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- Publisher
- American Society for Biochemistry and Molecular Biology
- Copyright
- Copyright © 2015 Elsevier Inc.
- ISSN
- 0021-9258
- eISSN
- 1083-351X
- DOI
- 10.1074/jbc.m114.618827
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Abstract
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 12, pp. 7736 –7746, March 20, 2015 © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Roles of the NH -terminal Domains of Cardiac Ryanodine Receptor in Ca Release Activation and Termination Received for publication, October 13, 2014, and in revised form, January 9, 2015 Published, JBC Papers in Press, January 27, 2015, DOI 10.1074/jbc.M114.618827 ‡1 ‡2 ‡ ‡ ‡ § ‡ ‡ Yingjie Liu ,BoSun , Zhichao Xiao , Ruiwu Wang , Wenting Guo , Joe Z. Zhang , Tao Mi , Yundi Wang , § ¶ ‡3 Peter P. Jones , Filip Van Petegem , and S. R. Wayne Chen From the Libin Cardiovascular Institute of Alberta, Departments of Physiology and Pharmacology and Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada, Department of Physiology and HeartOtago, University of Otago, Dunedin 9054, New Zealand, and Cardiovascular Research Group, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada Background: The NH -terminal region of cardiac ryanodine receptor (RyR2) contains three domains (A, B, and C) that harbor many disease-causing mutations. Results: Domains A, B, and C distinctively regulate the activation and termination of Ca release. Conclusion: Individual NH -terminal domains play distinct roles in RyR2 channel function. Significance: These data shed new insights into the actions of RyR2 NH -terminal disease mutations. The NH -terminal region (residues 1–543) of the cardiac relationship of the NH -terminal domains of RyR2 and the 2 2 ryanodine receptor (RyR2) harbors a large number of mutations action of NH -terminal disease mutations. associated with cardiac arrhythmias and cardiomyopathies. Functional studies have revealed that the NH -terminal region is involved in the activation and termination of Ca release. The cardiac ryanodine receptor (RyR2) is an essential player The three-dimensional structure of the NH -terminal region in excitation-contraction coupling in the heart. It governs the has recently been solved. It is composed of three domains (A, B, release of Ca from the sarcoplasmic reticulum that drives and C). However, the roles of these individual domains in Ca muscle contraction (1, 2). This RyR2-mediated sarcoplasmic release activation and termination are largely unknown. To reticulum Ca release also plays a critical role in the control of understand the functional significance of each of these NH - heart rhythm (1, 2). Consistent with its fundamental role in terminal domains, we systematically deleted these domains and cardiac function, naturally occurring mutations in RyR2 are 2 2 assessed their impact on caffeine- or Ca -induced Ca release associated with cardiac arrhythmias and cardiomyopathies and store overload-induced Ca release (SOICR) in HEK293 (2–5). Interestingly, most of the disease-associated RyR2 muta- cells. We found that all deletion mutants were capable of form- tions are clustered in three hot spots in the linear sequence of ing caffeine- and ryanodine-sensitive functional channels, indi- the channel: the NH -terminal, central, and COOH-terminal cating that the NH -terminal region is not essential for channel regions (5, 6). Although the functional impact of disease-linked gating. Ca release measurements revealed that deleting RyR2 mutations has been extensively studied, the molecular domain A markedly reduced the threshold for SOICR termina- basis of actions of these disease mutations is largely unknown. tion but had no effect on caffeine or Ca activation or the This is in part due to the lack of understanding of the structure- threshold for SOICR activation, whereas deleting domain B sub- function relationship in the RyR2 channel. stantially enhanced caffeine and Ca activation and lowered The recently solved crystal structures of the NH -terminal the threshold for SOICR activation and termination. Con- region of RyR have provided novel insights into the structural versely, deleting domain C suppressed caffeine activation, abol- basis of disease mechanisms associated with the NH -terminal ished Ca activation and SOICR, and diminished protein mutations (7–14). The three-dimensional structure of the expression. These results suggest that domain A is involved in NH -terminal region of RyR contains three domains: domain A channel termination, domain B is involved in channel suppres- (residues 1–217), domain B (residues 218–409), and domain C sion, and domain C is critical for channel activation and expres- (residues 410–543) (9). This NH -terminal region harbors sion. Our data shed new insights into the structure-function more than 50 disease mutations. Interestingly, almost all of the disease-causing mutations in this region are located at domain * This work was supported in part by research grants from the Canadian Insti- interfaces (9). Docking the NH -terminal structure into low tutes of Health Research; the Heart and Stroke Foundation of Alberta, resolution cryoelectron maps of the RyR1 structure places these Northwest Territories, and Nunavut; the Canada Foundation for Innova- NH -terminal domains at the top of the cytoplasmic assembly, tion; and the Heart and Stroke Foundation/Libin Professorship in Cardio- forming a ring structure around the 4-fold axis of the RyR chan- vascular Research (to S. R. W. C.). Recipient of an Alberta Innovates-Health Solutions (AIHS) graduate student- ship award. 2 4 Recipient of an AIHS fellowship award. The abbreviations used are: RyR2, cardiac ryanodine receptor; RyR, ryanodine An AIHS scientist. To whom correspondence should be addressed: Dept. of receptor; IP , inositol 1,4,5-trisphosphate; IP R, inositol 1,4,5-trisphosphate 3 3 Physiology and Pharmacology, University of Calgary, HRIC G48, 3330 Hos- receptor; SOICR, store overload-induced Ca release; SD, suppressor domain; pital Dr. N. W., Calgary, Alberta T2N 4N1, Canada. Tel.: 403-220-4235; Fax: IBC, IP binding core; KRH, Krebs-Ringer-Hepes; ER, endoplasmic reticulum; 403-270-0313; E-mail: [email protected]. CFP, cyan fluorescent protein; Del, deletion. 7736 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 12 • MARCH 20, 2015 This is an Open Access article under the CC BY license. Distinct Roles of NH -terminal Domains of RyR2 nel (9). This central ring structure is connected to the channel Flp-In T-REx Core kit from Invitrogen. Briefly, Flp-In T-REx pore-forming domain via inner branches (15). Furthermore, HEK293 cells were co-transfected with the inducible expres- this central region has been shown to undergo large conforma- sion vector pcDNA5/FRT (flippase recognition target)/TO tional changes upon channel activation (15). Based on these containing the mutant cDNAs and the pOG44 vector encoding observations, it has been hypothesized that disease mutations the Flp recombinase in 1:5 ratios using the calcium phosphate in the NH precipitation method. The transfected cells were washed with -terminal region destabilize domain interfaces, which in turn alters conformational changes in the NH -termi- phosphate-buffered saline (PBS; 137 mM NaCl, 8 mM Na HPO , 2 2 4 nal region that are important for channel gating (7, 9–12, 14). 1.5 mM KH PO , and 2.7 mM KCl, pH 7.4) 24 h after transfec- 2 4 Consistent with this hypothesis, NH -terminal disease muta- tion followed by a change into fresh medium for 24 h. The cells tions have been shown to enhance the activation of the RyR2 were then washed again with PBS, harvested, and plated on new channel (16–19). We have recently shown that a naturally dishes. After the cells had attached (4 h), the growth medium occurring deletion of exon 3, corresponding to residues Asn – was replaced with a selection medium containing 200 g/ml Gly within domain A in the NH -terminal region, markedly hygromycin (Invitrogen). The selection medium was changed every 3–4 days until the desired number of cells was grown. reduces the threshold at which Ca release terminates (18). However, it is unclear how mutations in the NH -terminal The hygromycin-resistant cells were pooled, aliquoted (1 ml), region of RyR2 alter the activation and/or termination of Ca and stored at 80 °C. These positive cells are believed to be release. isogenic because the integration of RyR2 cDNA is mediated by The structure of the NH the Flp recombinase at a single FRT site. -terminal region of RyR is remark- ably similar to that of the inositol 1,4,5-trisphosphate receptor Caffeine-induced Ca Release in HEK293 Cells (IP R) despite considerable differences in their amino acid sequences (9, 20). The IP RNH -terminal region is also com- The free cytosolic Ca concentration in transfected 3 2 posed of three domains: the suppressor domain (SD) (residues HEK293 cells was measured using the fluorescence Ca indi- 1–223), IP binding core- (IBC-) (residues 224–436), and cator dye Fluo-3 AM (Molecular Probes). HEK293 cells grown IBC- (residues 437–604), corresponding to domains A, B, and on 100-mm tissue culture dishes for 18–20 h after subculture C of RyR, respectively (20–23). Functional studies revealed that were transfected with 12–16 g of WT or deletion mutant domains IBC- and IBC- form the IP RyR2 cDNAs. Cells grown for 18–20 h after transfection were binding pocket, whereas the SD inhibits IP binding (20–22, 24, 25). Given the washed four times with PBS and incubated in Krebs-Ringer- structural similarities between the NH -terminal domains of Hepes (KRH) buffer 1 (125 mM NaCl, 5 mM KCl, 1.2 mM IP R and RyR, it is possible that individual NH -terminal KH PO ,6mM glucose, and 25 mM HEPES, pH 7.4 with NaOH) 3 2 2 4 domains of RyR2 may also play a distinct role in channel func- without MgCl and CaCl at room temperature for 40 min and 2 2 tion. To test this possibility, in the present study, we deleted at 37 °C for 40 min. After being detached from culture dishes by individual NH -terminal domains of RyR2 and assessed the pipetting, cells were collected by centrifugation at 1,000 rpm for impact of these deletions on the activation and termination of 2 min in a Beckman TH-4 rotor. Cell pellets were loaded with 10 M Fluo-3 AM in high glucose Dulbecco’s modified Eagle’s Ca release. We found that deletion of domain A markedly delayed the termination of Ca release, whereas deletion of medium at room temperature for 60 min followed by washing domain B significantly enhanced the activation of Ca release. with KRH buffer 1 plus 2 mM CaCl and 1.2 mM MgCl (KRH 2 2 Deletion of domain C drastically reduced the expression of the buffer) three times and resuspended in 150 l of KRH buffer plus 0.1 mg/ml BSA and 250 M sulfinpyrazone. The Fluo-3 channel protein. Our data suggest that individual NH -termi- nal domains of RyR2 are involved in distinct roles in channel AM-loaded cells were added to 2 ml (final volume) of KRH function. buffer in a cuvette. The fluorescence intensity of Fluo-3 AM at 530 nm was measured before and after repeated additions or EXPERIMENTAL PROCEDURES single additions of various concentrations of caffeine (0.025–5 Construction of NH -terminal Deletion Mutants of RyR2 mM) in an SLM-Aminco series 2 luminescence spectrometer with 480-nm excitation at 25 °C (SLM Instruments). For ryan- The NH -terminal deletions in mouse RyR2 were generated odine sensitivity studies, the RyR2 WT or mutant channels by the overlap extension method using PCR (26, 27). Briefly, an were first sensitized by a relatively low concentration of caffeine NheI/ClaI fragment containing deletion-B (Del-B), Del-C, Del- (0.1 or 0.25 mM). The caffeine-sensitized channels were then AB, or Del-ABC was obtained by overlapping PCR and used to treated with ryanodine (25 M). The ryanodine-treated chan- replace the corresponding wild type (WT) fragment in the nels were further activated by multiple additions of a relatively full-length RyR2 cDNA in pcDNA3, which was then subcloned high concentration of caffeine (1 mM). The peak levels of each into pcDNA5. An NheI/AflII fragment containing Del-A was caffeine-induced Ca release were determined and normal- obtained by overlapping PCR and was used to replace the cor- ized to the highest level (100%) of caffeine-induced Ca responding WT fragment. The sequences of all deletions were release for each experiment. confirmed by DNA sequencing. Generation of Stable, Inducible Cell Lines Expressing WT and Single Cell Ca Imaging 2 2 Deletion Mutants of RyR2 Cytosolic Ca Measurements—Cytosolic Ca levels in sta- Stable, inducible HEK293 cell lines expressing RyR2 Del-A, ble, inducible HEK293 cells expressing RyR2 WT or mutants Del-B, Del-C, Del-AB, and Del-ABC were generated using the were monitored using single cell Ca imaging and the fluores- MARCH 20, 2015 • VOLUME 290 • NUMBER 12 JOURNAL OF BIOLOGICAL CHEMISTRY 7737 Distinct Roles of NH -terminal Domains of RyR2 cent Ca indicator dye Fura-2 AM as described previously (16, Western Blotting 28). Briefly, cells grown on glass coverslips for 8–18 h after HEK293 cell lines grown for certain periods of time after induction (as indicated) by 1 g/ml tetracycline (Sigma) were induction were washed with PBS plus 2.5 mM EDTA and har- loaded with 5 M Fura-2 AM in KRH buffer 2 (125 mM NaCl, 5 vested in the same solution by centrifugation for 8 min at 700 mM KCl, 6 mM glucose, 1.2 mM MgCl , and 25 mM HEPES, pH g in an IEC Centra-CL2 centrifuge. The cells were then washed 7.4 with NaOH) plus 0.02% Pluronic F-127 and 0.1 mg/ml BSA with PBS without EDTA and centrifuged again at 700 g for 8 for 20 min at room temperature (23 °C). The coverslips were min. The PBS-washed cells were solubilized in a lysis buffer then mounted in a perfusion chamber (Warner Instruments) containing 25 mM Tris, 50 mM HEPES, pH 7.4, 137 mM NaCl, on an inverted microscope (Nikon TE2000-S). The cells were 1% CHAPS, 0.5% soy bean phosphatidylcholine, 2.5 mM DTT, perfused continuously with KRH buffer 2 containing increasing and a protease inhibitor mixture (1 mM benzamidine, 2 g/ml leupeptin, 2 g/ml pepstatin A, 2 g/ml aprotinin, and 0.5 mM extracellular Ca concentrations (0, 0.1, 0.2, 0.3, 0.5, 1.0, and PMSF). This mixture was incubated on ice for 1 h. Cell lysate 2.0 mM). Caffeine (10 mM) was applied at the end of each exper- was obtained by centrifuging twice at 16,000 g in a micro- iment to confirm the expression of active RyR2 channels. Time centrifuge at 4 °C for 30 min to remove unsolubilized mate- lapse images (0.25 frame/s) were captured and analyzed with rials. The RyR2 WT and mutant proteins were subjected to Compix Simple PCI 6 software. Fluorescence intensities were SDS-PAGE (6% gel) (32) and transferred onto nitrocellulose measured from regions of interest centered on individual cells. membranes at 90 V for 1.5 h at 4 °C in the presence of 0.01% Only cells that responded to caffeine were analyzed. The filters SDS (33). The nitrocellulose membranes containing the used for Fura-2 imaging were 340 26 and 387 11 nm ex transferred proteins were blocked for 30 min with PBS con- and 510 84 nm with a dichroic mirror (410 nm). em taining 0.5% Tween 20 and 5% (w/v) nonfat dried skimmed 2 2 Luminal Ca Measurements—Luminal Ca levels in milk powder. The blocked membrane was incubated with the HEK293 cells expressing RyR2 WT or mutants were measured anti-RyR antibody (34C) (1:1,000 dilution) and then incu- using single cell Ca imaging and the fluorescence resonance bated with the secondary anti-mouse IgG (heavy and light) energy transfer (FRET)-based endoplasmic reticulum (ER) antibodies conjugated to horseradish peroxidase (1:20,000 luminal Ca -sensitive chameleon protein D1ER as described dilution). After washing for 5 min three times, the bound previously (29, 30). The cells were grown to 95% confluence in a antibodies were detected using an enhanced chemilumines- 75-cm flask, passaged with PBS, and plated in 100-mm-diam- cence kit from Pierce. The intensity of each band was deter- eter tissue culture dishes at 10% confluence 18–20 h before mined from its intensity profile obtained using ImageQuant transfection with D1ER cDNA using the calcium phosphate LAS 4000 (GE Healthcare), analyzed using ImageJ software, precipitation method. After transfection for 24 h, the growth and normalized to that of -actin. medium was then changed to an induction medium containing 1 g/ml tetracycline. In intact cell studies, after induction for Statistical Analysis 22 h, the cells were perfused continuously with KRH buffer 2 All values shown are mean S.E. unless indicated otherwise. containing various concentrations of CaCl (0, 1, and 2 mM) and To test for differences between two groups, we used unpaired tetracaine (1 mM) for estimating the store capacity or caffeine Student’s t tests (two-tailed). A p value0.05 was considered to (20 mM) for estimating the minimum store level by depleting be statistically significant. the ER Ca stores at room temperature (23 °C). In permeabi- lized cells studies, the cells were first permeabilized by 50 g/ml RESULTS saponin (31) in incomplete intracellular-like medium (125 mM Construction and Expression of RyR2 NH -terminal Deletion KCl, 19 mM NaCl, and 10 mM HEPES, pH 7.4 with KOH) at Mutants—To understand the role of individual NH -terminal room temperature (23 °C) for 3–4 min. The cells were then domains (A, B, and C) in RyR2 function, we used a deletion switched to complete intracellular-like medium (incomplete approach in which NH -terminal domain A (residues 1–217), B intracellular-like medium plus 2 mM ATP, 2 mM MgCl , 0.05 (residues 218–409), C (residues 410–543), AB (residues mM EGTA, and 100 nM free Ca , pH7.4 with KOH) for 5–6 1–409), or ABC (residues 1–543) was deleted in the full-length min to remove saponin. The permeabilized cells were then per- RyR2 (Fig. 1A). The boundary of each domain was selected fused with various concentrations of Ca (0.1, 0.2, 0.4, 1, and based on the three-dimensional structure of the NH -terminal 10 M) followed by tetracaine (1 mM) for estimating the store region (residues 1–543) of RyR (9, 13). These deletion con- capacity and caffeine (10 mM) for estimating the minimum structs were generated by site-directed mutagenesis and store level by depleting the ER Ca stores. Images were cap- transiently expressed in HEK293 cells. Immunoblotting tured with Compix Simple PCI 6 software every 2 s using an analysis revealed that the expression level of Del-A was inverted microscope (Nikon TE2000-S) equipped with an reduced, whereas the expression level of Del-B was increased S-Fluor 20/0.75 objective. The filters used for D1ER imaging compared with that RyR2 WT. The expression levels of were 436 20 nm for CFP, 500 20 nm for YFP, Del-AB and WT were comparable. Conversely, the expres- ex ex 465 30 nm for CFP, and 535 30 nm for YFP sion level of Del-C or Del-ABC was markedly reduced com- em em with a dichroic mirror (500 nm). The amount of FRET was pared with that of WT (Fig. 1, B and C). Thus, deletion of determined from the ratio of the light emission at 535 and domain C considerably impaired the expression of the RyR2 465 nm. protein. 7738 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 12 • MARCH 20, 2015 Distinct Roles of NH -terminal Domains of RyR2 FIGURE 1. Construction and expression of RyR2 NH -terminal deletion mutants. A, the RyR2 WT and NH -terminal deletion mutants are depicted by 2 2 rectangles (constructs 1– 6). The relative positions of the deleted regions are indicated by solid lines. B, immunoblotting of RyR2 and -actin using stable, inducible HEK293 cell lines expressing WT or the deletion mutants. C, expression levels of deletion mutants normalized to that of WT. Data shown are mean S.E., and error bars represent S.E. (n 3) (*, p 0.05; **, p 0.01 versus WT; NS, not significant). The NH -terminal Deletion Mutants of RyR2 Form Caffeine- (34, 35). Thus, in the presence of ryanodine, the caffeine-acti- and Ryanodine-sensitive Functional Ca Release Channels— vated channels would be modified by ryanodine into a fully activated state, leading to a depletion of the intracellular Ca We first determined whether these NH -terminal deletion mutants are functional. HEK293 cells were transfected with store. Therefore, subsequent additions of caffeine yielded little RyR2 WT or Del-A, Del-B, Del-C, Del-AB, or Del-ABC or no Ca release in ryanodine-treated cells. However, in the mutants. The transfected HEK293 cells were loaded with the absence of ryanodine, a submaximal concentration of caffeine 2 2 fluorescent Ca indicator dye Fluo-3, AM, and the cytosolic induced only partial Ca release, a phenomenon known as 2 2 Ca level was monitored by using a luminescence spectrome- quantal Ca release (36–38). Importantly, similar to cells ter before and after the addition of caffeine or ryanodine. The expressing RyR2 WT, HEK293 cells expressing Del-A, Del-B, RyR2 WT or mutant channels were first sensitized by a rela- Del-C, Del-AB, or Del-ABC all exhibited quantal Ca release tively low concentration of caffeine (0.1 or 0.25 mM). The caf- induced by submaximal concentrations of caffeine in the feine-sensitized channels were then treated with ryanodine (25 absence of ryanodine (Fig. 2, B–F, top panels). Ryanodine pre- M). The ryanodine-treated channels were further activated by treatment rendered all these deletion mutant cells unrespon- multiple additions of a relatively high concentration of caffeine sive to repeated caffeine stimulations (Fig. 2, B–F, bottom pan- (1 mM). As shown in Fig. 2, the ryanodine-untreated (ryano- els). These observations indicate that all these NH -terminal dine) HEK293 cells expressing RyR2 WT responded to repeated deletion mutants are able to form caffeine- and ryanodine-sen- stimulations by submaximal concentrations of caffeine, each sitive functional Ca release channels. It is noted that there resulting in partial Ca release (Fig. 2A, top panel). In contrast, were immediate drops in the fluorescence level after additions M) WT-expressing HEK293 cells treated with ryanodine (25 of caffeine. This is due to fluorescence quenching by caffeine (ryanodine) only responded to the first subsequent stimula- (39, 40). tion by caffeine (Fig. 2A, bottom panel). It is known that ryan- Effect of NH -terminal Deletions on the Sensitivity of Caffeine odine only binds to the open RyR channel and that the binding Activation of RyR2—We next assessed whether NH -terminal of ryanodine converts the channel to a mainly fully open state deletions affect the sensitivity of the RyR2 channel to caffeine MARCH 20, 2015 • VOLUME 290 • NUMBER 12 JOURNAL OF BIOLOGICAL CHEMISTRY 7739 Distinct Roles of NH -terminal Domains of RyR2 FIGURE 2. The NH -terminal deletion mutants of RyR2 form caffeine- and ryanodine-sensitive Ca release channels. HEK293 cells were transfected with RyR2 WT (A), Del-A (B), Del-B (C), Del-C (D), Del-AB (E), or Del-ABC (F) cDNA. Fluorescence intensity of Fluo-3-loaded cells was measured continuously after addition of 0.1 or 0.25 mM caffeine, DMSO, or 25 M ryanodine followed by three doses of 1 mM caffeine (caff)(n 3– 4). Arrows indicate the presence of Ca release in ryanodine-untreated cells and the absence of Ca release in ryanodine-treated cells. Note that the immediate drops in fluorescence after the addition of caffeine were due to fluorescence quenching by caffeine. 2 2 activation. To this end, we determined the response of each of arrhythmogenic spontaneous Ca release during store Ca these deletion mutants to activation by increasing concentra- overload, a process also known as store overload-induced Ca tions of caffeine. As shown in Fig. 3, the level of Ca release in release (SOICR). It is of interest to assess whether deletion of HEK293 cells transfected with RyR2 WT increased progres- individual NH -terminal domains of RyR2 alters the propensity sively with each consecutive addition of caffeine (from 0.05 to for SOICR. To this end, we generated stable, inducible HEK293 1.0 mM) and then decreased with further additions of caffeine cell lines expressing the RyR2 WT and Del-A, Del-B, Del-C, (2.5 and 5 mM) likely due to the depletion of the intracellular Del-AB, and Del-ABC mutants. These HEK293 cells were per- 2 2 Ca stores by the prior additions of caffeine (0.025–1.0 mM) fused with elevating extracellular Ca (0–2.0 mM) to induce (Fig. 3A). The response to caffeine activation of HEK293 cells spontaneous Ca oscillations as described previously (16, 28). transfected with Del-A was similar to that of WT-expressing The resultant SOICR was then monitored by using a fluores- 2 2 cells (Fig. 3, B and G). Conversely, Del-B caused a significant cence Ca indicator, Fura-2 AM, and single cell Ca imaging. leftward shift in caffeine response (Fig. 3, C and G), whereas As shown in Fig. 4, HEK293 cells expressing the Del-A (Fig. 4B) Del-C (Fig. 3D) and Del-ABC (Fig. 3F) resulted in a significant and Del-AB (Fig. 4E) mutants exhibited a similar fraction of rightward shift (Fig. 3G). Del-AB slightly inhibited the caffeine cells that displayed spontaneous Ca oscillations as compared response (Fig. 3, E and G). Collectively, these data indicate that with WT cells (Fig. 4, G and H). In contrast, the Del-B (Fig. 4C) Del-A has no significant effect on the activation of RyR2 by mutant-expressing cells exhibited an increased fraction of caffeine and Del-B enhances it, whereas Del-C reduces it. oscillating cells (p 0.01) as compared with WT (Fig. 4G). NH -terminal Deletions of RyR2 Alter the Propensity for Conversely, HEK293 cells expressing Del-C (Fig. 4D) and Del- SOICR—Disease-causing mutations in the NH -terminal ABC (Fig. 4F) showed a caffeine response but no SOICR at all region of RyR2 have been shown to increase the propensity for (Fig. 4H). It is important to note that the enhanced SOICR 7740 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 12 • MARCH 20, 2015 Distinct Roles of NH -terminal Domains of RyR2 Del-C or Del-ABC is unlikely due to the reduced expression level of these mutants as SOICR still occurred in WT-express- ing HEK293 cells when the expression of the WT protein was reduced to a level similar to or less than that of Del-C or Del- ABC (Fig. 5, B and C). Thus, these results demonstrate that Del-A has no major impact on SOICR and Del-B enhances the propensity for SOICR, whereas Del-C abolishes SOICR. Effect of NH -terminal Deletions on the SOICR Activation and Termination Thresholds—To assess the impact of NH - terminal deletions on the activation and termination threshold for SOICR, we monitored the ER luminal Ca dynamics in HEK293 cells using a FRET-based ER luminal Ca -sensing protein, D1ER (29, 30). As shown in Fig. 6, elevating extracellu- 2 2 lar Ca from0to2mM induced spontaneous ER Ca oscil- lations in RyR2 WT-expressing HEK293 cells (depicted as downward deflections of the FRET signal). SOICR occurred when the ER luminal Ca content increased to a threshold level (F ) and terminated when the ER luminal Ca con- SOICR tent fell to another threshold level (F ) (Fig. 6A). The ER termi luminal Ca dynamics in Del-A-, Del-B-, and Del-AB-ex- pressing cells during SOICR is shown in Fig. 6, B, C, and D. The Del-A and Del-AB mutations markedly reduced the SOICR ter- mination threshold (34.7 2.3% in Del-A and 38.0 2.9% in Del-AB versus 59.4 1.0% in WT) (p 0.01) but had no sig- nificant effect on the SOICR activation threshold (93.2 0.4% in Del-A and 92.6 0.7% in Del-AB versus 93.1 0.5% in WT). As a result, the fractional Ca release during SOICR (activa- tion threshold termination threshold) in Del-A or Del-AB mutant cells (58.5 2.5% in Del-A and 54.7 3.6% in Del-AB) was significantly increased compared with that of the WT cells (33.7 0.9%) (p 0.01) (Fig. 6, E, F, and G). Conversely, the Del-B mutation substantially decreased the SOICR activation threshold (80.0 1.0 versus 93.1 0.5% in WT) (p 0.01), which is in agreement with its increased SOICR propensity (Fig. 4). The Del-B mutation also significantly reduced the SOICR termination threshold (41.6 1.4 versus 59.4 1.0% in WT) (p 0.01). The fractional Ca release in Del-B mutant cells (38.4 0.5%) was also significantly different from that of WT cells (33.7 0.9%) (p 0.01) (Fig. 6, E, F, and G). It should be noted that there was no significant difference in the store capacity (F F ) between RyR2 WT and deletion mutant max min cells (Fig. 6H). Consistent with their lack of SOICR activity (Fig. 4), no ER luminal Ca oscillations were observed in HEK293 cells expressing Del-C or Del-ABC (not shown). Furthermore, SOICR did not occur in control HEK293 cells expressing no FIGURE 3. Effect of NH -terminal deletions on the sensitivity of caffeine activation of RyR2. HEK293 cells were transfected with RyR2 WT (A), Del-A RyR2, and SOICR was not affected by the IP R inhibitor xesto- (B), Del-B (C), Del-C (D), Del-AB (E), or Del-ABC (F). Fluorescence intensity of the spongin C (18), indicating that SOICR is mediated by RyR2. Fluo-3-loaded transfected cells before and after additions of increasing con- Collectively, these data indicate that deletion of domain A only centrations of caffeine (0.025–5 mM) was monitored continuously. G,Ca release-cumulative caffeine concentration relationships in HEK293 cells affects the termination threshold for SOICR, whereas deletion transfected with RyR2 WT and NH -terminal deletion mutants. The amplitude of domain B alters both the SOICR activation and termination of each caffeine peak was normalized to that of the maximum peak for each experiment. Data shown are mean S.E., and error bars represent S.E. (n 7) thresholds. (*, p 0.05 versus WT). Effect of NH -terminal Deletions on the Cytosolic Ca Regu- lation of Ca Release—To determine the impact of NH -ter- activity observed in Del-B-expressing HEK293 cells is unlikely minal deletions on the regulation of Ca release by cytosolic 2 2 to result from its increased expression level because enhanced Ca , we measured the steady state ER Ca level in permea- SOICR activity was still observed in Del-B-expressing HEK293 bilized HEK293 cells (31) expressing the RyR2 WT or deletion cells when the expression of Del-B was reduced to a level less mutants in the presence of increasing cytosolic Ca concen- than that of WT (Fig. 5, A and C). Similarly, the lack of SOICR in M). The steady state ER Ca trations (0.1–10 level likely MARCH 20, 2015 • VOLUME 290 • NUMBER 12 JOURNAL OF BIOLOGICAL CHEMISTRY 7741 Distinct Roles of NH -terminal Domains of RyR2 FIGURE 4. NH -terminal deletion mutations of RyR2 alter the propensity for SOICR. Stable, inducible HEK-293 cells expressing RyR2 WT (A), Del-A (B), Del-B (C), Del-C (D), Del-AB (E), or Del-ABC (F) were loaded with 5 M Fura-2 AM in KRH buffer 2. The cells were then perfused continuously with KRH buffer 2 2 2 containing increasing levels of extracellular Ca (0 –2 mM) to induce SOICR. Fura-2 ratios were recorded using epifluorescence single cell Ca imaging. G and H, percentages of RyR2 WT (248 cells), Del-A (256 cells), Del-B (298 cells), Del-C (214 cells), Del-AB (287 cells), and Del-ABC (225 cells) cells that display Ca oscillations at various extracellular Ca concentrations. Data shown are mean S.E., and error bar represent S.E. (n 3– 4) (*, p 0.01 versus WT). Caff, caffeine. 2 2 reflects the equilibrium between ER Ca vation of RyR2. Cells expressing Del-AB exhibited reduced release and Ca 2 2 2 uptake. As shown in Fig. 7, elevating cytosolic Ca reduced the steady state ER Ca level at 200 nM cytosolic Ca as com- steady state ER Ca level in permeabilized HEK293 cells pared with that in WT cells (Fig. 7, D and G), suggesting that expressing RyR2 WT in a concentration-dependent manner Del-AB is able to sensitize RyR2 to cytosolic Ca activation. most likely due to increased Ca release as a result of en- However, Del-AB cells displayed increased steady state ER 2 2 2 hanced cytosolic Ca activation of the RyR2 channel (Fig. 7, A Ca levels at 1 and 10 M cytosolic Ca as compared with and G). HEK293 cells expressing Del-A showed a response to those in WT cells (Fig. 7, D and G), suggesting that Del-AB may 2 2 cytosolic Ca similar to that seen with the WT cells (Fig. 7, B also sensitize RyR2 to cytosolic Ca -dependent inactivation. and G). Conversely, HEK293 cells expressing Del-B showed a The steady state ER Ca level in HEK293 cells expressing 2 2 very different response to cytosolic Ca (Fig. 7, C and G). The Del-C or Del-ABC did not respond to increasing cytosolic Ca 2 2 steady state ER Ca level at resting cytosolic Ca (100 nM)in concentrations (100 nM–10 M) and was only slightly reduced Del-B cells was markedly reduced as compared with that in WT upon caffeine addition (Fig. 7, E and F). These data indicate that Del-C and Del-ABC diminish the cytosolic Ca cells (42.2 0.03% in Del-B versus 73.3 0.01% in WT) (p response and 0.001). This suggests that Del-B may enhance cytosolic Ca impair caffeine activation of RyR2. Taken together, our results activation of RyR2. Increasing cytosolic Ca from 100 to 200 suggest that the NH -terminal domains play an important role 2 2 nM reduced the steady state ER Ca level in Del-B cells simi- in cytosolic Ca activation and inactivation of RyR2. larly to that seen in WT cells. However, different from that seen DISCUSSION in WT cells, further elevation in cytosolic Ca concentration to 400 nM,1 M, and 10 M increased the steady state ER Ca The NH -terminal region of RyR2 is a hot spot of naturally level in Del-B cells (Fig. 7, C and G). These observations suggest occurring mutations associated with cardiac arrhythmias and that Del-B may also enhance cytosolic Ca -dependent inacti- cardiomyopathies (5, 6). We have recently shown that disease- 7742 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 12 • MARCH 20, 2015 Distinct Roles of NH -terminal Domains of RyR2 mutations A77V and R176Q and exon 3 deletion markedly reduce the termination threshold for Ca release (9). Interest- ingly, these mutations are located in the domain interface between domain A and the central electron-dense columns (also known as interface 4) (9, 15), suggesting that interface 4 may be involved in Ca release termination. The intra- and intersubunit interactions between domains A and B are believed to be important for stabilizing the closed state of the channel. Disease mutations located in interfaces between domains A and B would weaken these interactions, thus facilitating channel opening (7–14). Del-A would be expected to remove both intra- and intersubunit interactions between domains A and B, leading to destabilization of the closed state and channel activation. Surprisingly, Del-A did not significantly affect channel activation. The sensitivity to activa- tion by caffeine or Ca , the propensity for SOICR, or the SOICR activation threshold of the Del-A mutant were not sig- nificantly different from those of the WT. Conversely, deleting domain B (Del-B) significantly enhanced the sensitivity of RyR2 FIGURE 5. Enhanced SOICR activity of Del-B and reduced SOICR activity of 2 to caffeine, increased cytosolic Ca activation and the propen- Del-C and Del-ABC are not due to altered expression levels. A and B, sity for SOICR, and reduced the threshold for SOICR activa- immunoblotting of RyR2 using stable, inducible HEK293 cell lines after induc- tion for different periods of time (8, 14, or 18 h). C, percentages of RyR2 WT (8 tion. These observations suggest that disease mutations located or 18 h) and Del-B (14 h) cells that display Ca oscillations at various extra- in interfaces between domains A and B may enhance channel cellular Ca concentrations. No SOICR was detected in cells expressing Del-C or Del-ABC 18 h after induction. Data shown are mean S.E., and error bars activity by affecting the function of domain B. It should be represent S.E. (n 3) (*, p 0.05 versus WT at 18 h). WB, Western blot. noted that Del-B also reduced the threshold for Ca release termination, implying that domain B may also be involved in causing RyR2 mutations in the NH -terminal region alter the Ca release termination directly or indirectly via interaction activation and/or termination of Ca release (18). However, with domain A. Furthermore, Del-B also altered the cytosolic how the NH -terminal region regulates the activation and ter- Ca -dependent inactivation of RyR2. Thus, domain B plays an mination of Ca release and how mutations in this region important role in stabilizing the closed state of the RyR2 impair these processes are unclear. The NH -terminal region of channel. RyR2 encompasses three well defined domains: domain A (res- Del-A or Del-B resulted in gain of function either by delaying 2 2 idues 1–217), domain B (residues 218–409), and domain C Ca release termination or by sensitizing Ca release activa- (residues 410–543) (9, 13). In the present study, we assessed the tion. In contrast, deleting domain C (Del-C) suppressed caf- role of these individual domains in Ca release activation and feine activation of RyR2 and completely abolished cytosolic termination. Our data indicate that domain A is an important Ca activation and SOICR. Furthermore, unlike Del-A or determinant of Ca release termination, whereas domains B Del-B, Del-C drastically reduced the protein expression of and C play a critical role in Ca release activation. These RyR2. It should be noted that reducing the expression level of results provide novel insights into the structure-function rela- WT similar to or less than that of Del-C did not abolish SOICR tionship of the NH -terminal domains of RyR2 and the under- in WT-expressing cells. Thus, the lack of SOICR in Del-C-ex- standing of disease mechanisms. pressing cells is unlikely due solely to their reduced expression The NH -terminal domains (A, B, and C) of RyR have been level. These observations suggest that domain C is required for mapped to the central region around the 4-fold symmetry axis channel activation and expression. of the channel. There are extensive domain-domain interac- Docking the crystal structure of the NH -terminal domains tions in the NH -terminal region. Domains A and B through of RyR1 in the open and closed states of the cryo-EM structure intra- and intersubunit interactions form a central ring struc- of RyR1 revealed that the opening of the channel is associated ture at the top of the cytoplasmic assembly (9). This ring struc- with large conformational changes in the NH -terminal ture is connected to the transmembrane domain of the channel domains (14, 15). These have been confirmed in recent FRET- via some central electron-dense columns and to the peripheral based studies using conformational probes inserted into the “clamp” region via domain C (9, 15). To gain insights into the NH -terminal domains (41). During the transition from the functional significance of these domain-domain interactions, closed to the open state, the triangle-like structure formed by we determined the role of each NH -terminal domain in chan- domains A, B, and C within the same subunit appear to be tilted nel function. We found that removing domain A (Del-A) mark- upward and outward around a hinge located near domain C. As edly reduced the threshold for Ca release termination, sug- such, domains A and B rotated 7–8 Å, whereas domain C gesting that domain A is involved in the termination of Ca rotated 4 Å (14). Hence, part of domain C may act as a hinge release. Hence, it is possible that mutations that alter interac- and play an important structural role in mediating and control- tions with domain A may affect Ca release termination. We ling the movement of domains A and B during channel gating. have recently shown that cardiomyopathy-associated RyR2 Therefore, deleting domain C may affect the structure/folding MARCH 20, 2015 • VOLUME 290 • NUMBER 12 JOURNAL OF BIOLOGICAL CHEMISTRY 7743 Distinct Roles of NH -terminal Domains of RyR2 FIGURE 6. Effect of NH -terminal deletions on the SOICR activation and termination thresholds. Stable, inducible HEK293 cell lines expressing RyR2 WT (A), Del-A (B), Del-B (C), or Del-AB (D) were transfected with the FRET-based ER luminal Ca -sensing protein D1ER and induced using tetracycline before the experiment. The cells were perfused with KRH buffer 2 containing increasing levels of extracellular Ca (0 –2 mM) to induce SOICR. FRET recordings from representative cells (a total of 40 –75 cells each) are shown. To minimize the influence by CFP/YFP cross-talk, we used relative FRET measurements for calculating the activation threshold (E) and termination threshold (F) using the equations shown in A. F indicates the FRET level at which SOICR occurs, SOICR whereas F represents the FRET level at which SOICR terminates. The fractional Ca release (G) was calculated by subtracting the termination threshold termi from the activation threshold. The maximum FRET signal F is defined as the FRET level after tetracaine treatment. The minimum FRET signal F is defined max min as the FRET level after caffeine treatment. The store capacity (H) was calculated by subtracting F from F . Data shown are mean S.E., and error bars min max represent S.E. (n 3) (*, p 0.01 versus WT; NS, not significant). of this region, which may contribute to the markedly reduced Crystal structures of the NH -terminal region of the IP R 2 3 expression level of the Del-C or Del-ABC mutant protein. have also been solved recently. The overall structure of the We also determined the impact of deleting the first two NH - NH -terminal region of IP R is very similar to that of RyR (20– 2 2 3 terminal domains (Del-AB) or all three domains (Del-ABC) on 22). As with RyR, IP R contains three NH -terminal domains: 3 2 Ca release. Del-AB substantially reduced the termination the SD, IBC-, and IBC-, corresponding to domains A, B, and threshold for Ca release, which is consistent with the impact C in RyR, respectively. The functional role of individual NH - of Del-A or Del-B on Ca release termination. Del-AB also terminal domains of IP R has been well studied. IBC- and enhanced cytosolic Ca -dependent activation and inactiva- IBC- are involved in IP binding, whereas the SD is believed to tion of RyR2 similarly to Del-B. However, unlike Del-B, Del-AB clamp domains IBC- and IBC- in a conformation with did not significantly affect the activation of SOICR. One would reduced affinity for IP , thus acting as a suppressor of IP bind- 3 3 expect that Del-AB would have the combined effect of Del-A ing (20–25). Interestingly, it has recently been shown that the and Del-B, but this is not the case. The reason for this seemingly IP R SD and domain A of RyR are functionally interchangeable (20). An RyR-IP R chimeric channel in which the SD in the contradictory data is unclear. It is possible that the stimulating effect of Del-B on Ca release may require the presence of full-length IP R was replaced with domain A of RyR was still domain A. Del-ABC markedly inhibited caffeine activation, gated by IP . These observations suggest that the SD of IP R and 3 3 reduced protein expression, and completely abolished cytosolic domain A of RyR may share similar functional roles. However, Ca activation of RyR2 and SOICR, which are similar to the it is important to know that deletion of the SD in IP R com- effects of Del-C, suggesting that Del-C has a dominant impact pletely abolished IP -induced Ca release (25), whereas Del-A on channel function. These results also demonstrate that the or even the deletion of the entire NH -terminal region (Del- NH -terminal region is not essential for the gating of the RyR2 ABC) retained caffeine-induced Ca release. Thus, the respec- channel, although it plays an important role in regulating it. -terminal region plays a very different role in IP -de- tive NH 2 3 7744 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 12 • MARCH 20, 2015 Distinct Roles of NH -terminal Domains of RyR2 FIGURE 7. Effect of NH -terminal deletions on cytosolic Ca regulation of RyR2. Stable, inducible HEK293 cell lines expressing RyR2 WT (A), Del-A (B), Del-B (C), Del-AB (D), Del-C (E), or Del-ABC (F) were transfected with the FRET-based ER luminal Ca -sensing protein D1ER and induced using tetracycline. The transfected and induced cells were permeabilized with saponin, washed, and perfused with intracellular-like medium plus increasing levels of free Ca (0.1, 0.2, 0.4, 1 and 10 M). FRET recordings from representative cells (total 59 –91 cells each) are shown. To minimize the influence by CFP/YFP cross-talk, we used relative FRET measurements for calculating the steady state ER Ca level (defined in A)(G). The dashed lines (F –F ) indicate the steady state FRET levels after 0.1 10 perfusion with each Ca concentration (0.1, 0.2, 0.4, 1, or 10 M). The maximum FRET signal F is defined as the FRET level after tetracaine treatment. The max minimum FRET signal F is defined as the FRET level after caffeine treatment. Data shown are mean S.E., and error bars represent S.E. 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Uchida, K., Miyauchi, H., Furuichi, T., Michikawa, T., and Mikoshiba, K. 2051–2060 7746 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 12 • MARCH 20, 2015
Journal
Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology
Published: Mar 20, 2015
Keywords: Calcium Imaging; Calcium Intracellular Release; Cardiac Muscle; Ryanodine Receptor; Sarcoplasmic Reticulum (SR)