+ release events, Ca2+ sparks, has been found for a quarter of century. However, the molecular regulation of the spark generator, the ryanodine receptor (RyR) on the sarcoplasmic reticulum, remains obscure. Although each subunit of the RyR homotetramer has a site for FK506-binding protein (FKBP), the role of FKBPs in modifying RyR Ca2+ sparks has been debated for long. One of the reasons behind the controversy is that most previous studies detect spontaneous sparks, where the mixture with out-of-focus events and local wavelets prevents an accurate characterization of Ca2+ sparks. In the present study, we detected Ca2+ sparks triggered by single L-type Ca2+ channels (LCCs) under loose-seal patch clamp conditions in FK506-treated or FKBP12.6 knockout cardiomyocytes. We found that FKBP dissociation both by FK506 and by rapamycin decreased the Ca2+ spark amplitude in ventricular cardiomyocytes. This change was neither due to decreased releasable Ca2+ in the sarcoplasmic reticulum, nor explained by changed RyR sensitivity. Actually FK506 increased the LCC-RyR coupling probability and curtailed the latency for an LCC to trigger a RyR Ca2+ spark. FKBP12.6 knockout had similar effects as FK506/rapamycin treatment, indicating that the decreased spark amplitude was attributable to the dissociation of FKBP12.6 rather than FKBP12. We also explained how decreased amplitude of spontaneous sparks after FKBP dissociation sometimes appears to be increased or unchanged due to inappropriate data processing. Our results provided firm evidence that without the inter-RyR coordination by functional FKBP12.6, the RyR recruitment during a Ca2+ spark would be compromised despite the sensitization of individual RyRs. Ó 2017 Science China Press. Published by Elsevier B.V. and Science China Press. All rights reserved.

1. Introduction Ca2+ is a universal and versatile intracellular messenger regulating most biological processes, from gene expression to protein modification, and from cell proliferation to apoptosis [1–4]. Dysregulation of intracellular Ca2+ underlies a variety of severe diseases, including cancer, neural degeneration and cardiovascular diseases [5–10]. In heart cells, the Ca2+-induced Ca2+ release [11] between L-type Ca2+ channels (LCCs) in the cell membrane/ T-tubules and ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) determines the pace and strength of myocardial contraction [12,13]. FK506-binding proteins (FKBPs), including FKBP12 and FKBP12.6, are RyR accessory proteins that bind the immunosuppressant drug FK506 [14,15]. Although recent struc⇑ Corresponding author. 1

E-mail address: [email protected] (S.-Q. Wang). Authors contributed equally to this work.

tural studies confirmed that there is an FKBP-binding site on each subunit of the RyR homotetramer [16–19], the role of FKBPs in RyR function has been highly controversial for the past two decades [15,20–23]. One of the problems keeping the role of FKBP12.6 from clarification has been that RyRs are intracellular channels inaccessible to direct electrophysiological measurements. Although lipid bilayers have been widely used to study the interaction between FKBPs and RyRs [20,21], the experimental settings vary greatly from lab to lab. For example, an experiment supporting FKBP12.6modulation of RyR was done in the presence of Mg2+ and 50 mM trans(luminal)-side Ca2+ [20], while some contradicting data were collected with symmetrical high concentration of K+ without Mg2+ [21]. Despite the differences in ionic composition, lipid bilayer experiments cannot reconstruct the native environment of interacting proteins, such as calsequestrin, junction and triadin. Therefore, the results in lipid bilayers may not necessarily represent the in situ effect of FKBP12.6.

https://doi.org/10.1016/j.scib.2017.09.009 2095-9273/Ó 2017 Science China Press. Published by Elsevier B.V. and Science China Press. All rights reserved.

Please cite this article in press as: Zhao Y-T et al. Role of FK506-binding protein in Ca2+ spark regulation. Sci Bull (2017), https://doi.org/10.1016/j. scib.2017.09.009

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In intact cardiomyocytes, studies in FKBP12.6-knock mice showed that Ca2+ spark amplitude is either increased [21] or unchanged [24]. We noticed that, in reports showing increased Ca2+ spark amplitude, spark duration and spatial width were also increased [25,26]. Given that the RyR Ca2+ sensitivity was increased after FKBP12.6 knockout [23], FKBP12.6-null cells display overlapping sparks and local wavelets [25,26], which caused overestimation of Ca2+ spark amplitude, duration and spatial size. Also, the statistics of spontaneous Ca2+ sparks involves a mixture of in-focus and off-focus Ca2+ release events, making it difficult to reliably quantify RyR properties. In the present study, we studied the in situ role of FKBPs in regulating Ca2+ sparks with a more rigorous experimental design. We used loose-seal patch clamp combined with confocal Ca2+ imaging to detect Ca2+ sparks triggered by their native Ca2+ activators from a single LCC [27] in intact cardiomyocytes. Using this approach, which allows quantitative analysis of in-focus Ca2+ sparks, we found that both FK506/rapamycin treatment and FKBP12.6 knockout actually decreased, rather than increased, the amplitude of Ca2+ sparks despite the sensitization of RyR activation. This finding rectified previous intuition and unmasked the exact role of FKBP12.6 in coordinating the activation of multiple RyRs during a Ca2+ spark. 2. Materials and methods 2.1. Isolation of ventricular cardiomyocyte Single ventricular cardiomyocytes were isolated from threemonth-old wild-type and FKBP12.6-knockout 129 mice [25] or from adult Sprague-Dawley rats, and were loaded with fluo-4AM as previously described [23,28]. 2.2. Loose-seal patch clamp The loose-seal patch clamp was made at room temperature (
25 °C) using an EPC7 amplifier. Cells were bathed in extracellular solution containing (in mM) 135 NaCl, 4 KCl, 1 or 2 CaCl2, 1 MgCl2, 10 Hepes, 1.2 NaH2PO42H2O, 10 glucose, pH adjusted to 7.35 with NaOH. A glass pipette with a resistance (Rp) of 4–6 MX was filled with (in mM) 120 TEACl, 10 Hepes, 0.01 TTX, 20 CaCl2 and 0.01 FPL64176, pH adjusted to 7.4 with TEAOH, and was gently pressed onto the cell surface to form a low-resistance seal (Rs = 20–30 MX). The patch membrane voltage (VP) was determined based on the resting potential (RP) and command voltage (Vcom) by VP = RP  Vcom(Rs  Rp)/Rs. 2+

2.3. Confocal Ca

Research, Faversham, UK). A solution containing 10 mM caffeine (with 0 Na+ and 0 Ca2+ to block Na/Ca exchange, Na+ replaced by meglumine) was rapidly applied onto the cells to measure the SR Ca2+ load. Cytosolic [Ca2+] change was calculated as described [28].

2.5. Data analysis and statistics All data are expressed as mean ± SE unless otherwise indicated. Student’s t test or the v2 test were applied for unpaired samples to determine statistical differences. Curve fitting was performed using Sigmaplot software. Fitted data were compared using the u test. P < 0.05 was considered statistically significant.

3. Results 3.1. Effect of FK506 and rapamycin on triggered Ca2+ sparks in rat ventricular cardiomyocytes In order to quantify the effect of FKBP on the in situ RyR response to LCC Ca2+ influx, we recorded in-focus Ca2+ sparks by confocal imaging under the loose-seal patch clamp condition in ventricular cardiomyocytes from rats (Fig. 1a). Measurement of the Ca2+ sparks showed that 30 lmol/L FK506 decreased their amplitude (Fig. 1b), slightly curtailed their time-to-peak (Fig. 1c) but did not change their full width at half maximum (FWHM, Fig. 1d). To determine whether the decreased Ca2+ spark amplitude had to do with SR Ca2+ content, we measured the caffeine-induced Ca2+ transient in cardiomyocytes loaded with the Ca2+ probe indo-1 (Fig. 1e). The caffeine-induced Ca2+ transient exhibited the same amplitude in Control and FK506-treated groups (Fig. 1f), indicating the FK506 did not alter the SR Ca2+ load. Therefore, the decreased spark amplitude was not due to releasable Ca2+ content in the SR. FK506 has targets other than FKPBs, such as calcineurin [29]. We therefore tested the effect of 10 lmol/L rapamycin, which also dissociate FKBPs from RyRs [30]. We found that rapamycin also decreased the amplitude of Ca2+ sparks (Fig. 2a) without altering the time-to-peak (Fig. 2b) and FWHM (Fig. 2c), confirming that FKBP dissociation decreased spark amplitude.

3.2. Effect of FK506 on RyR activation probability in rat ventricular cardiomyocytes

imaging

Ca2+ imaging was recorded with a Zeiss LSM-510 inverted confocal microscope (Carl Zeiss) with 488 nm laser excitation and a 40  1.3 N.A. oil-immersion objective. All images were acquired along the long axis of cells in line-scan mode at a sampling rate of 0.768 ms/line for sparklet-spark experiments and 15.36 ms/line for other experiments. Local [Ca2+] was determined by the formula [Ca2+] = kdR/(kd/C0 + 1  R), where R is the relative fluo-4 fluorescence normalized by the resting level, kd  1.1 lmol/L is the apparent dissociation constant of fluo-4, and C0  100 nM is the resting Ca2+ concentration. 2.4. Indo-1-based Ca2+ measurement Rat cardiomyocytes loaded with indo-1AM (10 lM, Invitrogen) were excited by ultraviolet light. Indo-1 fluorescence was recorded by a Luca CCD camera (Andor, South Windsor, CT) at 405 and 485 nm separated by an Optosplit II emission splitter (Cairn

To determine whether the RyR responsivity is altered in the presence of FK506, we measured the hit index for a single LCC Ca2+ sparklet to trigger a Ca2+ spark. To visualize the onset of Ca2+ trigger from a single LCC, we included in the pipette electrode 20 mM Ca2+ and 10 lM FPL64176, an LCC agonist that prompts long openings [31] without altering the amplitude and kinetics of Ca2+ sparks [27,28]. When the patched membrane was depolarized from resting potential (70 mV) to 10 mV, line-scan imaging focused at the pipette tip detected two populations of local Ca2+ events (Fig. 3a): ryanodine-sensitive Ca2+ sparks from RyRs with a sharper takeoff and higher amplitude, and nifedipine-sensitive Ca2+ sparklets from LCCs with a blunter takeoff and lower amplitude [28,32]. Hit index is defined as the percentage of Ca2+ sparks that are apparently triggered by the first Ca2+ sparklet after depolarization [28,32]. We found that the hit index was significantly higher in FK506-treated cells (Fig. 3b). This result suggested that FK506 increased the probability of RyR activation.

Please cite this article in press as: Zhao Y-T et al. Role of FK506-binding protein in Ca2+ spark regulation. Sci Bull (2017), https://doi.org/10.1016/j. scib.2017.09.009

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Fig. 1. (Color online) Effect of FK506 on Ca2+ spark properties measured under the loose-patch confocal imaging configuration in rat cardiomyocytes. (a) Representative recordings showing that 50 mV depolarization from resting potential (RP, upper panels) triggered Ca2+ sparks (middle panels for images and lower panels for time profiles). (b) Amplitude, (c) time-to-peak and (d) full-width at half-maximum (FWHM) of Ca2+ sparks in Control and FK506-treated rat cardiomyocytes. Data were averages of 39 triggered Ca2+ sparks from 6 rats in each group. (e), SR Ca2+ load measured by indo-1 fluorescence ratio, and expressed as the amplitude of caffeine-induced Ca2+ transient in cytosol. Data from 15 cells from 4 rats in each group. *P < 0.05 vs control.

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3.3. Effect of FK506 on RyR activation kinetics in rat ventricular cardiomyocytes If the RyRs were indeed sensitized, their response to Ca2+ triggers would be accelerated. We therefore measured the kinetics of LCC-RyR intermolecular coupling. The LCC-RyR coupling latency was gauged as

the delay (t) from the onset of an LCC Ca2+ sparklet to the takeoff of a triggered RyR Ca2+ spark (Fig. 4a). In both Control and FK506-treated cardiomyocytes, the LCC-RyR coupling latency had an exponential distribution (Fig. 4b). Fitting the distributions with the formula t

N ¼ N 0 e s

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Fig. 4. (Color online) Kinetics of LCC-RyR coupling in rat cardiomyocytes. (a) An example of a confocal image (upper) and its time profile (lower) illustrating the measurement of LCC-RyR coupling latency. (b) Distributions (bars) and exponential fittings (curves) of the coupling latency in control (upper) and FK506-treated (lower) cardiomyocytes. (c) Effect of FK506 on the time constants of LCC-RyR coupling. Data from 43 recordings in cells from 6 rats in each group. *P < 0.05 vs control.

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Fig. 5. (Color online) Ca2+ spark properties in FKBP12.6 knockout mice. (a) Representative recordings showing that 50 mV depolarization from resting potential (RP, upper panels) triggered Ca2+ sparklets and Ca2+ sparks (middle panels for images and lower panels for time profiles) in a probabilistic manner. Images are from wild-type (WTM) and FKBP12.6 knockout (FKM) mouse groups. (b) Histograms and logarithmic normal fittings (red line for WTM and blue line for FKM) of spark amplitude. The insert shows that the spark amplitude distribution was shifted to the low-amplitude side after FKBP12.6 knockout. (c) time-to-peak and (D) FWHM of Ca2+ sparks in WTM and FKM cardiomyocytes. Data were averages of 128 triggered Ca2+ sparks from 15 mice in each group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

where N is the number of observations and N0 is the N when t = 0, showed that the time constant (s) for RyRs to respond to LCC Ca2+ sparklets was significantly briefer in the FK506-treated group than in the Control group (Fig. 4c), indicating that FKBP dissociation accelerated the kinetics of RyR activation. 3.4. Effect of FKBP12.6 knockout on triggered Ca2+ sparks in mice ventricular cardiomyocytes FKBP12 and FKBP12.6 are both expressed in cardiomyocytes, but FKBP12.6 binds RyR with higher affinity [14,22,33]. In order to determine which FKBP induced the decrease of spark amplitude, we measured the amplitude of triggered Ca2+ sparks in wild-type (WTM) and FKBP12.6 knockout (FKM) mice (Fig. 5a). We found that FKBP12.6 knockout significantly decreased the spark amplitude (Fig. 5b) without altering the time-to-peak (Fig. 5c) and FWHM (Fig. 5d). To determine whether the decreased Ca2+ spark amplitude had to do with SR Ca2+ content, we measured the caffeine-induced Ca2+

transients (Fig. 6a), which was indeed lower in FKM cells (Fig. 6b, grey bar). To adjust the SR load to the same level as that in Wildtype mouse cardiomyocytes, we elevated the extracellular Ca2+ from 1 mM to 2 mM in FKM cells. With comparable SR Ca2+ load (Fig. 6b, black bar), the amplitude of Ca2+ sparks was still lower in FKM cells than in WTM cells (Fig. 6c), confirming that the amplitude of Ca2+ sparks was indeed lower without FKBP12.6. 3.5. Effect of FKBP12.6 knockout on spontaneous Ca2+ sparks in mice ventricular cardiomyocytes Our above results do not agree with previous studies, which have shown that the amplitude of spontaneous Ca2+ sparks is either increased [21] or unchanged [24] in FKBP12.6-knock mice. We therefore imaged spontaneous Ca2+ sparks (Fig. 7a) using the same line-scan settings as above experiments. Most sparks are solitary sparks. However, associated with increased spark frequency in FKBP12.6-knock cardiomyocytes (Fig. 7b), there were often overlapped sparks and burst of sparks, leading to local propagating

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Fig. 6. (Color online) The relationship between SR Ca2+ load and spark amplitude in mouse cardiomyocytes. (a) Typical line-scan images of caffeine-induced Ca2+ transients in WTM and FKM cells with 1 mM extracellular Ca2+ and in FKM cells with 2 mM extracellular Ca2+. (b) The SR Ca2+ load was compared as the amplitude of fluorescence normalized by the resting level (F/F0) in the above 3 groups. Data were from 44 cells from 6 mice in each group. *P < 0.05 vs WTM. (c) Amplitude of Ca2+ sparks in WTM and FKM cardiomyocytes with 1 mM extracellular Ca2+ and in FKM cells with 2 mM extracellular Ca2+. Data were averages of 128 triggered Ca2+ sparks from 15 mice in each group. *P < 0.05 vs WTM.

Ca2+ wavelets (Fig. 7c). When the overlapping sparks and wavelets, which do not represent the real properties of Ca2+ sparks, were excluded from statistics, the amplitude (Fig. 7d) and time-topeak (Fig. 7e) of spontaneous Ca2+ sparks did not show significant difference between Wild-type and FKBP12.6-knockout cells. Base on the discussion below, measurements of spontaneous sparks did not reflect RyR behavior with sufficient accuracy.

4. Discussion Ca2+ sparks as the elementary intracellular Ca2+ signaling events has been found for a quarter of century [34]. However, the molecular mechanisms that regulate the generation of Ca2+ sparks remains elusive [35]. For example, it is still uncertain whether a few or a large number of RyRs are involved in the Ca2+ release during a Ca2+ spark [36,37]. FK506-binding proteins, including FKBP12 and FKBP12.6, are accessory proteins of RyRs [14,15,38]. In heart cells, FKBP12.6 binds RyRs with higher affinity than FKBP12 [14,22,33]. However, whether FKBPs play a role in modulating RyR function and spark generation has been highly controversial [15,20,21]. In the present study, by rigorous experimental designs, we provide firm evidence that both FKBP12.6 knockout and FK506/ rapamycin treatment decreased the amplitude of Ca2+ sparks in ventricular cardiomyocytes. This change was neither attributable to decreased releasable Ca2+ in the SR, nor explained by changes in the sensitivity of RyR activation. The decreased Ca2+ release in FKBP12.6-null cells suggested FKBP12.6 is important in coordinating the activation of multiple RyRs during a Ca2+ spark. The RyR is an intracellular ion channel inaccessible to direct electrophysiological measurements. Therefore, lipid bilayer recording has been a major tool to assess its molecular behavior. While lipid bilayer experiments provide detailed quantification of

RyR gating properties [14,39–41], the RyRs are reconstructed into artificial systems that are isolated from the physiological environment. As mentioned in the Introduction, the experimental settings differ among different laboratories leading to contradictory results. In the present study, we studied the effect of FK506/rapamycin on the in situ RyR behavior in intact rat heart cells. We showed that the probability for an LCC opening to activate RyR was increased after FK506/rapamycin treatment. In accordance, the response of RyRs to single LCC Ca2+ influx was accelerated by FK506/rapamycin. These results agreed well with our recent report that the RyR activation is sensitized after FKBP12.6 knockout [23]. In the literature, the amplitude of spontaneous Ca2+ sparks has been reported to be increased, decreased or unchanged after FKBP12.6 knockout or FK506-induced FKBP dissociation [21,24,39]. Intuitively, the sensitized RyR activation after FKBP dysfunction would support an increased spark amplitude because the sensitization would increase the response of RyRs to trigger signals. To the contrary, however, our results of trigged sparks showed that both FK506 and rapamycin treatments decreased the amplitude of Ca2+ sparks. In heart cells, FKBP12 is more abundant than FKBP12.6, although it has lower affinity in binging cardiac RyRs [33]. As the spark amplitude was changed similarly in FKBP12.6 knockout cells, the decreased spark amplitude should be attributable to FKBP12.6. FKBPs have been proposed for coordinating the allosteric change among adjacent RyRs [42,43]. FPBP12.6 is required for coupled gating of cardiac RyRs [42]. Model simulation have shown that the cooperative gating of multiple RyRs confers a steeper Ca2+ dependence for RyR activation compared with activation of rogue RyRs [44]. The Ca2+ release site can thus be made stable at resting state but highly responsive to trigger Ca2+ signals. Indeed, introducing cooperative gating among arrayed RyRs can effectively explain the diastolic low probability and systolic high probability of RyRs dur-

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Fig. 7. (Color online) Measurement of spontaneous Ca2+ sparks. (a) Representative recordings showing Ca2+ sparks occurring randomly in WTM (left) and FKM (right) groups. (b) Frequency of Ca2+ sparks in WTM and FKM cardiomyocytes. (c) Example of spontaneous Ca2+ releases in the form of overlapped Ca2+ sparks and wavelets. (d) Amplitude and (e) time-to-peak of spontaneous Ca2+ sparks. Data were from 73 cells from 10 mice in each group.

ing cardiac excitation-contraction coupling, which cannot otherwise be achieved concurrently by individual RyRs [45]. Based on the recently reported FKBP12-bound RyR1 structure [16,17], FKBP is inserted into the gap between JSol (handle) domain and SPRY triangle such that the BSol (HD2) domain can be anchored onto the SPRY2 domain of the neighboring protomer (for illustration see [22]). It is possible that this inter-protomer interaction, by stabilizing HD2, enabled the P2-P2 and HD2-HD2 interactions (Corresponding to Domains 6 and 8, respectively, in low-resolution RyR structure, [45,46]) between neighboring RyRs (Fig. 8a and C left), which might provide a mechanism for cooperative activation of multiple RyRs. In FKBP-dissociated RyR2, however, the HD2 domain became floating [19], and thus not possible to bridge inter-RyR cooperation (Fig. 8b and c right). In this scenario, the dissociation of FKBP12.6, by decoupling neighboring RyRs, leads to two consequences: 1) desynchronized activation of RyRs and 2) decreased number of activated RyRs and thus decreased Ca2+ release flux. Both of these consequences would predict decreased amplitude of Ca2+ sparks. Therefore, the decreased spark amplitude after FK506 treatment and FKBP12.6 knockout revealed the important role of FKBP12.6 in coordinating the activation of multiple RyRs during Ca2+ spark generation and excitation-contraction coupling. Then, why the decreased amplitude is not detected in spontaneous Ca2+ sparks? To answer this question, we imaged spontaneous Ca2+ sparks. However, we did not find significant difference, either, in spark amplitude between Wild-type and FKBP12.6-knockout cells (Fig. 7). However, during the data processing, we realized three problems that prevent the accurate esti-

mation of Ca2+ sparks: 1) overlapping sparks. Due to the sensitization of RyRs after FKBP12.6 dissociation, the frequency of spontaneous Ca2+ sparks is increased by several folds. This greatly increased the chance of ‘‘apparently overlapping” sparks, in which sparks from different focal levels summed up resulting in over-estimation of spark amplitude. 2) Local wavelets. Sensitization of RyRs and increase of spark frequency also promotes local Ca2+-induced Ca2+ release, leading to local wavelets [25,26] that looks like Ca2+ sparks but have wider size, longer duration and higher amplitude. If these wavelets are detected as Ca2+ sparks, the spark amplitude, FWHM and time-to-peak would be overestimated. We noticed that, in reports showing increased Ca2+ spark amplitude after FKBP12.6 dissociation, spark duration and FWHM were also increased [25]. 3) Threshold amplitude for spark detection. In the measurement of spontaneous Ca2+ sparks, most sparks are out-of-focus events. We need to set a standard for spark detection because many out-of-focus events are too weak to be discriminated from noise. Under the same threshold standard, low-amplitude sparks are more likely to be rejected, which will lead to over-estimation of spark amplitude in the smaller group. Indeed, even in triggered sparks, if we elevate the detection threshold to 50 nM, the difference between Wild-type and FKBP12.6knockout groups was eliminated (Fig. S1). In our detection of triggered sparks, all the events are in-focus events, and even small sparks are easily discriminated from the relatively clean background. Therefore, triggering Ca2+ sparks by loose-patch imaging provides a unique technique to study Ca2+ sparks and in situ RyR behavior with currently the best accuracy.

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Fig. 8. Illustration of possible role of FKBP in coordinating the allostery among RyRs (a) An update of the schematic model of RyR1 array [46] generated using the highresolution structural data of RyR1-FKBP12 complex [47]. The blue line denote the section positioning of the models in C. (b) The structure of FKBP-null RyR2 generated based on the original data from [18], showing that the deformation of FKBP-null RyR2 relative to FKBP12-bound RyR1 destroyed the structural basis for inter-RyR coordination. (c) A hypothetic scheme based on previous models [46] and recent structural information [18,47]. The cartoons illustrate that the putative P2-P2 and HD2-HD2 interactions provides a possible mechanism for coordinating the allosteric activity among RyRs in FKBP-bound RyRs. Without FKBP, however, the interactions become disabled due to shifted P2 domains and floating HD2 domains. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Conflict of interest The authors declare that they have no conflict of interest.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.scib.2017.09.009.

Acknowledgments This work was supported by the National Research and Development Program of China (2016YFA0500401), National Natural Science Foundation of China (31630035, 31571486, 81370203, 81461148026, 31271228 and 31327901) and the Project of Beijing Municipal Science and Technology Commission (Z141100000214006).

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