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J. Biol. Chem., Vol. 278, Issue 41, 39644-39652, October 10, 2003
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From the Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115
Received for publication, May 16, 2003 , and in revised form, July 25, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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An essential aspect yet to be solved in the understanding of the mechanism(s) that control EC coupling is the identification of the domains of the DHPR and RyR1 involved in the bidirectional signaling. Based on a yeast two-hybrid approach, it has been reported recently (9) that the RyR1 domain encompassing residues 1,837–2,168 can carry out physical interactions with the II–III loop of DHPR and, in addition, can restore weak skeletal type EC coupling when expressed as an RyR1/RyR2 chimera. In agreement with this report, Nakai et al. (10), using chimeric RyR1/RyR2 receptors, showed that an RyR1/RyR2 chimera encompassing amino acids 1,635–3,720 of RyR1 is able to rescue both DHPR Ca2+ currents and skeletal type EC coupling and suggested that this region was the critical component of RyR1 needed to restore bi-directional signaling between the two proteins. By using RyR1/RyR3 chimera we have recently defined two separate regions of RyR1 within amino acids 1,681–3,770 that are independently capable of restoring skeletal EC coupling to RyR3 (11). Interestingly, although this domain of RyR1 was able to restore EC coupling in the context of both RyR3- and RyR2-based chimera, important differences were observed between the two. In myotubes expressing RyR3-based chimeras, both the number of myotubes in which skeletal EC coupling was restored and the amplitude of the depolarization-induced Ca2+ transients seemed to be smaller than in myotubes expressing RyR2-based chimeras with similar RyR1 insertions. The reduced efficiency of RyR3-based chimeras in rescuing skeletal EC coupling suggests that in addition to the critical domains within RyR1 aa 1,681–3,770, other regions are involved in this interaction.
Leong and MacLennan (12) demonstrated that residues 922–1,117 of RyR1 were able to bind to the II–III loop of the DHPR 
1S subunit and suggested that this region may be involved in EC coupling. Yamazawa et al. (13) have reported that the deletion of the high divergence "D2 domain" from RyR1 (aa 1,303–1,406) resulted in the loss of depolarization-induced Ca2+ release in myotubes, and they suggested the possibility that this domain alone is required for skeletal EC coupling. Interestingly, the D2 region represents one of the most prominent differences among the three RyRs isoforms. This domain is present in both RyR1 (aa 1,342–1,403) and RyR2 (aa 1,316–1,400) but is almost completely absent in RyR3 (14).
In an attempt to map the critical domains of RyR1 that have functional relevance for EC coupling in this study, we have evaluated the ability of a series of RyR1/RyR3 chimeric receptors to restore skeletal EC coupling to RyR1 null 1B5 myotubes. We report that in addition to amino acids 1,681–3,770, the N-terminal domain of RyR1 (aa 1–1,680) also holds key determinants needed to support skeletal EC coupling. We show evidence that although the RyR1 D2 domain by itself was unable to restore skeletal type EC coupling to RyR3, the addition of this domain to RyR1/RyR3 chimeras significantly improved the efficiency of skeletal coupling restored by other regions of RyR1, revealing that this domain of RyR1 helps to optimize its interaction with DHPR.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Nucleotides 3,812–4,369 from RyR1, carrying divergence domain D2, were inserted in-frame to replace nucleotides 3,810–4,047 of RyR3 in both wtRyR3 to create chimera RyR3+D2 and Ch-4 to create chimera Ch4+D2. Similarly, nucleotides 3,810–4,047 of RyR3 were substituted with the corresponding nucleotides of wtRyR1 (fragment 3,812–4,369) to create chimera RyR1-D2.
Cell Culture and Calcium Imaging—Dyspedic 1B5 myoblasts (which lack expression of all RyRs) were cultured and differentiated in 96-well plates (Costar®, Corning Glass) as described previously (11, 16). Differentiated myotubes were then infected with 2.5 x 104 HSV-1 amplicon virion particles (17) containing either wt or chimeric cDNAs for 2 h, and Ca2+ imaging was performed during the stable phase of transduced protein expression in the myotubes 36–48-h post-infection. For imaging, cells were loaded for 30 min with 5 µM Fluo-4AM (Molecular Probes Inc., Eugene, OR) at 37 °C in imaging buffer (125 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1.2 mM MgSO4, 6 mM glucose, and 25 mM HEPES, pH 7.4, supplemented with 0.05% bovine serum albumin). To prevent calcium entry through the sarcolemma from adding to the signal observed, the cells were then washed with imaging buffer with no added Ca2+ (
7 µM free Ca2+) supplemented with 0.5 mM CdCl2 and 0.1 mM LaCl3. The ability of the constructs to support depolarization-induced Ca2+ release was tested by exposing the cells to chemical depolarization with a 10-s exposure to high K+ buffer (50 mM NaCl, 80 mM KCl, 1.2 mM MgSO4, 6 mM glucose, and 25 mM HEPES, pH 7.4) supplemented with Cd2+ and La3+ using a multivalve perfusion system (AutoMate Scientific, Inc., Oakland, CA) as described elsewhere (11). Imaging was performed collecting the fluorescence signal at 490–500 nm. The data were collected at 15 frames/second with an intensified 12-bit digital CCD (Stanford Photonics, Stanford, CA), and the data were analyzed using QED Camera Plug-inTM package (QED Imaging Inc., Pittsburgh, PA). Transfection efficiency of the myotubes and proper targeting of the expressed receptors were determined by immunofluorescence using monoclonal antibody 34C that recognizes all three RyR isoforms (18, 19). To rule out that any chimeric receptors that did not restore depolarization-induced EC coupling were either not expressed as full-length proteins or were completely functionally impaired, Western blot, [3H]ryanodine binding, and caffeine dose-response analyses were carried out as described previously (11). In order to evaluate the efficiency of EC coupling displayed by any given construct and to compare data from different experiments, we integrated the area under the curve of the KCl-induced Ca2+ transient (peak amplitude, Fi – Fo) and normalized this to the peak fluorescence amplitude induced by the maximal caffeine response (Fmax) of the same cell. Unless otherwise indicated, calcium fluorescence data are presented as means ± S.E. and include only those myotubes that responded to both caffeine and KCl. Statistically significant differences among the data were evaluated by using either unpaired Student's t test analysis with Welsh's correction or one-way analysis of variance (Kruckshal-Wallace) (GraphPad Software, San Diego, CA). N refers to the number of independent experiments for each construct.
Membrane Preparations and Immunoblotting—Crude membrane preparations were made 36 h after infection from 1B5 myotubes that had been allowed to differentiate for 5–6 days prior to transduction with virions carrying each of the different wt and chimeric constructs. Myotubes were harvested in harvest buffer (137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2, and 0.6 mM EDTA) from 10 to 15 100-mm plates and centrifuged for 10 min at 250 x g. The pellet was resuspended in buffer consisting of 250 mM sucrose, 10 mM HEPES, pH 7.4, supplemented with 1 mM EDTA, 10 µg/ml leupeptin, 0.7 µg/ml pepstatin A, 5 µg/ml aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride and then homogenized using a Polytron cell disrupter (Brinkmann Instruments, Westbury, NY). The whole cell homogenates were centrifuged for 20 min at 1,500 x g, and the supernatants were collected and re-centrifuged for 60 min at 100,000 x g at 4 °C. The membranes were finally resuspended in 250 mM sucrose, 20 mM HEPES, pH 7.4, frozen in liquid N2, and stored at –80 °C. SDS-PAGE (20) was performed on proteins from the crude homogenates as described previously. Immunoblots were incubated with monoclonal antibody 34C (provided by J. A. Airey and J. L. Sutko, University of Iowa), which recognizes both RyR1 and RyR3, and then incubated with horseradish peroxidase-conjugated goat anti-mouse secondary antibody. Immunoreactive proteins were developed with SuperSignal ultra-chemiluminescent substrate (Pierce).
Equilibrium [3H]Ryanodine Binding Assay—Crude membrane extracts (0.1 mg/ml) were incubated at 37 °C for 3 h in buffer containing 0.25 M KCl, 20 mM HEPES, pH 7.2, and 10 nM [3H]ryanodine (PerkinElmer Life Sciences) in the presence of a protein inhibitor mixture. Different free calcium concentrations were obtained by mixing 5 mM EGTA with specific amounts of CaCl2 according to calculation with the WEBMAXC Standard program (21). Nonspecific binding was determined by incubating the vesicles with 5 µM unlabeled ryanodine. Samples were filtrated in Whatman GF/B glass fiber filters using a BrandelTM cell harvester, and filters were washed with ice-cold buffer (20 mM Tris-HCl, pH 7.2). The [3H]ryanodine remaining on the filters was quantified by liquid scintillation spectrometry.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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To rule out a lack of expression or mis-targeting of Ch-4 as a possible explanation for its reduced efficiency for EC coupling, we carried out immunostaining of myotubes infected with equivalent amounts of virus containing R4 and Ch-4. Fig. 1C shows that antibody 34C, which recognizes an identical epitope in RyR1, R4, and Ch-4 (RyR1 residues 2,756–2803 (19)), revealed that all three constructs are expressed and have a punctate pattern of fluorescence. Because this punctate pattern has been characterized previously as the pattern displayed by RyRs when they are co-localized with the DHPR and other triadic proteins, and therefore indicates that they are properly targeted to the calcium release units (16, 22, 23), our results suggest that there are no differences in the targeting of chimeric receptors R4 and Ch-4 that could explain their functional differences.
In order to compare the efficiency of the EC coupling signaling restored by different chimera, we determined the average amplitude of the Ca2+ transient induced by depolarization with 80 mM KCl in each Fluo-4 loaded cell by integrating the signal for the full duration of the calcium transient. To compare data from different cells, the average amplitude of this Ca2+ transient was normalized to the average amplitude of the response obtained with 20 mM caffeine that always represented the maximal response for any construct (Fmax). Fig. 2C shows that, in the absence of extracellular Ca2+ ([Ca2+]o), upon KCl depolarization 82/82 myotubes expressing RyR1 (n = 6) displayed Ca2+ transients with average amplitude of the 111 ± 5.0% of the maximal response induced by caffeine (Fmax). By comparison, Ch-4 expressing myotubes presented a significant reduction in both the number of cells responding (55/137 myotubes, n = 6, p < 0.001 compared with wtRyR1) and the amplitude of the transient induced by KCl depolarization in the responding myotubes (29.9 ± 2.2% of Fmax, p < 0.001).
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In our previous study we showed that, with the exception of its ability to support skeletal type EC coupling, there were no differences in caffeine sensitivity, [3H]ryanodine binding, and molecular size detected between Ch-4 and wtRyR3 (11). Thus, it seems unlikely that the reduced ability of Ch-4 to restore EC coupling stems from an alteration of its overall protein conformation that would cause a change in its ability to function as a Ca2+ channel. Instead, these results suggest that although amino acids residues 1,681–3,770 of RyR1 partially conferred skeletal type EC coupling to RyR3, one or more additional region(s) of RyR1 are needed to fully restore wt skeletal type EC coupling.
To determine the region(s) necessary, we first generated the chimeric receptor Ch-1 that contains the whole putative foot domain of RyR1 (residues 1–3,770) with the C-terminal domain of RyR3 (residues 3,621–4,873; Fig. 2A). Unlike Ch-4, 100% of the myotubes expressing Ch-1 supported skeletal EC coupling (55/55 cells analyzed, n = 2). Moreover, the average Ca2+ transients induced by KCl on these myotubes reached 80.8 ± 1.5% of Fmax, a value 2.7-fold larger than the transients displayed by Ch-4 (p < 0.001) although smaller than wtRyR1 (p < 0.001). On the contrary, myotubes expressing chimeric receptor Ch-3 (Fig. 2, B and C) carrying only the C-terminal domain of RyR1, in an otherwise RyR3 background, was targeted correctly and responded to caffeine but did not display skeletal type EC coupling (0/29 myotubes, n = 2).
By having determined that there were modules in the N-terminal 1/3 of RyR1 that are needed to improve skeletal EC coupling efficiency, we then examined the ability of the chimera Ch-2, which contains only the N-terminal 1/3 of RyR1 (residues 1–1,680, Fig. 2A), to support skeletal type EC coupling. The number of cells expressing Ch-2 responding to K+ (45/50 cells, n = 3) was not significantly different from wtRyR1 (p > 0.05). Analysis of the average amplitude of the KCl-induced Ca2+ transients (Fig. 2C) showed that myotubes expressing chimera Ch-2 displayed a 50% larger than average Ca2+ transient than Ch-4 expressing myotubes but an 60% smaller transient than Ch-1 expressing myotubes (47.7 ± 3.5% (Ch-2) versus 29.9 ± 2.2% (Ch-4) versus 80.8 ± 1.5% (Ch-1) of Fmax). This indicates that the first 1,680 amino acids of the N-terminal domain of RyR1 carry an important element or elements that support skeletal type EC coupling.
The N-terminal Domain of RyR1 Contains Essential Determinant(s) for Skeletal Type EC Coupling—To compare the relative contributions of the Ch-4 and Ch-2 regions of RyR1 to EC coupling signaling, we constructed reverse chimeras in which the corresponding regions of RyR3 were inserted into an RyR1 sequence (Fig. 3A). We found that although the substitution of residues 1,681–3,770 of RyR1 with the corresponding region of RyR3 significantly diminished the ability of Ch-4rev to support skeletal type EC coupling, it did not completely abolish its capacity to do so. In 21/29 myotubes expressing Ch-4rev (n = 2), the average peak amplitude of KCl-induced Ca2+ transients was 15% less than wtRyR1 (84.7 ± 10.7% of Fmax, p < 0.05; Fig. 3B). On the other hand, myotubes expressing chimera Ch-2rev, containing residues 1–1,576 of RyR3 in an RyR1 background, were unable to support skeletal type EC coupling (0/28 myotubes, n = 2; Fig. 3B). These results suggest, at least in regards to supporting skeletal type EC coupling, that the N-terminal domain of RyR1 is more critical than the central portion of the molecule.
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Role of D2 Domain in EC Coupling—Analysis of the primary amino acid sequence between the three RyR isoforms of reveals that one of the most striking differences within the N-terminal region of these channels is the high divergence region D2, which is present in RyR1 and RyR2 but almost completely absent in RyR3 (Fig. 4A). To evaluate whether this domain alone accounts for the ability of the Ch-2 to restore depolarization-induced EC coupling, we constructed the chimeric receptors pictured in Fig. 4B. In these chimeras the region of RyR1 containing the D2 domain (amino acids 1,271–1,456) was replaced with the corresponding region of RyR3, which almost completely lacks the D2 domain (chimera RyR1-D2), and vice versa (chimera RyR3+D2). Fig. 4 shows that, unlike wtRyR1, chimeric RyR1 receptors containing the RyR3 D2 domain demonstrated no skeletal type EC coupling capacity (0/36, n = 2; Fig. 4, C and D). However, no depolarization induced Ca2+ release was observed in RyR3+D2 expressing myotubes (0/59, n = 2; Fig. 4, C and D) as well. These results suggest that although the presence of the D2 domain of RyR1 seems to play a critical role in supporting skeletal type EC coupling by RyR1, this domain by itself is insufficient to support skeletal type EC coupling in the context of RyR3 and must combine with another domain(s) in the N-terminal third to confer skeletal type EC coupling to Ch-2.
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To rule out that the absence of skeletal EC coupling observed in myotubes expressing RyR1-D2, RyR3+D2, and Ch-2rev was because they were either not expressed or had lost their ability to function as Ca2+ release channels, we performed a series of tests to evaluate the functionality of all expressed proteins. As shown in Fig. 5A, Western blot analysis indicates that all chimeric receptors expressed in 1B5 myotubes displayed a molecular size comparable with the wt receptors. Fig. 5B shows representative records of intracellular Ca2+ transients induced by caffeine in myotubes expressing either RyR1-D2, RyR3+D2, or Ch-2rev, demonstrating that these three chimeric receptors behaved as Ca2+ release channels responding to caffeine in a dose-response manner similar to wt receptors. To test the ability of the chimeric constructs to respond to endogenous modulators, [3H]ryanodine binding assays were performed to evaluate the sensitivity of the channels to Ca2+, ATP, and ruthenium red. Fig. 5C shows that all the constructs presented high affinity [3H]ryanodine binding and, like control membranes from myotubes expressing wtRyR1 and wtRyR3, the binding was Ca2+-sensitive. At 100 µM free Ca2+, the addition of5mM ATP induced further increase in [3H]ryanodine binding in all constructs tested and subsequent addition of 10 µM ruthenium red resulted in the inhibition of ryanodine binding. Thus, with the exception of their ability to support skeletal EC coupling, chimeric receptors RyR1-D2, RyR3+D2, and Ch-2rev presented normal Ca2+ release channel behavior.
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D2 Domain Enhances Skeletal Type EC Coupling to RyR1/RyR3 Chimeras—Based on the above results, we hypothesized that the absence of the D2 domain alone could explain the difference in the efficiency of Ch-1 and Ch-4 in restoring skeletal type EC coupling (Fig. 2). To test this hypothesis, we constructed a double chimera shown in Fig. 6A, in which the D2 domain from RyR1 (residues 1,271–1,456 of RyR1) was added to chimera Ch-4. In comparison to myotubes transduced with Ch-4, myotubes transduced with chimera Ch4+D2 showed a significant improvement in both the number of cells responding to KCl-depolarization (from 55/137 in Ch-4 to 40/43 cells in Ch-4+D2, p < 0.001) and the strength of their average depolarization-induced Ca2+ transient amplitude (Fig. 6). Fig, 6, B and C, shows that the presence of D2 domain in Ch4+D2 enhanced the peak Ca2+ transient induced by KCl in 2.0-fold, from 29.9 ± 2.2% of Fmax in Ch-4 to 64.7 ± 4.6% in Ch4 + D2 (p < 0.001). However, this peak amplitude was significantly smaller than the peak amplitude observed in myotubes expressing Ch-1 (80.8 ± 1.5% of Fmax, p < 0.005). Previously, we have shown that Ch-4 region of RyR1 contains at least two separated regions, Ch-21 and Ch-19, that independently can partially restore skeletal EC coupling to RyR1/RyR3 chimeras (11). In order to verify the importance of all these domains, we constructed a chimeric receptor (chimera 2119+D2, Fig. 6A) containing all three domains of RyR1 that, so far, have been found to play some role in skeletal EC coupling. Fig. 6B shows that chimera 2119+D2 was just as efficient as Ch4+D2 in restoring skeletal type EC coupling (19/24 myotubes tested, n = 2, p > 0.05) and had a similar average KCl-induced Ca2+ transient (51.4 ± 4.8% of Fmax, p > 0.05) to the response shown by Ch4+D2 (Fig. 6C). Thus, although the presence of the D2 domain significantly enhances the efficiency of skeletal EC coupling of Ch-4 or its active counterparts Ch-21 and Ch-19, it was not as efficient as Ch-1, indicating that another domain(s) in the N-terminal region is required.
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N-terminal Domain Is More Efficient than D2 Enhancing Skeletal EC Coupling to Key RyR1 Domains—To confirm that the presence of additional domains of the N-terminal region of RyR1 is important for skeletal EC coupling, we constructed a chimeric receptor combining the domains Ch-21, Ch-19, and the whole N-terminal end of RyR1 (2119+Nt, Fig. 6A). As shown in Fig. 6, B and C, chimera 2119+Nt not only was capable of restoring EC coupling to dyspedic myotubes but also displayed significantly more frequent (36/36 myotubes analyzed, n = 2) and stronger (78.5 ± 3.7% of Fmax n = 2) coupling than chimera Ch4+D2 (p < 0.05), 2119+D2 (p < 0.005) and Ch-2 (p < 0.001) and had a peak amplitude similar to the amplitude observed in Ch-1 expressing cells (p > 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Here we present evidence that in addition to domains lying within aa 1,681–3,770, which have been identified in previous studies (10, 11, 16), other domains within aa 1–1,680 of RyR1 contribute one or more elements of structure important for optimizing RyR1/DHPRS interactions in vivo. Replacement of amino acids 1–1,680 of RyR1 with the corresponding region of RyR3 totally eliminates the ability of the chimeric receptor to support any detectable level of depolarization-induced EC coupling but does not affect the capacity of myotubes expressing this chimera to respond to caffeine, or an appropriate modulation of [3H]ryanodine binding in response to Ca2+, ATP, and ruthenium red. Accordingly, replacement of amino acids 1–1,680 of RyR3 with the corresponding region of RyR1 partially restores skeletal EC coupling. We have also shown that a sub-domain lying within this N-terminal end of RyR1, divergence domain D2, is a critical component needed to optimally engage skeletal EC coupling but that other not yet defined domains in the N-terminal region are also needed for "normal" skeletal EC coupling.
Based on immunostaining analysis of myotubes expressing RyR1/RyR2 (23) and RyR1/RyR3 chimeras, we conclude that the reduced efficiency in EC coupling observed between RyR2 and RyR3 based chimeras is not the result of a difference in the level or pattern of expression of the chimeric constructs (R4 and Ch-4 shown as examples). These results are consistent with our previous study in which we have shown no evidence of functional distress in the expressed RyR1/RyR3 receptors, based on [3H]ryanodine binding and Ca2+ release in response to caffeine (11). Furthermore, they suggest that unlike RyR3, there are domains in the N-terminal region of RyR2, although not identical to RyR1, that are sufficiently similar to support skeletal type EC coupling in response to K+ depolarization. These data give further support to the hypothesis that several domains of RyR1 are needed to fully restore RyR1-DHPR signaling.
Interestingly, the previous work of Nakai et al. (10) using RyR1/RyR2 chimeras reported that replacement of amino acids 1–1,622 of RyR2 for the corresponding domain of RyR1 (residues 1–1,631, chimera R2) did not restore skeletal EC coupling to dyspedic myotubes. The explanation for these seemingly divergent results can be found in the experimental conditions used to determine restoration of skeletal type EC coupling in the two studies. Whereas Nakai et al. (10) used electrically evoked Ca2+ release, we used 5–10-s pulses of 80 mM KCl to trigger depolarization, a condition previously proven to be more effective than electrical stimulation to observe weak interactions between the chimeric RyRs and the DHPR (11, 16). In fact, RyR1/RyR2 chimera R2 was tested under the same experimental conditions as the ones described in this study (KCl depolarization), restoring weak skeletal type EC coupling similar to Ch-2 (data not shown).
The importance of the N-terminal domains of RyR1 and RyR2 to skeletal EC coupling has been previously shown. Yamazawa et al. (13) reported that although substitution of divergent domain D2 of RyR1 with the equivalent portion of RyR2 had no apparent change in phenotype, the deletion of residues 1,303–1,406 of RyR1, containing the D2 domain, led to the complete loss of electrically evoked Ca2+ release in dyspedic myotubes. Accordingly, our chimeric construct in which the D2 domain from RyR1 had been replaced with the corresponding domain of RyR3 (RyR1-D2) resulted in the loss of skeletal EC coupling. Our data also show that although the D2 domain by itself could not restore EC coupling to RyR3, the insertion of this region dramatically enhanced the level of EC coupling restored by chimeras containing all or part of the middle third of RyR1 (Ch-4 and Ch-2119). Chimeras Ch4+D2 and 2119+D2 presented a nearly 2.5–5-fold stronger KCl-induced Ca2+ transient than their counterparts without D2 and had a level of response equivalent to 85–90% of the response observed by chimera Ch-1, which contains the whole putative foot domain of RyR1. Furthermore, the level of response of these two D2 containing chimera was similar to that reported for RyR1/RyR2 chimera R4, which contains nearly the same region of RyR1 (aa 1,635–3,720) and the cardiac D2 region (16). By taking into consideration that (i) that addition of the D2 domain significantly improved the EC coupling recovered by chimera Ch-4 and Ch-2119 but did not restore EC coupling by itself to RyR3, (ii) that cardiac and skeletal D2 domains can be interchanged without noticeable alteration in phenotype, and (iii) that the three-dimensional reconstitution of RyR3 revealed that the lack of D2 region in RyR3 resulted in a significant ultrastructure perturbation of the "clamp" domain, the region thought to face the DHPR in the triadic junction (27), it seems likely that D2 domain plays a structural rather than functional role in supporting skeletal EC coupling. We hypothesize that its role is to provide a suitable protein conformation to allow proper exposure of other critical regions that are needed to interact with the DHPR. This hypothesis is supported by the findings of Leong and MacLennan (12, 28) who have shown that what they term the F4 region (amino acids 1,220–1,614) that contains the D2 domain did not bind to either II–III loop or III–IV loop affinity columns. Because there is compelling evidence suggesting that the II–III loop of the 1S subunit is the primary determinant of the DHPR needed to allow bi-directional signaling with RyR1 and to define skeletal or cardiac type EC coupling (29–31), the fact that the D2 domain appears not to interact with the II–III loop somehow makes it unlikely that this divergence domain plays a direct role defining skeletal or cardiac type EC coupling.
From our results with chimera 2119-Nt, it is obvious that there are other regions of the N-terminal third of RyR1 in addition to the D2 domain that enhance the efficiency of skeletal EC coupling restored by domains Ch-19 and Ch-21. Leong and MacLennan (12) have shown that peptide fragment F3, containing aa 922–1,112 of RyR1, interacts in vitro with the skeletal but not cardiac DHPR II–III loop, suggesting that a domain within this region of RyR1 might be involved in mediating specific bi-directional signaling between these two proteins. The actual domains in the N-terminal region that are responsible for the enhancement of skeletal EC coupling remain to be determined.
The selective loss of EC coupling function in chimera Ch-2rev was surprising because the smaller chimera Ch4, which contains the central portion of RyR1 without the C-terminal region of RyR1, has the ability to support skeletal EC coupling, albeit relatively weakly. Furthermore, myotubes expressing Ch-1, holding N-terminal domain of RyR3, showed that the peak Ca2+ transient induced by KCl reached only 88% of the average peak Ca2+ transient displayed by the RyR1 expressing myotubes. On the other hand Ch-4rev, which contains the N- and C-terminal regions of RyR1, had a 1.5-fold larger transient than Ch-2 expressing myotubes that contain only the N-terminal domain of RyR1. These data suggest that the C-terminal portion of RyR1 interacts with the N-terminal region and that this interaction can have both salutatory and inhibitory effects during EC coupling in myotubes expressing chimeric RyRs. The mechanism of this interaction and its role in EC coupling remains an open question. One thing is for certain, our results with chimera Ch-3 indicate that whatever important determinant(s) for EC coupling lay within the aa 3,771–5,038 of RyR1, this domain is not sufficient to independently restore skeletal EC coupling. The fact that several central core disease-associated mutations that lie within the intra-luminal domain (within aa 4,889–4,913) of RyR1 significantly disturb the orthograde but not the retrograde signal between RyR1 and 1S DHPR (32, 33) seems to support at least some indirect role of this region in EC coupling signaling.
Overall, these data further support our previous hypothesis that multiple regions of RyR1 are needed in order to sustain normal bi-directional signaling with the DHPR during EC coupling. Some of these regions, like divergence domain D2, may play structural role whereas others, like the proposed determinants identified within amino acids 922–1,112 (12, 28) and the regions Ch-19 (aa 2,644–3,223) and Ch21 (aa 1,924–2,446), may play a more direct role in interacting with the DHPR and allowing skeletal EC coupling.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Anesthesiology, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. Tel.: 617-732-6881; Fax: 617-732-6927; E-mail: cperez{at}zeus.bwh.harvard.edu.
1 The abbreviations used are: EC, excitation-contraction; RyR, ryanodine receptor; DHPR, dihydropyridine receptor; aa, amino acids; a.u., arbitrary units; wt, wild type.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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A. M. Hurne, J. J. O'Brien, D. Wingrove, G. Cherednichenko, P. D. Allen, K. G. Beam, and I. N. Pessah Ryanodine Receptor Type 1 (RyR1) Mutations C4958S and C4961S Reveal Excitation-coupled Calcium Entry (ECCE) Is Independent of Sarcoplasmic Reticulum Store Depletion J. Biol. Chem., November 4, 2005; 280(44): 36994 - 37004. [Abstract] [Full Text] [PDF]
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X. Altafaj, W. Cheng, E. Esteve, J. Urbani, D. Grunwald, J.-M. Sabatier, R. Coronado, M. De Waard, and M. Ronjat Maurocalcine and Domain A of the II-III Loop of the Dihydropyridine Receptor Cav 1.1 Subunit Share Common Binding Sites on the Skeletal Ryanodine Receptor J. Biol. Chem., February 11, 2005; 280(6): 4013 - 4016. [Abstract] [Full Text] [PDF]
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C. Paolini, J. D. Fessenden, I. N. Pessah, and C. Franzini-Armstrong Evidence for conformational coupling between two calcium channels PNAS, August 24, 2004; 101(34): 12748 - 12752. [Abstract] [Full Text] [PDF]
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