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Ca2+ release induced by cADP-ribose is mediated by FKBP12.6 proteins in mouse bladder smooth muscle
Cell Calcium 47 (2010) 449–457
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Ca2+ release induced by cADP-ribose is mediated by FKBP12.6 proteins in mouse bladder smooth muscle Ji Zheng a,1 , Bi Wenzhi b,1 , Lin Miao c,1 , Yumin Hao c , Xu Zhang c , Wenxuan Yin c , Jinhong Pan a , Zengqiang Yuan c,∗ , Bo Song a,∗ , Guangju Ji c,d,∗∗ a
Urological Surgery Research Institute, Southwest Hospital, Third Military Medical University, Gao Tanyan Rd. 30, Chongqing 400038, China Clinical Division of Surgery, General Hospital of PLA, China National Laboratory of Biomacromolecules, Institute of Biophysics of Chinese Academy of Sciences, 15 Datun Rd, Beijing 100101, China d Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853 USA b c
a r t i c l e
i n f o
Article history: Received 3 July 2009 Received in revised form 22 March 2010 Accepted 26 March 2010 Available online 7 May 2010 Keywords: cADP ribose Ca2+ spark FKBP12.6 proteins Smooth muscle
a b s t r a c t We examined the role and molecular mechanism of cADPR action on Ca2+ spark properties in mouse bladder smooth muscle. Dialysis of cADPR with patch pipettes increased frequency and amplitude of spontaneous transient out currents (STOCs) to 6.1 ± 0.87 currents/min from 1.2 ± 0.36 currents/min (control) and to 179.8 ± 48.7 pA from 36.4 ± 22.6 pA (control), respectively, in wildtype (WT) cells, and the effects of cADPR on STOCs were significantly blocked by JVT-591, a RYR2 stabilizer. In contrast, no significant changes were observed in FKBP12.6 null cells. Further studies indicated that Ca2+ spark properties were altered by cADPR in WT but not FKBP12.6 null cells, namely, Ca2+ spark frequency was increased by about 3.4-fold, peak Ca2+ (F/F0) increased to 1.72 ± 0.57 from 1.56 ± 0.13, size increased to 2.86 ± 0.26 M from 1.92 ± 0.14 M, rise time and half-time decay were prolonged 1.6-fold and 2.3-fold, respectively, in WT cells. Furthermore, in the presence of thapsigargin cADPR still altered Ca2+ spark properties, and cADPR increased F/F0 without affecting Ca2+ spark decay time in voltage clamping cells. Dissociation studies demonstrated that application of cADPR resulted in significant removal of FKBP12.6 proteins from sarcoplasmic reticulum (SR) microsomes, and that treatment of the RyR2 immunoprecipitation complexes with cADPR or FK506 disrupted the interaction between RyR2 and FKBP12.6. Finally, cADPR altered SR Ca2+ load in WT myocytes but not in FKBP12.6-null myocytes. Taken together, these results suggest that Ca2+ release induced by cADPR is mediated by FKBP12.6 proteins in mouse bladder smooth muscle. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Ca2+ release from intracellular Ca2+ stores (the endoplasmic/sarcoplasmic reticulum (ER/SR)) is one of the key signal transduction mechanisms that play a pivotal role in the regulation of numerous cellular functions [1]. In smooth myocytes, Ca2+ release from intracellular Ca2+ stores to the cytoplasm is controlled by ryanodine Ca2+ release receptors subtype 2 (RYR2), and occurs in the form of spontaneous SR Ca2+ release events, or Ca2+ sparks [2]. Ca2+ release is triggered by the influx of Ca2+ through sarcolemmal ion channels, often termed Ca2+ induced Ca2+ release
∗ Corresponding authors. ∗∗ Corresponding author at: Institute of Biophysics, Chinese Academy of Sciences, Beijing Datun Rd. 15, Beijing 100101, China. Tel.: +86 10 64889873; fax: +86 10 64846720. E-mail addresses: [email protected] (Z. Yuan), [email protected] (B. Song), [email protected] (G. Ji). 1 These authors contributed equally to this manuscript. 0143-4160/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.ceca.2010.03.006
(CICR) [3]. FKBP12.6, a 12.6 kDa protein, specifically binds with and regulates RYR2 in cardiomyocytes and smooth muscle [4,5,6]. It has been reported that FKBP12.6 stabilizes the closed state of the RyR2. The amount of FKBP12.6 bound to RYR2 is decreased in heart failure and consequently leads to instability of the FKBP12.6RYR2 complex and results in SR Ca2+ leakage [7,8]. Patch clamp and confocal microscopy combined studies have demonstrated that both FKBP12.6 deficient [9,6] and FKBP12.6 overexpressing animals exhibit a significant alteration in Ca2+ spark properties [10,11]. These findings strongly suggest that FKBP12.6 is pivotal in regulating calcium release from the SR, possibly via its association with RyR2 cADPR, a Ca2+ mobilizer, has been shown to be a potential regulator of RYR activity in myocytes despite the fact that the role and mechanisms of cADPR regulation of Ca2+ release remain unclear and very controversial. For instance, some studies suggest that cADPR specifically binds to FKBP12.6 and regulates Ca2+ release [12–14,9,15]. However, some studies are inconsistent with the above observation. It has been demonstrated that cADPR does not
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Fig. 1. cADPR regulates STOCs in wildtype but not in FKBP12.6 null smooth myocytes. Dialysis of WT myocytes with cADPR (5 M) causes increases in both frequency and amplitude of spontaneous transient outward currents and this effect of cADPR was abolished by pre-treatment of the cells with JTV-519, a RYR complex stabilizer (A, n = 7, P < 0.05); in FKBP12.6 deleted myocytes, dialysis of cADPR failed to alter the properties of STOCs (B, n = 9, P > 0.05). (C and D) Summary data for frequency and amplitude of STOCs in WT and FKBP12.6 KO myocytes.
alter Ca2+ release in cardiac and skeletal myocytes [16,17,18]. As for smooth muscles, although cADPR increases Ca2+ release in pulmonary [19], vascular [20] and tracheal myocytes [1], it fails to cause Ca2+ release in other types of smooth muscle, including aortic, vas deferens, coronary, bronchial, cerebral arterial, and intestinal types [21]. Moreover, SERCA may also be involved in the action of cADPR on Ca2+ release [22]. In the present study we sought to examine the effect and mechanism of cADPR on Ca2+ release in mouse bladder smooth muscle. We have used FKBP12.6 null mice to test directly if cADPR action on Ca2+ sparks is through FKBP12.6 disassociation from the RYR2 complex. We found that cADPR significantly alters the properties of STOCs and Ca2+ sparks, and that the underlying mechanism of cADPR action is related to the dissociation of FKBP12.6 proteins from the RYR2 complex in mouse bladder smooth muscle.
2. Materials and methods 2.1. Cell isolation Mice were anesthetized and killed in accordance with an approved laboratory animal protocol and single bladder cells were prepared as previously described [6]. Briefly, the urinary bladder was removed, dissected in an ice-cold oxygenated Ca2+ -free solution containing (mM): 80 Na-glutamate, 55 NaCl, 6 KCl, 2 MgCl2 , 10 HEPES, and 10 glucose. The detrusor muscle was minced and incubated for 20 min at 37 ◦ C in dissociation solution containing 1 mg/ml dithioerythreitol, 1 mg/ml papain, and 1 mg/ml bovine serum albumin (Sigma–Aldrich), and the partially digested tissue
was then transferred to a solution containing 1 mg/ml collagenase type II (Worthington Biochemical), 1 mg/ml bovine serum albumin, and 100 M Ca2+ . The tissue was incubated for 10 min, and then triturated with a wide-bore Pasteur pipette, and passed through a 125-mm nylon mesh. Cells were concentrated by low-speed centrifugation, washed with fresh medium, resuspended, and stored at 4 ◦ C. All animal procedures described in this study were performed in adherence with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996), and with approval from the Institute of Biophysics Committee on Animal Care. 2.2. Measurement of Ca2+ fluorescence Myocytes were incubated with 10 M Fluo-4 AM (Molecular Probes) for 10 min at room temperature and transferred into a recording chamber mounted on an inverted microscope (IX81; Olympus). Cells were allowed to adhere to the bottom of the recording chamber for 15 min and were then perfused with extracellular solution (see above) for 40 min. Cells were excited with 488 nm light from a krypton/argon laser and linescan images were collected using a laser scanning confocal head (FV1000; Olympus) attached to an inverted microscope (IX81; Olympus). Linescans were obtained at an interval of 1.33 or 0.833 ms per line. Images were processed and analyzed using MATLAB 7.1 software (MathWorks). For linescan images, F0 was obtained by averaging the fluorescence for each pixel (× dimension) for a period preceding activation of a Ca2+ spark, and the fluorescence of all pixels (F) was divided by F0.
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the pellet was resuspended in buffer A. SR membranes were also incubated with 10 M FK506 as a positive control. The protein concentration was determined by the BCA method. 2.5. Western blot analysis Western blot analysis was performed as previously described [13]. Briefly, 30 g protein from each sample was separated by SDS-polyacrylamide gel electrophoresis using 15% gels. The resolved proteins were transferred to PVDF membranes (Millipore) at 100 mA for 70 min. The membranes were blocked for 2 h with Tris-buffered saline-Tween 20 (TBST) containing 5% BSA at room temperature. The blocked membranes were immunoblotted with polyclonal goat anti-mouse FKBP12.6 antibody (R&D systems) for 2 h at room temperature. After washing 3 times in TBST, the membranes were incubated in TBST containing 5% BSA and the secondary antibody conjugated to horseradish peroxidase. Final detection was performed using enhanced chemiluminescence detection solution 1 and 2 (1:1) (ECL; Millipore). 2.6. Co-immunoprecipitation assays 2+
Fig. 2. cADPR alters both STOCs and Ca sparks in WT myocytes. Cells were dialyzed with cADPR (5 M) through patch pipettes, and linescan images were collected after 6 min of whole cell configuration obtained. (A) A representative sample of simultaneous recordings of STOCs and Ca2+ release in a wildtype cell, indicating that with the dialysis of cADPR both STOCs and Ca2+ sparks were altered. The upper panel is the linescan image, and the lower panel is fluorescence profile (taken from the above image as indicated by arrow) of Ca2+ (red) and STOCs (black) recorded simultaneously. (B) A sample experiment of linescan images obtained from FKBP12.6-deficient myocyte. It is notable that there was no significant alternation in both STOCs and Ca2+ sparks with dialysis of cADPR. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
In SR Ca2+ load experiments, short applications of 10 mM caffeine (Sigma) by pressure ejection from a glass pipette were used to estimate SR Ca2+ content as previously described [23]. All experiments were carried out at room temperature. 2.3. Patch-clamp recording Membrane currents were recorded at room temperature using whole-cell voltage clamp configuration. To examine STOCs, cells were clamped at −20 mV. To test the voltage-clamping induced Ca2+ spark cells were clamped at −60 mV and depolarized to 0 mV with 200 ms duration. The intracellular solution was (mM): 130 KCl, 1.8 MgCl2 , 1.0 Na2ATP, 0.05 CaCl2 , 0.1 EGTA (pH 7.3). The extracellular solution was (mM): 137 NaCl, 5.4 KCl, 1.8 CaCl2 , 1.0 MgCl2 , 10 glucose, 10 HEPES and (pH 7.4). Currents were filtered at 500 Hz and digitized at 2 kHz. 2.4. Sarcoplasmic reticulum preparation Sarcoplasmic reticulum (SR) microsomes were prepared from mouse bladders as previously described [14]. Briefly, mouse bladders were removed and the inside and outside layers were carefully cleaned. The bladders were then cut into small pieces and homogenized in 3-(N-morpholino) propanesulfonic acid (MOPS) buffer containing 0.9% NaCl, 10 mM MOPS (pH 7.0) and phenylmethylsulfonyl fluoride (PMSF). The homogenate was centrifuged at 14,000 × g for 25 min at 4 ◦ C. The supernatant was then collected and centrifuged at 100,000 × g for 90 min at 4 ◦ C. The pellet, which contained SR membranes-enriched microsomes, was resuspended in a buffer containing 0.9% NaCl, 0.3 M sucrose, and PMSF. In order to determine if cADPR can induce FKBP12.6 release from RyRs, SR membranes were incubated with cADPR in buffer at 37 ◦ C for 15 min. The mixture was then centrifuged at 100,000 × g for 90 min at 4 ◦ C. The supernatant was stored at −80 ◦ C until required and
The total protein from mice bladder was prepared in a usual way. Briefly, bladder tissues dissected from mice were cut into small pieces and homogenized in RIPA lysis buffer containing (in mM) 50 Tris (pH 7.4), 150 NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS and phenylmethylsulfonyl fluoride (PMSF). The homogenate was centrifuged at 12,000 × g for 15 min at 4 ◦ C. The supernatant was then collected. In order to determine whether cADPR induces release of FKBP12.6 from the RyR2 complex, cADPR was incubated with the resultant supernatant at 37 ◦ C for 15 min. Then the supernatant was stored at −80 ◦ C until use. The resultant supernatant was also incubated with 10 M FK506, which was used as positive control. The concentration of protein was determined using BCA method. Samples were suspended in RIPA buffer and incubated with RYR2 antibody (CHEMICON, USA) O/N at 4 ◦ C (IgG alone was used as a negative control, data not shown). Protein G sepharose deads were added, incubated at 4 ◦ C for 1 h, and washed with RIPA for three times (each time for 10 min), then centrifuged and denatured for loading. Immunoblots were performed using antiFKBP12.6 (1:1000) and anti-RYR2 (1:1000). After three washes, membranes were incubated with peroxide-conjugated rabbit antigoat or -horse anti-rabbit IgG antiserum (1:4000) for 60 min at room temperature and developed with an enhanced chemiluminescence (ELC, Amersham). 2.7. Data analysis Image processing and data analysis were carried out with custom software written in MATLAB. Ca2+ sparks were counted manually and with a spark-counting software algorithm to verify the results objectively. To display the linescan images in a consistent way, F0 was obtained by averaging the fluorescence for each pixel (× dimension) for a period preceding activation of a Ca2+ spark, and the fluorescence of all pixels (F) was divided by F0 on a pixel by pixel basis. Linescans were then displayed with a colormap ranging from 0.5 to 3 F/F0. Profiles were constructed by averaging the pixels bisecting a Ca2+ spark for each time point in the scan. Ca2+ sparks and Ca2+ transients were fitted to a function with six free parameters (F0, start time, rise time, peak F/F0, halftime decay and final offset) and four free parameters (F0, rise time, peak F/F0, and half-time decay), respectively using a LevenbergMarquardt nonlinear least squares fitting routine. STOC rise time and peak current were measured by hand from the raw current recordings. Results are expressed as means ± SE where applicable.
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Fig. 3. cADPR induced alteration of Ca2+ spark properties. The experiments were conducted in smooth myocytes from wildtype mice, and cells were dialyzed with cADP5 (5 M). (A) Representative linescan images recorded from a wildtype mouse bladder cell. The upper panel shows the control (without cADPR), and the lower panel shows the cADPR treatment. (B) Linescan images obtained from FKBP12.6 knockout myocytes, demonstrating that in the presence (upper) and absence (lower) of cADPR the properties of Ca2+ sparks were not altered. (C) Summary data of Ca2+ spark properties. Ca2+ spark frequency increased in wildtype but not in FKBP12.6 null cells (a). Similarly, F/F0 (b), FWHM (c), rise time (d) and half-time decay (e) were significantly altered by cADPR only in wildtype cells. *P < 0.05, and **P < 0.01.
Data from the WT and FKBP12.6 −/− groups were compared by one-way, repeated measures of ANOVA and significant differences between groups were determined by the Student–Newman–Keuls (SNK) test for pairwise comparisons. 3. Results 3.1. cADPR regulates STOCs in wildtype but not in FKBP12.6 null mouse urinary bladder myocytes Our previous studies indicated that RYR2 Ca2+ release has a central functional role and that spontaneous and evoked Ca2+ sparks are closely associated with STOCs in smooth muscle [6]. The selective interaction of FKBP12.6 with RYR2 [12,5,9] allowed us to use FKBP12.6-deficient mice to determine whether cADPR regulates Ca2+ release by regulating the interaction of FKBP12.6 and RyR2. As STOCs are a convenient indicator of spontaneous SR Ca2+ release events [23,24,25,26], we initially examined the effect of cADPR on the frequency and amplitude of STOCs in urinary bladder myocytes from WT and FKBP12.6 −/− mice. On dialysis of 5 M cADPR into bladder smooth muscle cells via patch pipettes, both the frequency and amplitude of STOCs were significantly altered in WT cells. As
shown in Fig. 1, the frequency and amplitude of STOCs increased to 6.1 ± 0.87 currents/min from 1.2 ± 0.36 currents/min (control) and to 179.8 ± 48.7 pA from 36.4 ± 22.6 pA (control), respectively, in WT cells. To test if the effects of cADPR on STOCs were related to the dissociation of FKBP12.6 proteins from RYR complex, a RYR stabilizer, JVT-519, was used in the study. As shown in Fig. 1A (lower), in the presence of JVT-519 the stimulatory effects of cADPR on STOCs were abolished. In contrast to WT cells, the cells from FKBP12.6 null mice did not respond to cADPR stimulation (Fig. 1B–D), suggesting that the alteration of Ca2+ -release properties induced by cADPR was mediated by FKBP12.6 proteins in mouse bladder smooth muscle. It was notable that deletion of FKBP12.6 gave rise to significant increases in both STOC frequency and amplitude compared with WT myocytes (5.16 ± 2.03 currents/min vs. 1.2 ± 0.36 currents/min, 125 ± 50.6 pA vs. 36.4 ± 22.6 pA; Fig. 1C and D). 3.2. cADPR alters Ca2+ spark properties in smooth muscle To further test our hypothesis that alteration of STOCs induced by cADPR reflects changes in the underlying Ca2+ release events, we measured Ca2+ sparks in voltage-clamped urinary bladder myocytes from WT and FKBP12.6−/− mice. Fig. 2 is representative
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Fig. 4. Effects Ca2+ uptake pump inhibitor on cADPR action of Ca2+ sparks. Cells from WT mice were pre-treated by Thapsigargin (0.1 M, 10 min), and then were dialyzed with cADPR (5 M). (A) shows a sample of experiments demonstrating that the alteration of Ca2+ spark properties by thapsigargin was not significant (b), and that in the presence of thapsigargin cADPR still altered Ca2+ spark properties (d) compared to controls (a). (B) Summary data of Ca2+ spark properties. The increase in Ca2+ spark frequency (a), peak Ca2+ (b), size (c), and half-time decay (d) induced by dialysis of cADPR was not significantly affected by the presence of thapsigargin. The Ca2+ spark number measured (n) was 186; *P < 0.05, and **P < 0.01.
of simultaneous recordings of STOCs and Ca2+ sparks from the two cell types. On dialysis of wildtype cells with cADPR, both STOCs and Ca2+ sparks were markedly altered (n = 7). In contrast, dialysis of FKBP12.6 knockout cells with cADPR resulted in a negligible change in the properties of STOCs and Ca2+ sparks (n = 9). RYR Ca2+ release channels display stochastic gating behavior and consequently Ca2+ sparks vary substantially in frequency, amplitude, and duration. Next, we characterized the range of Ca2+ spark properties observed in control and FKBP12.6 null mouse bladder smooth muscle. Examples of individual Ca2+ sparks at high time resolution, and a summary of the number of Ca2+ sparks, the mean amplitude, spread (full width at half-maximum, FWHM), rise time and half-time decay for the different cell types are shown in Fig. 3. After dialysis of cADPR, marked alteration of Ca2+ spark properties was observed in WT cells (Fig. 3A) but not in FKBP12.6 KO cells (Fig. 3B). Ca2+ spark frequency was increased by about 3.4-fold (from 1.42 ± 0.43 to 4.78 ± 1.13, n = 8, P < 0.05) in wildtype but not in FKBP12.6 null cells (Fig. 3Ca). Similarly, on dialysis of wildtype cells with cADPR, peak Ca2+ increased to 1.72 ± 0.57 from 1.56 ± 0.13 (Fig. 3Cb), FWHM increased to 2.86 ± 0.26 M from 1.92 ± 0.14 M (Fig. 3Cc); rise time (Fig. 3Cd) and half-time decay (Fig. 3Ce) were prolonged 1.6-fold (35.6 ± 2.8 ms vs. 22.3 ± 1.9 ms) and 2.3-fold (68.7 ± 8.9 ms vs. 29.6 ± 3.2 ms), respectively. However, cADPR did not alter Ca2+ spark properties in FKBP12.6 knockout cells. Taken together, the present results showing that cADPR alters STOCs and Ca2+ spark properties in wildtype but not in FKPBP12.6-deficient myocytes suggesting that the action of cADPR is mediated by FKBP12.6 proteins. 3.3. Effect of cADPR on Ca2+ uptake in smooth muscle In the next experiments we tested whether SERCA activity is necessary for cADPR-induced increase in Ca2+ sparks, as it has been reported that cADPR stimulates SR Ca2+ uptake and potentiates Ca2+ release in cardiomyocytes [22]. Thapsigargin, a specific inhibitor of the SR Ca2+ pump was used in this study. As shown in Fig. 4, cADPR
significantly altered Ca2+ spark properties even in the presence of thapsigargin(0.1 M). A summary of Ca2+ spark properties for the three groups is shown in Fig. 4B. It has been known that activation of SERCA speeds up the re-uptake of Ca2+ into the SR, shortens the [Ca2+ ]i transient and reduces the Ca2+ spark duration [27]. To further test if SERCA activity involved in the action of cADPR on Ca2+ spark here we also carried out experiments in voltage clamping cells. As shown in Fig. 5, cADPR, as expected, increased the amplitude of Ca2+ spark significantly in 7 experiments (P < 0.05) but there was no significant effect on half time decay of Ca2+ spark, suggesting that the alteration of Ca2+ spark properties induced by cADPR was not relevant to the SERCA activity in mouse bladder smooth muscle. 3.4. FKBP12.6 mediates cADPR-induced Ca2+ release in smooth muscle To further confirm that the effect of cADPR on Ca2+ spark properties is directly dependent on the dissociation of FKBP12.6 from the RYR2 complex, Western blotting analysis was performed on SR microsomes from mouse bladder smooth muscle. As shown in Fig. 6A, the endogenous FKBP12.6 was released from SR microsomes by incubation with cADPR and practically all of the FKBP12.6 was bound to SR microsomes in the pellet. We also incubated the SR fraction with FK506 (10 M) as a positive control. On addition of FK506, the FKBP12.6 was all released from the SR fraction into the supernatant. Concomitantly, no corresponding FKBP12.6 band was detected in the pellet containing the SR microsomes (Fig. 6Aa). After treatment of SR microsomes with cADPR (5 M), FKBP12.6 was not detected in pellet microsomes but was recovered in the supernatant in four individual experiments (Fig. 6Ab). The percentage of FKBP12.6 dissociation (supernatant FKBP12.6/Total FKBP12.6) induced by cADPR was 97.2 ± 2.8% (n = 5). To further confirm our finding that cADPR dissociated FKBP12.6 from RYR complexes, co-immunoprecipitation and western blot analysis were carried out. Lysates of mouse SR treated with FK506
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Fig. 5. Ca2+ spark property alteration induced by cADPR was independent of SERCA. Experiments were conducted in voltage-clamped myocytes from wildtype mice. Cells were clamped at −60 mV and depolarized to a test potential of 0 mV with duration of 200 ms. (A) Linescan images collected in the presence (left) and absence of cADPR (right). Currents and Ca2+ fluorescence profiles were shown the bottom of the images. (B) Summary data of Ca2+ spark peak amplitude, rise time, and half time decay. It was notable that the half time decay of the Ca2+ spark was not significantly changed by cADPR.
or cADPR or vehicle control were immunoprecipitated with antiRyR2 antibody and blotted with the antibody to RyR2 or FKBP12.6. As shown in Fig. 6B, treatment of FK506 or cADPR disrupted the interaction between RyR2 and FKBP12.6, confirming our hypothesis that cADPR altered Ca2+ spark properties via dissociation of FKBP12.6 from RYR2 complexes. To demonstrate that the band of FKBP12.6 is absent in knockout mice immunoblot of mouse bladder homogenate from WT and KO mice was carried out and the results were shown in Fig. 6C. 3.5. cADPR alters SR Ca2+ content through FKBP12.6 To determine whether cADPR affected SR Ca2+ content, caffeine (10 mM) was used to estimate SR Ca2+ load in myocytes of WT and FKBP12.6-null mice before and after cADPR treatment. As shown in Fig. 7, cADPR significantly altered SR Ca2+ load in WT myocytes but not in FKBP12.6-null myocytes. Ca2+ transients induced by caffeine were markedly reduced in cADPR (5 M) pre-treated WT cells. However, cADPR pre-treatment did not markedly affect Ca2+ transients induced by caffeine in FKBP12.6 null myocytes though it did affect the load in some extent (Fig. 7Aa). Fig. 7Ab shows the averaged amplitude of Ca2+ transients (F/F0) induced by caffeine. The averaged F/F0 of 2.71 ± 0.09 in the WT control group, was markedly reduced to 2.03 ± 0.11 by pre-treatment with cADPR (Fig. 7Ba left, P < 0.01). There was no significant difference in F/F0 before and after treatment with cADPR in FKBP12.6 null cells (1.84 ± 0.12 vs. 1.73 ± 0.15, P > 0.05). Similarly, the rise time of the Ca2+ transient was also significantly reduced by cADPR in only the wildtype cells (from 117.9 ± 10.4 ms to 87.2 ± 14.2 ms). We noted that the halftime decay in all groups was not affected by cADPR treatment (1029.1 ± 98.2 ms vs. 1023.0 ± 95.5 ms in WT and 999.2 ± 135.1 ms vs. 1015.2 ± 106.4 ms in FKBP12.6 null cells (data not shown). Fig. 7B shows that thapsigargin decreased SR Ca2+ load and that
Fig. 6. Dissociation of FKBP12.6 from Sarcoplasmic reticulum microsomes by cADPR. (A) Western blotting analysis of FKBP12.6 binding and release. SR was incubated 30 min at 37 ◦ C in control conditions or in the presence of FK506 (positive control), cADPR, or 8-Br-cADPR (negative control). Released FKBP12.6 proteins appear in the supernatant (Ab), whereas FKBP12.6 bounds are remained in the pellets for control condition or in the presence of 8-Br-cADPR (Aa). (B) Extracts from SR of 6 wildtype mouse bladder smooth muscle were used to measure the expression of the indicated proteins and for immunoprecipitation of RYR2. The immunoprecipitates were analyzed for co-immunoprecipitation of RYR2 and FKBP12.6. Lysates of mouse bladder SR treated with FK506 or cADPR or vehicle control were immunoprecipitated with anti-RyR2 antibody and blotted with the antibody to RyR2 (top panel) or FKBP12.6 (middle panel). Total FKBP12.6 protein level was shown in the bottom panel. Treatment of FK506 or cADPR disrupted the interaction between RyR2 and FKBP12.6. These results are from three independent experiments. The molecular weight of this protein is about 12 kDa. (C) Immunoblot of mouse bladder homogenate from WT and KO mice. Lanes 1–3 are hela pcmv, hela pcmv FKBP12, and hela pcmv FKBP12.6, respectively; lanes 1 and 2 are mouse bladder homogenate from FKBP12.6 KO and FKBP12.6 WT mice.
in the presence of thapsigargin cADPR could not further significantly reduce the SR Ca2+ content though it did reduce the content in some extent, suggesting SERCA is not the main target of cADPR action. Additionally, we also examined the effects of thapsigargin in FKBP12.6 null cells, and the results indicated that thapsigargin significantly reduced the Ca2+ load and that cADPR, as expected, did not markedly affect the Ca2+ load (Fig. 7C). It was notable that the FKBP12.6 protein deletion caused a reduction in Ca2+ content in the SR though statistically not significant (Fig. 7), suggesting that a Ca2+ “leaky” state exists due to instability of RYRs [7,8].
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Fig. 7. Reduction of SR Ca2+ by cADPR in smooth myocytes. (Aa) Representative Ca2+ transients induced by 10 mM caffeine from linescan images recorded from wildtype cells (blue), wildtype cells pretreated with cADPR (purple), FKBP12.6 null cells (yellow), and FKBP12.6 null cells pretreated with cADPR (green). cADPR significantly reduced Ca2+ transients induced by caffeine in WT myocytes. (Ab) Summary data of Ca2+ release properties. It is notable that mutation of FKBP12.6 proteins caused a reduction in Ca2+ content. (Ba and b) Thapsigargin alone significantly reduced SR Ca2+ content, and cADPR failed to significantly alter SR Ca2+ in the presence of thapsigargin, however in wildtype cells. (Ca and b) Experiments carried out in FKBP12.6−/− cells. *P < 0.05 and **P < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Taken together, these data demonstrate that cADPR significantly reduces SR Ca2+ content by causing dissociation of FKBP12.6 from the RyR2 complexes. 4. Discussion It is commonly believed that FKBP12.6 is a subunit that stabilizes the closed state of RyR2 and plays an important regulatory role in Ca2+ release in cardiomyocytes [7,9,28] and smooth muscle [6,15]. As a Ca2+ mobilizing agent, cADPR is capable of inducing Ca2+ release from SR in myocytes though the role and the underlying mechanisms of cADPR regulation of Ca2+ release remain unclear and very controversial. In the present study we have used FKBP12.6 knockout mice to test whether the regulation of Ca2+ release by cADPR is mediated by FKBP12.6 via RyR2. Our results demonstrate that cADPR significantly increased the frequency and amplitude of STOCs in wildtype smooth myocytes (Fig. 1A), and the effect of cADPR on STOCs was abolished by pre-treatment of the cells with JTV-519, the RYR complex stabilizer (Fig. 1A). In contrast, cADPR did not cause marked alterations of STOCs in FKBP12.6 null cells (Fig. 1B). Thus, our findings suggest that cADPR regulation of Ca2+ release from SR is mediated by FKBP12.6 proteins in mouse bladder smooth myocytes, and this is further confirmed by data from simultaneous recordings of STOCs and Ca2+ sparks before and after dialysis of cADPR (Fig. 2). FK506, a ligand that binds to FKBP12.6 proteins, dissociates FKBP12.6 from RyR2 and results in increases in Ca2+ release from SR [29]. Our findings indicating that exposure of wildtype cells to cADPR significantly increases Ca2+ spark frequency, peak Ca2+ , FWHM, rise time and half time decay (Fig. 3), which is con-
sistent with reports showing similar effects mediated by FK506 [30,29,22,15] and opposite effects mediated by FKBP12.6 overexpression [11,31], and increase in FKBP12.6 binding to RyR2 [9,7]. Our present data and previous studies [15,32,33] indicate that alteration of Ca2+ release by cADPR is mediated by the dissociation of FKBP12.6 from the RyR2 complex and that FKBP12.6 proteins are required for cADPR to release Ca2+ from SR, in contrast to previous reports that cADPR binds directly to and activates RYRs [32,34,35]. However, evidence also points to the conclusion that dissociation of FKBP12.6 from RyRs may not be the only underlying mechanism by which cADPR regulates Ca2+ release from SR. cADPR induces Ca2+ release from SR through both RYRs and via a mechanism independent of RYRs [36]. Lukyanenko et al. reported that the primary target of cADPR is the SR Ca2+ pump (SERCA) and that potentiation of Ca2+ release by cADPR is mediated by increasing the accumulation of Ca2+ in the SR in ventricular myocytes [22]. Here we showed that the effect of cADPR on Ca2+ spark properties was not significantly affected by SERCA inhibitor, thapsigargin (Fig. 4), suggesting that stimulation of SR Ca2+ uptake did not play an important role in cADPR regulation of Ca2+ release from SR in smooth muscle [13,32]. This was further implied by the experiments that cADPR failed to alter the half time decay of Ca2+ spark properties in voltageclamped cells (Fig. 5), and that cADPR did not significantly reduce the SR Ca2+ content in the presence of thapsigargin (Fig. 7B and C). Although our data and previous studies have suggested that cADPR may activate RyRs by causing dissociation of FKBP12.6 from RyR2, there is a lack of corresponding evidence to confirm this hypothesis in smooth muscle. It has been reported that FKBP12.6 binds to RyR2 selectively and that on addition of FK506, FKBP12.6 proteins dissociate from RyRs resulting in the formation of FK506FKBP12.6 complexes [37,28]. In the present study we show that
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cADPR causes significant dissociation of FKBP12.6 from the SR microsomes and the RYR2 complexes of mouse bladder smooth myocytes (Fig. 6A and B). This is in line with observations in islet microsomes and cardiomyocytes [13,33]. Since cADPR binds to and reverses the effects of FKBP12.6 on RyRs, an increase in the probability of open RyR2s would result in an augmentation of Ca2+ release from the SR. Thus, it is reasonable to hypothesise that cADPR alters SR Ca2+ load in smooth myocytes by affecting RyR2 activity in mouse bladder smooth myocytes. It has been reported that FKBP12.6 over-expression significantly increase the SR Ca2+ load and reduce the SR Ca2+ content in cardiac myocytes [10,11,38]. However, it has also been reported that FK506 fails to alter SR Ca2+ content in mouse ventricular myocytes [39] and even increased SR Ca2+ content in rat cardiac myocytes [30]. In the present study, we demonstrate that cADPR significantly reduces SR Ca2+ content in wildtype but not in FKBP12.6 knockout cells, implying that the reduction of SR Ca2+ caused by cADPR was mediated by FKBP12.6 proteins in mouse bladder smooth muscle. cADPR fails to induce Ca2+ release in other smooth muscle types [21] suggesting the existence of variation in the response to cADPR in different tissues and species. Our results also indicate that SR Ca2+ content is lower in FKBP12.6 null myocytes compared to that of wildtype cells (Fig. 7), suggesting that a Ca2+ “leaky” state also exists in smooth muscle due to the deletion of FKBP12.6 proteins. In summary, our study demonstrates that the alteration of Ca2+ release properties by cADPR is mediated by FKBP12.6 proteins in mouse bladder smooth muscle.
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
Conflict of interest statement [21]
None declared. [22]
Acknowledgements We thank Dr. Peace Cheng for critical reading of the manuscript and Dr. Andrew Marks for the gift of JTV-519 Sources of funding: Supported by the National Basic Research Program of China (2007CB512100 and 2009CB918701), the Knowledge Innovation Program of the Chinese Academy of Sciences (Grant No: KSCX2-YW-R-50), the National Foundation of Science and Technology (#30670505), and the ‘863’ research program (2006AA02A106).
[23]
[24] [25]
[26]
[27]
References [28] [1] M.J. Berridge, Inositol trisphosphate and calcium signalling, Nature 361 (1993) 315–325. [2] M.T. Nelson, H. Cheng, M. Rubart, L.F. Santana, A.D. Bonev, H.J. Knot, W.J. Lederer, Relaxation of arterial smooth muscle by calcium sparks, Science 270 (1995) 633–637. [3] M.L. Collier, G. Ji, Y. Wang, M.I. Kotlikoff, Calcium-induced calcium release in smooth muscle: loose coupling between the action potential and calcium release, J. Gen. Physiol. 115 (5) (2000) 653–662. [4] E. Lam, M.M. Martin, A.P. Timerman, C. Sabers, S. Fleischer, T. Lukas, R.T. Abraham, S.J. O’Keefe, E.A. O’Neill, G.J. Wiederrecht, A Novel FK506 binding protein can mediate the immunosuppressive effects of FK506 and is associated with the cardiac ryanodine receptor, J. Biol. Chem. 270 (44) (1995) 26511–26522. [5] H.B. Xin, K. Rogers, Y. Qi, T. Kanematsu, S. Fleischer, Three amino acid residues determine selective binding of FK506-binding protein 12.6 to the cardiac ryanodine receptor, J. Biol. Chem. 274 (1999) 15315–15319. [6] G. Ji, M.E. Feldman, K.S. Greene, V. Sorrentino, H.B. Xin, M.I. Kotlikoff, RYR2 proteins contribute to the formation of Ca2+ sparks in smooth muscle, J. Gen. Physiol. 123 (4) (2004) 377–386. [7] M. Yano, S. Kobayashi, M. Kohno, M. Doi, T. Tokuhisa, S. Okuda, M. Suetsugu, T. Hisaoka, M. Obayashi, T. Ohkusa, M. Kohno, M. Matsuzaki, FKBP12.6-mediated stabilization of calcium-release channel (ryanodine receptor) as a novel therapeutic strategy against heart failure, Circulation 107 (3) (2003) 477–484. [8] F. Huang, J. Shan, S. Reiken, X.H. Wehrens, A.R. Marks, Analysis of calstabin2 (FKBP12.6)-ryanodine receptor interactions: rescue of heart failure by calstabin2 in mice, Proc. Natl. Acad. Sci. U.S.A. 103 (9) (2006) 3456–3461. [9] H.B. Xin, T. Senbonmatsu, D.S. Cheng, Y.X. Wang, J.A. Copello, G.J. Ji, M.L. Collier, K.Y. Deng, L.H. Jeyakumar, M.A. Magnuson, T. Inagami, M.I. Kotlikoff, S. Fleis-
[29]
[30]
[31]
[32]
[33]
[34]
[35]
cher, Oestrogen protects FKBP12. 6 null mice from cardiac hypertrophy, Nature 416 (2002) 334–338. J. Prestle, P.M. Janssen, A.P. Janssen, O. Zeitz, S.E. Lehnart, L. Bruce, G.L. Smith, G. Hasenfuss, Overexpression of FK506-binding protein FKBP12.6 in cardiomyocytes reduces ryanodine receptor-mediated Ca2+ leak from the sarcoplasmic reticulum and increases contractility, Circ. Res. 88 (2) (2001) 188–194. A.M. Gómez, I. Schuster, J. Fauconnier, J. Prestle, G. Hasenfuss, S. Richard, FKBP12.6 overexpression decreases Ca2+ spark amplitude but enhances [Ca2+ ]i transient in rat cardiac myocytes, Am. J. Physiol. Heart Circ. Physiol. 287 (5) (2004) H1987–H1993. A.P. Timerman, H. Onoue, H.B. Xin, S. Barg, J. Copello, G. Wiederrecht, S. Fleischer, Selective binding of FKBP12.6 by the cardiac ryanodine receptor, J. Biol. Chem. 271 (1996) 20385–20391. N. Noguchi, S. Takasawa, K. Nata, A. Tohgo, I. Kato, F. Ikehata, H. Yonekura, H. Okamoto, Cyclic ADP-ribose binds to FK506-binding protein 12.6 to release Ca2+ from islet microsomes, J. Biol. Chem. 272 (1997) 3133–3136. W.X. Tang, Y.F. Chen, A.P. Zou, W.B. Campbell, P.L. Li, Role of FKBP12.6 in cADPRinduced activation of reconstituted ryanodine receptors from arterial smooth muscle, Am. J. Physiol. Heart Circ. Physiol. 282 (2002) H1304–H1310. Y.X. Wang, Y.M. Zheng, Q.B. Mei, Q.S. Wang, M.L. Collier, S. Fleischer, H.B. Xin, M.I. Kotlikoff, FKBP12.6 and cADPR regulation of Ca2+ release in smooth muscle cells, Am. J. Physiol. Cell Physiol. 286 (2004) C538–C546. B.R. Fruen, J.R. Mickelson, N.H. Shomer, P. Velez, C.F. Louis, Cyclic ADP-ribose does not affect cardiac or skeletal muscle ryanodine receptors, FEBS Lett. 352 (2) (1994) 123–126. X. Guo, M.A. Laflamme, P.L. Becker, Cyclic ADP-ribose does not regulate sarcoplasmic reticulum Ca2+ release in intact cardiac myocytes, Circ. Res. 79 (1) (1996) 147–151. J.A. Copello, Y. Qi, L.H. Jeyakumar, E. Ogunbunmi, S. Fleischer, Lack of effect of cADP-ribose and NAADP on the activity of skeletal muscle and heart ryanodine receptors, Cell Calcium 30 (4) (2001) 269–284. H.L. Wilson, M. Dipp, J.M. Thomas, C. Lad, A. Galione, A.M. Evans, Adp-ribosyl cyclase and cyclic ADP-ribose hydrolase act as a redox sensor. A primary role for cyclic ADP-ribose in hypoxic pulmonary vasoconstriction, J. Biol. Chem. 276 (14) (2001) 11180–11188. D.W. Cheung, Modulation of spontaneous transient Ca2+ -activated K+ channel currents by cADP-ribose in vascular smooth muscle cells, Eur. J. Pharmacol. 458 (1–2) (2003) 57–59. J.G. McCarron, S. Chalmers, K.N. Bradley, D. MacMillan, T.C. Muir, Ca2+ microdomains in smooth muscle, Cell Calcium 40 (5–6) (2006) 461– 493. V. Lukyanenko, I. Györke, T.F. Wiesner, S. Györke, Potentiation of Ca2+ release by cADP-ribose in the heart is mediated by enhanced SR Ca2+ uptake into the sarcoplasmic reticulum, Circ Res. 89 (7) (2001) 614–622. N. Fritz, J.L. Morel, L.H. Jeyakumar, S. Fleischer, P.D. Allen, J. Mironneau, N. Macrez, RyR1-specific requirement for depolarization-induced Ca2+ sparks in urinary bladder smooth muscle, J. Cell Sci. 120 (Pt 21) (2007) 3784–3791. J.V. Walsh Jr., J.J. Singer, Ca++ activated K+ channels in vertebrate smooth muscle cells, Cell Calcium 4 (1983) 321–330. U. Trieschmann, G. Isenberg, Ca2+ activated K+ channels contribute to the resting potential of vascular myocytes. Ca2+ sensitivity is increased by intracellular Mg2+ ions, Pflugers Arch. 414 (1989) S183–S184. Y.X. Wang, B.K. Fleischmann, M.I. Kotlikoff, Modulation of maxi-K+ channels by voltage-dependent Ca2+ channels and methacholine in single airway myocytes, Am. J. Physiol. 272 (1997) C1151–C1159. A.M. Gomez, H.P. Cheng, W.J. Lederer, D.M. Bers, Ca2+ diffusion and sarcoplasmic reticulum transport both contribute to [Ca2+]i decline during Ca2+ sparks in rat wentricular myocytes, J. Physiol. 462 (1996) 575–581. S.E. Lehnart, C. Terrenoire, S. Reiken, X.H. Wehrens, L.S. Song, E.J. Tillman, S. Mancarella, J. Coromilas, W.J. Lederer, R.S. Kass, A.R. Marks, Stabilization of cardiac ryanodine receptor prevents intracellular calcium leak and arrhythmias, Proc. Natl. Acad. Sci. U.S.A. 103 (20) (2006) 7906–7910. R.P. Xiao, H.H. Valdivia, K. Bogdanov, C. Valdivia, E.G. Lakatta, H. Cheng, The immunophilin FK506-binding protein modulates Ca2+ release channel closure in rat heart, J. Physiol. 500 (1997) 343–354. E. McCall, L. Li, H. Satoh, T.R. Shannon, L.A. Blatter, D.M. Bers, Effects of FK-506 on contraction and Ca2+ transients in rat cardiac myocytes, Circ. Res. 79 (6) (1996) 1110–1121. C.M. Loughrey, T. Seidler, S.L. Miller, J. Prestle, K.E. MacEachern, D.F. Reynolds, G. Hasenfuss, G.L. Smith, Over-expression of FK506-binding protein FKBP12.6 alters excitation-contraction coupling in adult rabbit cardiomyocytes, J. Physiol. 556 (Pt 3) (2004) 919–934. K. Morita, T. Kitayama, S. Kitayama, T. Dohi, Cyclic ADP-ribose requires FK506binding protein to regulate intracellular Ca2+ dynamics and catecholamine release in acetylcholine-stimulated bovine adrenal chromaffin cells, J. Pharmacol. Sci. 101 (1) (2006) 40–51. X. Zhang, Y.N. Tallini, Z. Chen, L. Gan, B. Wei, R. Doran, L. Miao, H.B. Xin, M.I. Kotlikoff, G. Ji, Dissociation of FKBP12.6 from ryanodine receptor type 2 is regulated by cyclic ADP-ribose but not beta-adrenergic stimulation in mouse cardiomyocytes, Cardiovasc. Res. 84 (2) (2009) 253–262. A. Galione, H.C. Lee, W.B. Busa, Ca2+ -induced Ca2+ release in sea urchin egg homogenates: modulation by cyclic ADP-ribose, Science 253 (1991) 1143–1146. L.G. Mészáros, J. Bak, A. Chu, Cyclic ADP-ribose as an endogenous regulator of the non-skeletal type ryanodine receptor Ca2+ channel, Nature 364 (1993) 76–79.
J. Zheng et al. / Cell Calcium 47 (2010) 449–457 [36] P.L. Li, W.X. Tang, H.H. Valdivia, A.P. Zou, W.B. Campbell, cADPribose activates reconstituted ryanodine receptors fromcoronary arterial smooth muscle, Am. J. Physiol. Heart Circ. Physiol. 280 (2001) H208–H215. [37] Y.S. Prakash, M. Kannan, T.F. Walseth, G.C. Sieck, cADPR ribose and [Ca2+ ]i regulation in rat cardiac myocytes, Am. J. Physiol. Heart Circ. Physiol. 279 (2000) 1482–1489.
457
[38] A.B. Brillantes, K. Ondrias, A. Scott, E. Kobrinsky, E. Ondriasová, M.C. Moschella, T. Jayaraman, M. Landers, B.E. Ehrlich, A.R. Marks, Stabilization of calcium release channel (ryanodine receptor) function by FK506-binding protein, Cell 77 (4) (1994) 513–523. [39] Z. Su, K. Sugishita, F. Li, M. Ritter, W.H. Barry, Effects of FK506 on [Ca2+ ]i differ in mouse and rabbit ventricular myocytes, J. Pharmacol. Exp. Ther. 304 (1) (2003) 334–341.
Contents lists available at ScienceDirect
Cell Calcium journal homepage: www.elsevier.com/locate/ceca
Ca2+ release induced by cADP-ribose is mediated by FKBP12.6 proteins in mouse bladder smooth muscle Ji Zheng a,1 , Bi Wenzhi b,1 , Lin Miao c,1 , Yumin Hao c , Xu Zhang c , Wenxuan Yin c , Jinhong Pan a , Zengqiang Yuan c,∗ , Bo Song a,∗ , Guangju Ji c,d,∗∗ a
Urological Surgery Research Institute, Southwest Hospital, Third Military Medical University, Gao Tanyan Rd. 30, Chongqing 400038, China Clinical Division of Surgery, General Hospital of PLA, China National Laboratory of Biomacromolecules, Institute of Biophysics of Chinese Academy of Sciences, 15 Datun Rd, Beijing 100101, China d Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853 USA b c
a r t i c l e
i n f o
Article history: Received 3 July 2009 Received in revised form 22 March 2010 Accepted 26 March 2010 Available online 7 May 2010 Keywords: cADP ribose Ca2+ spark FKBP12.6 proteins Smooth muscle
a b s t r a c t We examined the role and molecular mechanism of cADPR action on Ca2+ spark properties in mouse bladder smooth muscle. Dialysis of cADPR with patch pipettes increased frequency and amplitude of spontaneous transient out currents (STOCs) to 6.1 ± 0.87 currents/min from 1.2 ± 0.36 currents/min (control) and to 179.8 ± 48.7 pA from 36.4 ± 22.6 pA (control), respectively, in wildtype (WT) cells, and the effects of cADPR on STOCs were significantly blocked by JVT-591, a RYR2 stabilizer. In contrast, no significant changes were observed in FKBP12.6 null cells. Further studies indicated that Ca2+ spark properties were altered by cADPR in WT but not FKBP12.6 null cells, namely, Ca2+ spark frequency was increased by about 3.4-fold, peak Ca2+ (F/F0) increased to 1.72 ± 0.57 from 1.56 ± 0.13, size increased to 2.86 ± 0.26 M from 1.92 ± 0.14 M, rise time and half-time decay were prolonged 1.6-fold and 2.3-fold, respectively, in WT cells. Furthermore, in the presence of thapsigargin cADPR still altered Ca2+ spark properties, and cADPR increased F/F0 without affecting Ca2+ spark decay time in voltage clamping cells. Dissociation studies demonstrated that application of cADPR resulted in significant removal of FKBP12.6 proteins from sarcoplasmic reticulum (SR) microsomes, and that treatment of the RyR2 immunoprecipitation complexes with cADPR or FK506 disrupted the interaction between RyR2 and FKBP12.6. Finally, cADPR altered SR Ca2+ load in WT myocytes but not in FKBP12.6-null myocytes. Taken together, these results suggest that Ca2+ release induced by cADPR is mediated by FKBP12.6 proteins in mouse bladder smooth muscle. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Ca2+ release from intracellular Ca2+ stores (the endoplasmic/sarcoplasmic reticulum (ER/SR)) is one of the key signal transduction mechanisms that play a pivotal role in the regulation of numerous cellular functions [1]. In smooth myocytes, Ca2+ release from intracellular Ca2+ stores to the cytoplasm is controlled by ryanodine Ca2+ release receptors subtype 2 (RYR2), and occurs in the form of spontaneous SR Ca2+ release events, or Ca2+ sparks [2]. Ca2+ release is triggered by the influx of Ca2+ through sarcolemmal ion channels, often termed Ca2+ induced Ca2+ release
∗ Corresponding authors. ∗∗ Corresponding author at: Institute of Biophysics, Chinese Academy of Sciences, Beijing Datun Rd. 15, Beijing 100101, China. Tel.: +86 10 64889873; fax: +86 10 64846720. E-mail addresses: [email protected] (Z. Yuan), [email protected] (B. Song), [email protected] (G. Ji). 1 These authors contributed equally to this manuscript. 0143-4160/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.ceca.2010.03.006
(CICR) [3]. FKBP12.6, a 12.6 kDa protein, specifically binds with and regulates RYR2 in cardiomyocytes and smooth muscle [4,5,6]. It has been reported that FKBP12.6 stabilizes the closed state of the RyR2. The amount of FKBP12.6 bound to RYR2 is decreased in heart failure and consequently leads to instability of the FKBP12.6RYR2 complex and results in SR Ca2+ leakage [7,8]. Patch clamp and confocal microscopy combined studies have demonstrated that both FKBP12.6 deficient [9,6] and FKBP12.6 overexpressing animals exhibit a significant alteration in Ca2+ spark properties [10,11]. These findings strongly suggest that FKBP12.6 is pivotal in regulating calcium release from the SR, possibly via its association with RyR2 cADPR, a Ca2+ mobilizer, has been shown to be a potential regulator of RYR activity in myocytes despite the fact that the role and mechanisms of cADPR regulation of Ca2+ release remain unclear and very controversial. For instance, some studies suggest that cADPR specifically binds to FKBP12.6 and regulates Ca2+ release [12–14,9,15]. However, some studies are inconsistent with the above observation. It has been demonstrated that cADPR does not
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Fig. 1. cADPR regulates STOCs in wildtype but not in FKBP12.6 null smooth myocytes. Dialysis of WT myocytes with cADPR (5 M) causes increases in both frequency and amplitude of spontaneous transient outward currents and this effect of cADPR was abolished by pre-treatment of the cells with JTV-519, a RYR complex stabilizer (A, n = 7, P < 0.05); in FKBP12.6 deleted myocytes, dialysis of cADPR failed to alter the properties of STOCs (B, n = 9, P > 0.05). (C and D) Summary data for frequency and amplitude of STOCs in WT and FKBP12.6 KO myocytes.
alter Ca2+ release in cardiac and skeletal myocytes [16,17,18]. As for smooth muscles, although cADPR increases Ca2+ release in pulmonary [19], vascular [20] and tracheal myocytes [1], it fails to cause Ca2+ release in other types of smooth muscle, including aortic, vas deferens, coronary, bronchial, cerebral arterial, and intestinal types [21]. Moreover, SERCA may also be involved in the action of cADPR on Ca2+ release [22]. In the present study we sought to examine the effect and mechanism of cADPR on Ca2+ release in mouse bladder smooth muscle. We have used FKBP12.6 null mice to test directly if cADPR action on Ca2+ sparks is through FKBP12.6 disassociation from the RYR2 complex. We found that cADPR significantly alters the properties of STOCs and Ca2+ sparks, and that the underlying mechanism of cADPR action is related to the dissociation of FKBP12.6 proteins from the RYR2 complex in mouse bladder smooth muscle.
2. Materials and methods 2.1. Cell isolation Mice were anesthetized and killed in accordance with an approved laboratory animal protocol and single bladder cells were prepared as previously described [6]. Briefly, the urinary bladder was removed, dissected in an ice-cold oxygenated Ca2+ -free solution containing (mM): 80 Na-glutamate, 55 NaCl, 6 KCl, 2 MgCl2 , 10 HEPES, and 10 glucose. The detrusor muscle was minced and incubated for 20 min at 37 ◦ C in dissociation solution containing 1 mg/ml dithioerythreitol, 1 mg/ml papain, and 1 mg/ml bovine serum albumin (Sigma–Aldrich), and the partially digested tissue
was then transferred to a solution containing 1 mg/ml collagenase type II (Worthington Biochemical), 1 mg/ml bovine serum albumin, and 100 M Ca2+ . The tissue was incubated for 10 min, and then triturated with a wide-bore Pasteur pipette, and passed through a 125-mm nylon mesh. Cells were concentrated by low-speed centrifugation, washed with fresh medium, resuspended, and stored at 4 ◦ C. All animal procedures described in this study were performed in adherence with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996), and with approval from the Institute of Biophysics Committee on Animal Care. 2.2. Measurement of Ca2+ fluorescence Myocytes were incubated with 10 M Fluo-4 AM (Molecular Probes) for 10 min at room temperature and transferred into a recording chamber mounted on an inverted microscope (IX81; Olympus). Cells were allowed to adhere to the bottom of the recording chamber for 15 min and were then perfused with extracellular solution (see above) for 40 min. Cells were excited with 488 nm light from a krypton/argon laser and linescan images were collected using a laser scanning confocal head (FV1000; Olympus) attached to an inverted microscope (IX81; Olympus). Linescans were obtained at an interval of 1.33 or 0.833 ms per line. Images were processed and analyzed using MATLAB 7.1 software (MathWorks). For linescan images, F0 was obtained by averaging the fluorescence for each pixel (× dimension) for a period preceding activation of a Ca2+ spark, and the fluorescence of all pixels (F) was divided by F0.
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the pellet was resuspended in buffer A. SR membranes were also incubated with 10 M FK506 as a positive control. The protein concentration was determined by the BCA method. 2.5. Western blot analysis Western blot analysis was performed as previously described [13]. Briefly, 30 g protein from each sample was separated by SDS-polyacrylamide gel electrophoresis using 15% gels. The resolved proteins were transferred to PVDF membranes (Millipore) at 100 mA for 70 min. The membranes were blocked for 2 h with Tris-buffered saline-Tween 20 (TBST) containing 5% BSA at room temperature. The blocked membranes were immunoblotted with polyclonal goat anti-mouse FKBP12.6 antibody (R&D systems) for 2 h at room temperature. After washing 3 times in TBST, the membranes were incubated in TBST containing 5% BSA and the secondary antibody conjugated to horseradish peroxidase. Final detection was performed using enhanced chemiluminescence detection solution 1 and 2 (1:1) (ECL; Millipore). 2.6. Co-immunoprecipitation assays 2+
Fig. 2. cADPR alters both STOCs and Ca sparks in WT myocytes. Cells were dialyzed with cADPR (5 M) through patch pipettes, and linescan images were collected after 6 min of whole cell configuration obtained. (A) A representative sample of simultaneous recordings of STOCs and Ca2+ release in a wildtype cell, indicating that with the dialysis of cADPR both STOCs and Ca2+ sparks were altered. The upper panel is the linescan image, and the lower panel is fluorescence profile (taken from the above image as indicated by arrow) of Ca2+ (red) and STOCs (black) recorded simultaneously. (B) A sample experiment of linescan images obtained from FKBP12.6-deficient myocyte. It is notable that there was no significant alternation in both STOCs and Ca2+ sparks with dialysis of cADPR. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
In SR Ca2+ load experiments, short applications of 10 mM caffeine (Sigma) by pressure ejection from a glass pipette were used to estimate SR Ca2+ content as previously described [23]. All experiments were carried out at room temperature. 2.3. Patch-clamp recording Membrane currents were recorded at room temperature using whole-cell voltage clamp configuration. To examine STOCs, cells were clamped at −20 mV. To test the voltage-clamping induced Ca2+ spark cells were clamped at −60 mV and depolarized to 0 mV with 200 ms duration. The intracellular solution was (mM): 130 KCl, 1.8 MgCl2 , 1.0 Na2ATP, 0.05 CaCl2 , 0.1 EGTA (pH 7.3). The extracellular solution was (mM): 137 NaCl, 5.4 KCl, 1.8 CaCl2 , 1.0 MgCl2 , 10 glucose, 10 HEPES and (pH 7.4). Currents were filtered at 500 Hz and digitized at 2 kHz. 2.4. Sarcoplasmic reticulum preparation Sarcoplasmic reticulum (SR) microsomes were prepared from mouse bladders as previously described [14]. Briefly, mouse bladders were removed and the inside and outside layers were carefully cleaned. The bladders were then cut into small pieces and homogenized in 3-(N-morpholino) propanesulfonic acid (MOPS) buffer containing 0.9% NaCl, 10 mM MOPS (pH 7.0) and phenylmethylsulfonyl fluoride (PMSF). The homogenate was centrifuged at 14,000 × g for 25 min at 4 ◦ C. The supernatant was then collected and centrifuged at 100,000 × g for 90 min at 4 ◦ C. The pellet, which contained SR membranes-enriched microsomes, was resuspended in a buffer containing 0.9% NaCl, 0.3 M sucrose, and PMSF. In order to determine if cADPR can induce FKBP12.6 release from RyRs, SR membranes were incubated with cADPR in buffer at 37 ◦ C for 15 min. The mixture was then centrifuged at 100,000 × g for 90 min at 4 ◦ C. The supernatant was stored at −80 ◦ C until required and
The total protein from mice bladder was prepared in a usual way. Briefly, bladder tissues dissected from mice were cut into small pieces and homogenized in RIPA lysis buffer containing (in mM) 50 Tris (pH 7.4), 150 NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS and phenylmethylsulfonyl fluoride (PMSF). The homogenate was centrifuged at 12,000 × g for 15 min at 4 ◦ C. The supernatant was then collected. In order to determine whether cADPR induces release of FKBP12.6 from the RyR2 complex, cADPR was incubated with the resultant supernatant at 37 ◦ C for 15 min. Then the supernatant was stored at −80 ◦ C until use. The resultant supernatant was also incubated with 10 M FK506, which was used as positive control. The concentration of protein was determined using BCA method. Samples were suspended in RIPA buffer and incubated with RYR2 antibody (CHEMICON, USA) O/N at 4 ◦ C (IgG alone was used as a negative control, data not shown). Protein G sepharose deads were added, incubated at 4 ◦ C for 1 h, and washed with RIPA for three times (each time for 10 min), then centrifuged and denatured for loading. Immunoblots were performed using antiFKBP12.6 (1:1000) and anti-RYR2 (1:1000). After three washes, membranes were incubated with peroxide-conjugated rabbit antigoat or -horse anti-rabbit IgG antiserum (1:4000) for 60 min at room temperature and developed with an enhanced chemiluminescence (ELC, Amersham). 2.7. Data analysis Image processing and data analysis were carried out with custom software written in MATLAB. Ca2+ sparks were counted manually and with a spark-counting software algorithm to verify the results objectively. To display the linescan images in a consistent way, F0 was obtained by averaging the fluorescence for each pixel (× dimension) for a period preceding activation of a Ca2+ spark, and the fluorescence of all pixels (F) was divided by F0 on a pixel by pixel basis. Linescans were then displayed with a colormap ranging from 0.5 to 3 F/F0. Profiles were constructed by averaging the pixels bisecting a Ca2+ spark for each time point in the scan. Ca2+ sparks and Ca2+ transients were fitted to a function with six free parameters (F0, start time, rise time, peak F/F0, halftime decay and final offset) and four free parameters (F0, rise time, peak F/F0, and half-time decay), respectively using a LevenbergMarquardt nonlinear least squares fitting routine. STOC rise time and peak current were measured by hand from the raw current recordings. Results are expressed as means ± SE where applicable.
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Fig. 3. cADPR induced alteration of Ca2+ spark properties. The experiments were conducted in smooth myocytes from wildtype mice, and cells were dialyzed with cADP5 (5 M). (A) Representative linescan images recorded from a wildtype mouse bladder cell. The upper panel shows the control (without cADPR), and the lower panel shows the cADPR treatment. (B) Linescan images obtained from FKBP12.6 knockout myocytes, demonstrating that in the presence (upper) and absence (lower) of cADPR the properties of Ca2+ sparks were not altered. (C) Summary data of Ca2+ spark properties. Ca2+ spark frequency increased in wildtype but not in FKBP12.6 null cells (a). Similarly, F/F0 (b), FWHM (c), rise time (d) and half-time decay (e) were significantly altered by cADPR only in wildtype cells. *P < 0.05, and **P < 0.01.
Data from the WT and FKBP12.6 −/− groups were compared by one-way, repeated measures of ANOVA and significant differences between groups were determined by the Student–Newman–Keuls (SNK) test for pairwise comparisons. 3. Results 3.1. cADPR regulates STOCs in wildtype but not in FKBP12.6 null mouse urinary bladder myocytes Our previous studies indicated that RYR2 Ca2+ release has a central functional role and that spontaneous and evoked Ca2+ sparks are closely associated with STOCs in smooth muscle [6]. The selective interaction of FKBP12.6 with RYR2 [12,5,9] allowed us to use FKBP12.6-deficient mice to determine whether cADPR regulates Ca2+ release by regulating the interaction of FKBP12.6 and RyR2. As STOCs are a convenient indicator of spontaneous SR Ca2+ release events [23,24,25,26], we initially examined the effect of cADPR on the frequency and amplitude of STOCs in urinary bladder myocytes from WT and FKBP12.6 −/− mice. On dialysis of 5 M cADPR into bladder smooth muscle cells via patch pipettes, both the frequency and amplitude of STOCs were significantly altered in WT cells. As
shown in Fig. 1, the frequency and amplitude of STOCs increased to 6.1 ± 0.87 currents/min from 1.2 ± 0.36 currents/min (control) and to 179.8 ± 48.7 pA from 36.4 ± 22.6 pA (control), respectively, in WT cells. To test if the effects of cADPR on STOCs were related to the dissociation of FKBP12.6 proteins from RYR complex, a RYR stabilizer, JVT-519, was used in the study. As shown in Fig. 1A (lower), in the presence of JVT-519 the stimulatory effects of cADPR on STOCs were abolished. In contrast to WT cells, the cells from FKBP12.6 null mice did not respond to cADPR stimulation (Fig. 1B–D), suggesting that the alteration of Ca2+ -release properties induced by cADPR was mediated by FKBP12.6 proteins in mouse bladder smooth muscle. It was notable that deletion of FKBP12.6 gave rise to significant increases in both STOC frequency and amplitude compared with WT myocytes (5.16 ± 2.03 currents/min vs. 1.2 ± 0.36 currents/min, 125 ± 50.6 pA vs. 36.4 ± 22.6 pA; Fig. 1C and D). 3.2. cADPR alters Ca2+ spark properties in smooth muscle To further test our hypothesis that alteration of STOCs induced by cADPR reflects changes in the underlying Ca2+ release events, we measured Ca2+ sparks in voltage-clamped urinary bladder myocytes from WT and FKBP12.6−/− mice. Fig. 2 is representative
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Fig. 4. Effects Ca2+ uptake pump inhibitor on cADPR action of Ca2+ sparks. Cells from WT mice were pre-treated by Thapsigargin (0.1 M, 10 min), and then were dialyzed with cADPR (5 M). (A) shows a sample of experiments demonstrating that the alteration of Ca2+ spark properties by thapsigargin was not significant (b), and that in the presence of thapsigargin cADPR still altered Ca2+ spark properties (d) compared to controls (a). (B) Summary data of Ca2+ spark properties. The increase in Ca2+ spark frequency (a), peak Ca2+ (b), size (c), and half-time decay (d) induced by dialysis of cADPR was not significantly affected by the presence of thapsigargin. The Ca2+ spark number measured (n) was 186; *P < 0.05, and **P < 0.01.
of simultaneous recordings of STOCs and Ca2+ sparks from the two cell types. On dialysis of wildtype cells with cADPR, both STOCs and Ca2+ sparks were markedly altered (n = 7). In contrast, dialysis of FKBP12.6 knockout cells with cADPR resulted in a negligible change in the properties of STOCs and Ca2+ sparks (n = 9). RYR Ca2+ release channels display stochastic gating behavior and consequently Ca2+ sparks vary substantially in frequency, amplitude, and duration. Next, we characterized the range of Ca2+ spark properties observed in control and FKBP12.6 null mouse bladder smooth muscle. Examples of individual Ca2+ sparks at high time resolution, and a summary of the number of Ca2+ sparks, the mean amplitude, spread (full width at half-maximum, FWHM), rise time and half-time decay for the different cell types are shown in Fig. 3. After dialysis of cADPR, marked alteration of Ca2+ spark properties was observed in WT cells (Fig. 3A) but not in FKBP12.6 KO cells (Fig. 3B). Ca2+ spark frequency was increased by about 3.4-fold (from 1.42 ± 0.43 to 4.78 ± 1.13, n = 8, P < 0.05) in wildtype but not in FKBP12.6 null cells (Fig. 3Ca). Similarly, on dialysis of wildtype cells with cADPR, peak Ca2+ increased to 1.72 ± 0.57 from 1.56 ± 0.13 (Fig. 3Cb), FWHM increased to 2.86 ± 0.26 M from 1.92 ± 0.14 M (Fig. 3Cc); rise time (Fig. 3Cd) and half-time decay (Fig. 3Ce) were prolonged 1.6-fold (35.6 ± 2.8 ms vs. 22.3 ± 1.9 ms) and 2.3-fold (68.7 ± 8.9 ms vs. 29.6 ± 3.2 ms), respectively. However, cADPR did not alter Ca2+ spark properties in FKBP12.6 knockout cells. Taken together, the present results showing that cADPR alters STOCs and Ca2+ spark properties in wildtype but not in FKPBP12.6-deficient myocytes suggesting that the action of cADPR is mediated by FKBP12.6 proteins. 3.3. Effect of cADPR on Ca2+ uptake in smooth muscle In the next experiments we tested whether SERCA activity is necessary for cADPR-induced increase in Ca2+ sparks, as it has been reported that cADPR stimulates SR Ca2+ uptake and potentiates Ca2+ release in cardiomyocytes [22]. Thapsigargin, a specific inhibitor of the SR Ca2+ pump was used in this study. As shown in Fig. 4, cADPR
significantly altered Ca2+ spark properties even in the presence of thapsigargin(0.1 M). A summary of Ca2+ spark properties for the three groups is shown in Fig. 4B. It has been known that activation of SERCA speeds up the re-uptake of Ca2+ into the SR, shortens the [Ca2+ ]i transient and reduces the Ca2+ spark duration [27]. To further test if SERCA activity involved in the action of cADPR on Ca2+ spark here we also carried out experiments in voltage clamping cells. As shown in Fig. 5, cADPR, as expected, increased the amplitude of Ca2+ spark significantly in 7 experiments (P < 0.05) but there was no significant effect on half time decay of Ca2+ spark, suggesting that the alteration of Ca2+ spark properties induced by cADPR was not relevant to the SERCA activity in mouse bladder smooth muscle. 3.4. FKBP12.6 mediates cADPR-induced Ca2+ release in smooth muscle To further confirm that the effect of cADPR on Ca2+ spark properties is directly dependent on the dissociation of FKBP12.6 from the RYR2 complex, Western blotting analysis was performed on SR microsomes from mouse bladder smooth muscle. As shown in Fig. 6A, the endogenous FKBP12.6 was released from SR microsomes by incubation with cADPR and practically all of the FKBP12.6 was bound to SR microsomes in the pellet. We also incubated the SR fraction with FK506 (10 M) as a positive control. On addition of FK506, the FKBP12.6 was all released from the SR fraction into the supernatant. Concomitantly, no corresponding FKBP12.6 band was detected in the pellet containing the SR microsomes (Fig. 6Aa). After treatment of SR microsomes with cADPR (5 M), FKBP12.6 was not detected in pellet microsomes but was recovered in the supernatant in four individual experiments (Fig. 6Ab). The percentage of FKBP12.6 dissociation (supernatant FKBP12.6/Total FKBP12.6) induced by cADPR was 97.2 ± 2.8% (n = 5). To further confirm our finding that cADPR dissociated FKBP12.6 from RYR complexes, co-immunoprecipitation and western blot analysis were carried out. Lysates of mouse SR treated with FK506
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Fig. 5. Ca2+ spark property alteration induced by cADPR was independent of SERCA. Experiments were conducted in voltage-clamped myocytes from wildtype mice. Cells were clamped at −60 mV and depolarized to a test potential of 0 mV with duration of 200 ms. (A) Linescan images collected in the presence (left) and absence of cADPR (right). Currents and Ca2+ fluorescence profiles were shown the bottom of the images. (B) Summary data of Ca2+ spark peak amplitude, rise time, and half time decay. It was notable that the half time decay of the Ca2+ spark was not significantly changed by cADPR.
or cADPR or vehicle control were immunoprecipitated with antiRyR2 antibody and blotted with the antibody to RyR2 or FKBP12.6. As shown in Fig. 6B, treatment of FK506 or cADPR disrupted the interaction between RyR2 and FKBP12.6, confirming our hypothesis that cADPR altered Ca2+ spark properties via dissociation of FKBP12.6 from RYR2 complexes. To demonstrate that the band of FKBP12.6 is absent in knockout mice immunoblot of mouse bladder homogenate from WT and KO mice was carried out and the results were shown in Fig. 6C. 3.5. cADPR alters SR Ca2+ content through FKBP12.6 To determine whether cADPR affected SR Ca2+ content, caffeine (10 mM) was used to estimate SR Ca2+ load in myocytes of WT and FKBP12.6-null mice before and after cADPR treatment. As shown in Fig. 7, cADPR significantly altered SR Ca2+ load in WT myocytes but not in FKBP12.6-null myocytes. Ca2+ transients induced by caffeine were markedly reduced in cADPR (5 M) pre-treated WT cells. However, cADPR pre-treatment did not markedly affect Ca2+ transients induced by caffeine in FKBP12.6 null myocytes though it did affect the load in some extent (Fig. 7Aa). Fig. 7Ab shows the averaged amplitude of Ca2+ transients (F/F0) induced by caffeine. The averaged F/F0 of 2.71 ± 0.09 in the WT control group, was markedly reduced to 2.03 ± 0.11 by pre-treatment with cADPR (Fig. 7Ba left, P < 0.01). There was no significant difference in F/F0 before and after treatment with cADPR in FKBP12.6 null cells (1.84 ± 0.12 vs. 1.73 ± 0.15, P > 0.05). Similarly, the rise time of the Ca2+ transient was also significantly reduced by cADPR in only the wildtype cells (from 117.9 ± 10.4 ms to 87.2 ± 14.2 ms). We noted that the halftime decay in all groups was not affected by cADPR treatment (1029.1 ± 98.2 ms vs. 1023.0 ± 95.5 ms in WT and 999.2 ± 135.1 ms vs. 1015.2 ± 106.4 ms in FKBP12.6 null cells (data not shown). Fig. 7B shows that thapsigargin decreased SR Ca2+ load and that
Fig. 6. Dissociation of FKBP12.6 from Sarcoplasmic reticulum microsomes by cADPR. (A) Western blotting analysis of FKBP12.6 binding and release. SR was incubated 30 min at 37 ◦ C in control conditions or in the presence of FK506 (positive control), cADPR, or 8-Br-cADPR (negative control). Released FKBP12.6 proteins appear in the supernatant (Ab), whereas FKBP12.6 bounds are remained in the pellets for control condition or in the presence of 8-Br-cADPR (Aa). (B) Extracts from SR of 6 wildtype mouse bladder smooth muscle were used to measure the expression of the indicated proteins and for immunoprecipitation of RYR2. The immunoprecipitates were analyzed for co-immunoprecipitation of RYR2 and FKBP12.6. Lysates of mouse bladder SR treated with FK506 or cADPR or vehicle control were immunoprecipitated with anti-RyR2 antibody and blotted with the antibody to RyR2 (top panel) or FKBP12.6 (middle panel). Total FKBP12.6 protein level was shown in the bottom panel. Treatment of FK506 or cADPR disrupted the interaction between RyR2 and FKBP12.6. These results are from three independent experiments. The molecular weight of this protein is about 12 kDa. (C) Immunoblot of mouse bladder homogenate from WT and KO mice. Lanes 1–3 are hela pcmv, hela pcmv FKBP12, and hela pcmv FKBP12.6, respectively; lanes 1 and 2 are mouse bladder homogenate from FKBP12.6 KO and FKBP12.6 WT mice.
in the presence of thapsigargin cADPR could not further significantly reduce the SR Ca2+ content though it did reduce the content in some extent, suggesting SERCA is not the main target of cADPR action. Additionally, we also examined the effects of thapsigargin in FKBP12.6 null cells, and the results indicated that thapsigargin significantly reduced the Ca2+ load and that cADPR, as expected, did not markedly affect the Ca2+ load (Fig. 7C). It was notable that the FKBP12.6 protein deletion caused a reduction in Ca2+ content in the SR though statistically not significant (Fig. 7), suggesting that a Ca2+ “leaky” state exists due to instability of RYRs [7,8].
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Fig. 7. Reduction of SR Ca2+ by cADPR in smooth myocytes. (Aa) Representative Ca2+ transients induced by 10 mM caffeine from linescan images recorded from wildtype cells (blue), wildtype cells pretreated with cADPR (purple), FKBP12.6 null cells (yellow), and FKBP12.6 null cells pretreated with cADPR (green). cADPR significantly reduced Ca2+ transients induced by caffeine in WT myocytes. (Ab) Summary data of Ca2+ release properties. It is notable that mutation of FKBP12.6 proteins caused a reduction in Ca2+ content. (Ba and b) Thapsigargin alone significantly reduced SR Ca2+ content, and cADPR failed to significantly alter SR Ca2+ in the presence of thapsigargin, however in wildtype cells. (Ca and b) Experiments carried out in FKBP12.6−/− cells. *P < 0.05 and **P < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Taken together, these data demonstrate that cADPR significantly reduces SR Ca2+ content by causing dissociation of FKBP12.6 from the RyR2 complexes. 4. Discussion It is commonly believed that FKBP12.6 is a subunit that stabilizes the closed state of RyR2 and plays an important regulatory role in Ca2+ release in cardiomyocytes [7,9,28] and smooth muscle [6,15]. As a Ca2+ mobilizing agent, cADPR is capable of inducing Ca2+ release from SR in myocytes though the role and the underlying mechanisms of cADPR regulation of Ca2+ release remain unclear and very controversial. In the present study we have used FKBP12.6 knockout mice to test whether the regulation of Ca2+ release by cADPR is mediated by FKBP12.6 via RyR2. Our results demonstrate that cADPR significantly increased the frequency and amplitude of STOCs in wildtype smooth myocytes (Fig. 1A), and the effect of cADPR on STOCs was abolished by pre-treatment of the cells with JTV-519, the RYR complex stabilizer (Fig. 1A). In contrast, cADPR did not cause marked alterations of STOCs in FKBP12.6 null cells (Fig. 1B). Thus, our findings suggest that cADPR regulation of Ca2+ release from SR is mediated by FKBP12.6 proteins in mouse bladder smooth myocytes, and this is further confirmed by data from simultaneous recordings of STOCs and Ca2+ sparks before and after dialysis of cADPR (Fig. 2). FK506, a ligand that binds to FKBP12.6 proteins, dissociates FKBP12.6 from RyR2 and results in increases in Ca2+ release from SR [29]. Our findings indicating that exposure of wildtype cells to cADPR significantly increases Ca2+ spark frequency, peak Ca2+ , FWHM, rise time and half time decay (Fig. 3), which is con-
sistent with reports showing similar effects mediated by FK506 [30,29,22,15] and opposite effects mediated by FKBP12.6 overexpression [11,31], and increase in FKBP12.6 binding to RyR2 [9,7]. Our present data and previous studies [15,32,33] indicate that alteration of Ca2+ release by cADPR is mediated by the dissociation of FKBP12.6 from the RyR2 complex and that FKBP12.6 proteins are required for cADPR to release Ca2+ from SR, in contrast to previous reports that cADPR binds directly to and activates RYRs [32,34,35]. However, evidence also points to the conclusion that dissociation of FKBP12.6 from RyRs may not be the only underlying mechanism by which cADPR regulates Ca2+ release from SR. cADPR induces Ca2+ release from SR through both RYRs and via a mechanism independent of RYRs [36]. Lukyanenko et al. reported that the primary target of cADPR is the SR Ca2+ pump (SERCA) and that potentiation of Ca2+ release by cADPR is mediated by increasing the accumulation of Ca2+ in the SR in ventricular myocytes [22]. Here we showed that the effect of cADPR on Ca2+ spark properties was not significantly affected by SERCA inhibitor, thapsigargin (Fig. 4), suggesting that stimulation of SR Ca2+ uptake did not play an important role in cADPR regulation of Ca2+ release from SR in smooth muscle [13,32]. This was further implied by the experiments that cADPR failed to alter the half time decay of Ca2+ spark properties in voltageclamped cells (Fig. 5), and that cADPR did not significantly reduce the SR Ca2+ content in the presence of thapsigargin (Fig. 7B and C). Although our data and previous studies have suggested that cADPR may activate RyRs by causing dissociation of FKBP12.6 from RyR2, there is a lack of corresponding evidence to confirm this hypothesis in smooth muscle. It has been reported that FKBP12.6 binds to RyR2 selectively and that on addition of FK506, FKBP12.6 proteins dissociate from RyRs resulting in the formation of FK506FKBP12.6 complexes [37,28]. In the present study we show that
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cADPR causes significant dissociation of FKBP12.6 from the SR microsomes and the RYR2 complexes of mouse bladder smooth myocytes (Fig. 6A and B). This is in line with observations in islet microsomes and cardiomyocytes [13,33]. Since cADPR binds to and reverses the effects of FKBP12.6 on RyRs, an increase in the probability of open RyR2s would result in an augmentation of Ca2+ release from the SR. Thus, it is reasonable to hypothesise that cADPR alters SR Ca2+ load in smooth myocytes by affecting RyR2 activity in mouse bladder smooth myocytes. It has been reported that FKBP12.6 over-expression significantly increase the SR Ca2+ load and reduce the SR Ca2+ content in cardiac myocytes [10,11,38]. However, it has also been reported that FK506 fails to alter SR Ca2+ content in mouse ventricular myocytes [39] and even increased SR Ca2+ content in rat cardiac myocytes [30]. In the present study, we demonstrate that cADPR significantly reduces SR Ca2+ content in wildtype but not in FKBP12.6 knockout cells, implying that the reduction of SR Ca2+ caused by cADPR was mediated by FKBP12.6 proteins in mouse bladder smooth muscle. cADPR fails to induce Ca2+ release in other smooth muscle types [21] suggesting the existence of variation in the response to cADPR in different tissues and species. Our results also indicate that SR Ca2+ content is lower in FKBP12.6 null myocytes compared to that of wildtype cells (Fig. 7), suggesting that a Ca2+ “leaky” state also exists in smooth muscle due to the deletion of FKBP12.6 proteins. In summary, our study demonstrates that the alteration of Ca2+ release properties by cADPR is mediated by FKBP12.6 proteins in mouse bladder smooth muscle.
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
Conflict of interest statement [21]
None declared. [22]
Acknowledgements We thank Dr. Peace Cheng for critical reading of the manuscript and Dr. Andrew Marks for the gift of JTV-519 Sources of funding: Supported by the National Basic Research Program of China (2007CB512100 and 2009CB918701), the Knowledge Innovation Program of the Chinese Academy of Sciences (Grant No: KSCX2-YW-R-50), the National Foundation of Science and Technology (#30670505), and the ‘863’ research program (2006AA02A106).
[23]
[24] [25]
[26]
[27]
References [28] [1] M.J. Berridge, Inositol trisphosphate and calcium signalling, Nature 361 (1993) 315–325. [2] M.T. Nelson, H. Cheng, M. Rubart, L.F. Santana, A.D. Bonev, H.J. Knot, W.J. Lederer, Relaxation of arterial smooth muscle by calcium sparks, Science 270 (1995) 633–637. [3] M.L. Collier, G. Ji, Y. Wang, M.I. Kotlikoff, Calcium-induced calcium release in smooth muscle: loose coupling between the action potential and calcium release, J. Gen. Physiol. 115 (5) (2000) 653–662. [4] E. Lam, M.M. Martin, A.P. Timerman, C. Sabers, S. Fleischer, T. Lukas, R.T. Abraham, S.J. O’Keefe, E.A. O’Neill, G.J. Wiederrecht, A Novel FK506 binding protein can mediate the immunosuppressive effects of FK506 and is associated with the cardiac ryanodine receptor, J. Biol. Chem. 270 (44) (1995) 26511–26522. [5] H.B. Xin, K. Rogers, Y. Qi, T. Kanematsu, S. Fleischer, Three amino acid residues determine selective binding of FK506-binding protein 12.6 to the cardiac ryanodine receptor, J. Biol. Chem. 274 (1999) 15315–15319. [6] G. Ji, M.E. Feldman, K.S. Greene, V. Sorrentino, H.B. Xin, M.I. Kotlikoff, RYR2 proteins contribute to the formation of Ca2+ sparks in smooth muscle, J. Gen. Physiol. 123 (4) (2004) 377–386. [7] M. Yano, S. Kobayashi, M. Kohno, M. Doi, T. Tokuhisa, S. Okuda, M. Suetsugu, T. Hisaoka, M. Obayashi, T. Ohkusa, M. Kohno, M. Matsuzaki, FKBP12.6-mediated stabilization of calcium-release channel (ryanodine receptor) as a novel therapeutic strategy against heart failure, Circulation 107 (3) (2003) 477–484. [8] F. Huang, J. Shan, S. Reiken, X.H. Wehrens, A.R. Marks, Analysis of calstabin2 (FKBP12.6)-ryanodine receptor interactions: rescue of heart failure by calstabin2 in mice, Proc. Natl. Acad. Sci. U.S.A. 103 (9) (2006) 3456–3461. [9] H.B. Xin, T. Senbonmatsu, D.S. Cheng, Y.X. Wang, J.A. Copello, G.J. Ji, M.L. Collier, K.Y. Deng, L.H. Jeyakumar, M.A. Magnuson, T. Inagami, M.I. Kotlikoff, S. Fleis-
[29]
[30]
[31]
[32]
[33]
[34]
[35]
cher, Oestrogen protects FKBP12. 6 null mice from cardiac hypertrophy, Nature 416 (2002) 334–338. J. Prestle, P.M. Janssen, A.P. Janssen, O. Zeitz, S.E. Lehnart, L. Bruce, G.L. Smith, G. Hasenfuss, Overexpression of FK506-binding protein FKBP12.6 in cardiomyocytes reduces ryanodine receptor-mediated Ca2+ leak from the sarcoplasmic reticulum and increases contractility, Circ. Res. 88 (2) (2001) 188–194. A.M. Gómez, I. Schuster, J. Fauconnier, J. Prestle, G. Hasenfuss, S. Richard, FKBP12.6 overexpression decreases Ca2+ spark amplitude but enhances [Ca2+ ]i transient in rat cardiac myocytes, Am. J. Physiol. Heart Circ. Physiol. 287 (5) (2004) H1987–H1993. A.P. Timerman, H. Onoue, H.B. Xin, S. Barg, J. Copello, G. Wiederrecht, S. Fleischer, Selective binding of FKBP12.6 by the cardiac ryanodine receptor, J. Biol. Chem. 271 (1996) 20385–20391. N. Noguchi, S. Takasawa, K. Nata, A. Tohgo, I. Kato, F. Ikehata, H. Yonekura, H. Okamoto, Cyclic ADP-ribose binds to FK506-binding protein 12.6 to release Ca2+ from islet microsomes, J. Biol. Chem. 272 (1997) 3133–3136. W.X. Tang, Y.F. Chen, A.P. Zou, W.B. Campbell, P.L. Li, Role of FKBP12.6 in cADPRinduced activation of reconstituted ryanodine receptors from arterial smooth muscle, Am. J. Physiol. Heart Circ. Physiol. 282 (2002) H1304–H1310. Y.X. Wang, Y.M. Zheng, Q.B. Mei, Q.S. Wang, M.L. Collier, S. Fleischer, H.B. Xin, M.I. Kotlikoff, FKBP12.6 and cADPR regulation of Ca2+ release in smooth muscle cells, Am. J. Physiol. Cell Physiol. 286 (2004) C538–C546. B.R. Fruen, J.R. Mickelson, N.H. Shomer, P. Velez, C.F. Louis, Cyclic ADP-ribose does not affect cardiac or skeletal muscle ryanodine receptors, FEBS Lett. 352 (2) (1994) 123–126. X. Guo, M.A. Laflamme, P.L. Becker, Cyclic ADP-ribose does not regulate sarcoplasmic reticulum Ca2+ release in intact cardiac myocytes, Circ. Res. 79 (1) (1996) 147–151. J.A. Copello, Y. Qi, L.H. Jeyakumar, E. Ogunbunmi, S. Fleischer, Lack of effect of cADP-ribose and NAADP on the activity of skeletal muscle and heart ryanodine receptors, Cell Calcium 30 (4) (2001) 269–284. H.L. Wilson, M. Dipp, J.M. Thomas, C. Lad, A. Galione, A.M. Evans, Adp-ribosyl cyclase and cyclic ADP-ribose hydrolase act as a redox sensor. A primary role for cyclic ADP-ribose in hypoxic pulmonary vasoconstriction, J. Biol. Chem. 276 (14) (2001) 11180–11188. D.W. Cheung, Modulation of spontaneous transient Ca2+ -activated K+ channel currents by cADP-ribose in vascular smooth muscle cells, Eur. J. Pharmacol. 458 (1–2) (2003) 57–59. J.G. McCarron, S. Chalmers, K.N. Bradley, D. MacMillan, T.C. Muir, Ca2+ microdomains in smooth muscle, Cell Calcium 40 (5–6) (2006) 461– 493. V. Lukyanenko, I. Györke, T.F. Wiesner, S. Györke, Potentiation of Ca2+ release by cADP-ribose in the heart is mediated by enhanced SR Ca2+ uptake into the sarcoplasmic reticulum, Circ Res. 89 (7) (2001) 614–622. N. Fritz, J.L. Morel, L.H. Jeyakumar, S. Fleischer, P.D. Allen, J. Mironneau, N. Macrez, RyR1-specific requirement for depolarization-induced Ca2+ sparks in urinary bladder smooth muscle, J. Cell Sci. 120 (Pt 21) (2007) 3784–3791. J.V. Walsh Jr., J.J. Singer, Ca++ activated K+ channels in vertebrate smooth muscle cells, Cell Calcium 4 (1983) 321–330. U. Trieschmann, G. Isenberg, Ca2+ activated K+ channels contribute to the resting potential of vascular myocytes. Ca2+ sensitivity is increased by intracellular Mg2+ ions, Pflugers Arch. 414 (1989) S183–S184. Y.X. Wang, B.K. Fleischmann, M.I. Kotlikoff, Modulation of maxi-K+ channels by voltage-dependent Ca2+ channels and methacholine in single airway myocytes, Am. J. Physiol. 272 (1997) C1151–C1159. A.M. Gomez, H.P. Cheng, W.J. Lederer, D.M. Bers, Ca2+ diffusion and sarcoplasmic reticulum transport both contribute to [Ca2+]i decline during Ca2+ sparks in rat wentricular myocytes, J. Physiol. 462 (1996) 575–581. S.E. Lehnart, C. Terrenoire, S. Reiken, X.H. Wehrens, L.S. Song, E.J. Tillman, S. Mancarella, J. Coromilas, W.J. Lederer, R.S. Kass, A.R. Marks, Stabilization of cardiac ryanodine receptor prevents intracellular calcium leak and arrhythmias, Proc. Natl. Acad. Sci. U.S.A. 103 (20) (2006) 7906–7910. R.P. Xiao, H.H. Valdivia, K. Bogdanov, C. Valdivia, E.G. Lakatta, H. Cheng, The immunophilin FK506-binding protein modulates Ca2+ release channel closure in rat heart, J. Physiol. 500 (1997) 343–354. E. McCall, L. Li, H. Satoh, T.R. Shannon, L.A. Blatter, D.M. Bers, Effects of FK-506 on contraction and Ca2+ transients in rat cardiac myocytes, Circ. Res. 79 (6) (1996) 1110–1121. C.M. Loughrey, T. Seidler, S.L. Miller, J. Prestle, K.E. MacEachern, D.F. Reynolds, G. Hasenfuss, G.L. Smith, Over-expression of FK506-binding protein FKBP12.6 alters excitation-contraction coupling in adult rabbit cardiomyocytes, J. Physiol. 556 (Pt 3) (2004) 919–934. K. Morita, T. Kitayama, S. Kitayama, T. Dohi, Cyclic ADP-ribose requires FK506binding protein to regulate intracellular Ca2+ dynamics and catecholamine release in acetylcholine-stimulated bovine adrenal chromaffin cells, J. Pharmacol. Sci. 101 (1) (2006) 40–51. X. Zhang, Y.N. Tallini, Z. Chen, L. Gan, B. Wei, R. Doran, L. Miao, H.B. Xin, M.I. Kotlikoff, G. Ji, Dissociation of FKBP12.6 from ryanodine receptor type 2 is regulated by cyclic ADP-ribose but not beta-adrenergic stimulation in mouse cardiomyocytes, Cardiovasc. Res. 84 (2) (2009) 253–262. A. Galione, H.C. Lee, W.B. Busa, Ca2+ -induced Ca2+ release in sea urchin egg homogenates: modulation by cyclic ADP-ribose, Science 253 (1991) 1143–1146. L.G. Mészáros, J. Bak, A. Chu, Cyclic ADP-ribose as an endogenous regulator of the non-skeletal type ryanodine receptor Ca2+ channel, Nature 364 (1993) 76–79.
J. Zheng et al. / Cell Calcium 47 (2010) 449–457 [36] P.L. Li, W.X. Tang, H.H. Valdivia, A.P. Zou, W.B. Campbell, cADPribose activates reconstituted ryanodine receptors fromcoronary arterial smooth muscle, Am. J. Physiol. Heart Circ. Physiol. 280 (2001) H208–H215. [37] Y.S. Prakash, M. Kannan, T.F. Walseth, G.C. Sieck, cADPR ribose and [Ca2+ ]i regulation in rat cardiac myocytes, Am. J. Physiol. Heart Circ. Physiol. 279 (2000) 1482–1489.
457
[38] A.B. Brillantes, K. Ondrias, A. Scott, E. Kobrinsky, E. Ondriasová, M.C. Moschella, T. Jayaraman, M. Landers, B.E. Ehrlich, A.R. Marks, Stabilization of calcium release channel (ryanodine receptor) function by FK506-binding protein, Cell 77 (4) (1994) 513–523. [39] Z. Su, K. Sugishita, F. Li, M. Ritter, W.H. Barry, Effects of FK506 on [Ca2+ ]i differ in mouse and rabbit ventricular myocytes, J. Pharmacol. Exp. Ther. 304 (1) (2003) 334–341.