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J. Biol. Chem., Vol. 278, Issue 31, 28727-28735, August 1, 2003
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From the
Department of Anesthesia Research,
Brigham and Women's Hospital, Boston, Massachusetts 02115 and the
¶Department of Molecular Biosciences, School of
Veterinary Medicine, University of California, Davis, California 95616
Received for publication, April 11, 2003 , and in revised form, May 16, 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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RyRs are modulated by numerous exogenous and endogenous compounds. The principal endogenous modulator of the RyR is Ca2+, which, at micromolar levels, can activate the RyR via Ca2+-induced Ca2+ release. Moreover, studies of [3H]ryanodine binding to sarcoplasmic reticulum membranes (2) and single RyRs fused into lipids bilayer (3) indicate that Ca2+ has a biphasic effect since millimolar concentrations of the ion can inhibit the channel activity. Other endogenous RyR modulators include magnesium (an inhibitor) (4–6) and adenine nucleotides (activators) (4, 5) as well as the cellular oxidation/reduction state, which has complex effects on channel activity (7, 8).
In addition, exogenous modulators that can alter RyR activity have been discovered. A number of these agents, including ryanodine, caffeine, and ruthenium red, have become specific tools used to pharmacologically identify intracellular signaling pathways involving RyRs (9). In addition, the RyR inhibitor dantrolene is used to treat episodes of malignant hyperthermia (MH), a human skeletal muscle disorder that can result in uncontrolled Ca2+ release and sustained muscle contraction (10).
Recently, a new RyR activator has been discovered, 4-chloro-m-cresol (4-CmC) (11–13). Originally used as a preservative in commercial preparations of some intravenous drugs, including succinylcholine, 4-CmC can directly activate the RyR with 10–25-fold higher potency compared with the more commonly used RyR activator, caffeine (14–16). In addition, the sensitivity to activation by 4-CmC is increased for RyR1 channels containing point mutations that have been linked to MH (11, 17, 18). Indeed, because of its higher potency relative to caffeine, 4-CmC has been recommended as a supplemental test substance in the in vitro contracture test for diagnosing MH in humans (12).
Although little is known about the molecular mechanism of action of 4-CmC,
it has been suggested that this compound acts similarly to caffeine because
both compounds increase the sensitivity of the RyR to activation by
Ca2+
(15). In addition, studies on
isolated RyR1 channels indicate that 4-CmC acts preferentially on the luminal
side of the channel (15).
4-CmC can cause contractures of both ventricular
(19) and skeletal
(20) muscles, thus suggesting
that it can activate at least two of the three RyR isoforms, RyR1 (skeletal)
and RyR2 (cardiac). Interestingly, 4-CmC can also activate RyR3 with an
EC50 of 
1.5 mM
(21), thus indicating that
4-CmC can activate this isoform, albeit with a 10-fold lower potency compared
with its ability to activate RyR1. These findings have been confirmed by
recent studies indicating that concentrations of 4-CmC up to 0.5 mM
can fully activate Ca2+ release in dyspedic 1B5 myotubes
expressing recombinant RyR1 and RyR2, but not RyR3
(1,
22). One hypothesis that may
explain these findings is that sequence divergence within RyR3 could lower the
affinity and/or efficacy for activation by 4-CmC compared with RyR1 and
RyR2.
To determine the site(s) on RyR1 necessary for activation by 4-CmC, we have taken advantage of these isoform-specific differences in 4-CmC sensitivity and constructed RyR1-RyR3 chimeric proteins. If 4-CmC acts on a discrete region of RyR1, then substitution of this region into RyR3 should restore 4-CmC sensitivity to the chimeric protein. In contrast, substitution of RyR3 sequences into this critical region of RyR1 should disrupt 4-CmC-induced activation of RyR1. Through the construction of a successive series of chimeric proteins, we have identified a 173-amino acid segment in RyR1 that mediates activation by 4-CmC.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The DNA sequences of the RyR1 primers used to generate the restriction sites indicated by asterisks were as follows: AscI site (position 11302), 5'-CTCTACCAGCAGGCGCGCCTGCACACGC (forward) and 5'-GCGTGTGCAGGCGCGCCTGCTGGTAGAG (reverse); AvrII site (position 12543), 5'-CCTAGGCCGCATCGAGATCATGG-3' (forward) and 5'-CCTAGGTAGGGCCGGAAGTAC-3' (reverse); and PstI site (position 13895), 5'-GTATACTACTTCCTGCAGGAGAGCACGGGCTACAT-3' (forward). The DNA sequences of the RyR3 primers used to generate the restriction sites indicated by asterisks are as follows: AscI site (position 10862), 5'-CTATCAGCAGGCGCGCCTGCATGAGCGC-3' (forward) and 5'-GCGCTCATGCAGGCGCGCCTGCTGATAG-3' (reverse); XhoI site (position 11577), 5'-CTCGAGTCAGATCGAGCTGCTGAAGG-3' (forward) and 5'-CTCGAGTCCTGAGAGAGTTTCATCTGC-3' (reverse); and SphI site (position 12711), 5'-GCATGCCTGACCCAACCCAGTTTGG-3' (forward) and 5'-GCATGCCACCCAGGATCTTAGTCACTC-3' (reverse). PCR products generated using Pfu DNA polymerase (Promega, Madison, WI) were cloned into TOPO-TA vectors (Invitrogen) and confirmed using bidirectional DNA sequencing.
Cell Culture—1B5 myotubes were prepared as described (1, 24). Briefly, 1B5 myoblasts were propagated in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 20% (v/v) fetal bovine serum (Hyclone Laboratories, Logan, UT), 100 units/ml penicillin G, 100 µg/ml streptomycin sulfate, and 2 mM L-glutamine at 37 °C at 10% CO2. Cells were seeded onto a 96-well plate and cultured until they attained 50% confluence. Differentiation was initiated by changing the medium to Dulbecco's modified Eagle's medium supplemented with 5% (v/v) heat-inactivated horse serum and L-glutamine/antibiotics as described above at 37 °C at 18% CO2. After 7 days, differentiated myotubes were transduced with the RyR constructs using equivalent amounts of HSV virions (23) containing the cDNAs encoding the RyRs. After a 2-h incubation at 37 °C in antibiotic-free medium, the virus was removed, and the myotubes were incubated for 2 days to allow full expression of the RyRs before testing.
Calcium Imaging—Changes in intracellular Ca2+ in 1B5 myotubes were measured using the Ca2+ indicator dye Fluo-4. 1B5 myotubes were loaded in the presence of 5 µM Fluo-4/AM (Molecular Probes, Inc., Eugene, OR) for 30 min at 37 °C in imaging solution (125 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 6mM glucose, 2 mM CaCl2, and 25 mM HEPES, pH 7.4, with NaOH). After extensive washing with imaging solution, changes in intracellular Ca2+ were monitored using an Olympus U-PlanApo/340 40x objective lens and a Nikon Diaphot 300 microscope. Myotubes were illuminated using a DeltaRam light source (Photon Technology International, Monmouth Junction, NJ) set at 495 nm. Fluorescence emitted from the 1B5 myotubes was measured using an XR/Mega-12 ICCD camera (Stanford Photonics Inc., Palo Alto, CA), and resultant images were monitored using QED imaging software on a Macintosh G4 computer. Images were acquired at the rate of 12 images/s, and changes in fluorescence of individual cells were monitored using regions of interest. Record traces from individual cells were stored in spreadsheets and analyzed using Microsoft Excel (see below).
Myotubes transduced with RyR-encoding viruses were challenged with successive additions of 80 mM KCl, 40 mM caffeine, and 0.5 mM 4-CmC dissolved in imaging solution. These substances were delivered to the myotubes using a ValveBank 8/II pressure-driven perfusion system (Automate Scientific, Inc., Foster City, CA). Results from a given RyR construct are presented as a single data trace obtained by averaging data traces from all individual measurements of 1B5 myotubes transduced with that construct. Bars represent the S.E. of the measurements at the peaks of the Ca2+ transients induced by the RyR activators.
Quantification of Ca2+ Transients—The relative amount of Ca2+ released in response to each RyR agonist was estimated by calculating the average change in Fluo-4 fluorescence during application of the agonist. This mean value was then normalized to the resting fluorescence of the cell obtained 10 s prior to addition of the agonist. Comparison of 4-CmC-elicited responses from cells transduced with various RyR constructs was accomplished by expressing the magnitude of each 4-CmC-elicited response as a percentage of the magnitude of the 40 mM caffeine-elicited Ca2+ transient obtained from the same cell. These normalized values from each RyR chimera were then compared with the values obtained from the 4-CmC-induced transients from wtRyR-expressing cells. This comparison was made using two-way analysis of variance with Dunnett's post-test. A significant difference was inferred at p < 0.05.
Binding Assay Sample Preparation and Immunoblotting—Crude membrane preparations used for [3H]ryanodine binding studies were prepared and characterized using Western blot techniques as described previously (25). Briefly, 2 days after transduction with RyR cDNA-containing HSV virions, 1B5 myotubes were harvested in harvest buffer (137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, and 0.6 mM EDTA, pH 7.2) and pelleted at 250 x g. The myotubes were then homogenized in buffer consisting of 250 mM sucrose and 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 using a Polytron cell disrupter (Brinkmann Instruments). After clearing the homogenates at 1500 x g for 30 min, the supernatant was centrifuged at 100,000 x g for 1 h at 4 °C. The resulting pellet was resuspended in 10% sucrose and 20 mM HEPES, pH 7.4; divided into aliquots; and frozen in liquid N2 until used.
The RyRs contained in these membrane preparations were characterized using Western blot procedures. Fifty micrograms of crude membranes from 1B5 myotubes transduced with wtRyR3, chimera 1, and chimera 1rev [PDB] were resolved on a 6% SDS-polyacrylamide gel before being transferred to a polyvinylidene difluoride membrane, which was then probed with anti-RyR monoclonal antibody 34C and horseradish peroxidase-conjugated goat anti-mouse secondary antibody. Immunoreactive proteins were visualized using SuperSignal chemiluminescent substrate (Pierce).
[3H]Ryanodine Binding Assays—[3H]Ryanodine (specific activity of 56 Ci/mmol) was purchased from PerkinElmer Life Sciences. Specific binding of [3H]ryanodine to sarcoplasmic reticulum preparations closely followed the method reported by Pessah et al. (4). The ability of recombinant and skeletal muscle sarcoplasmic reticulum preparations to bind 10 nM [3H]ryanodine (specific activity of 28 Ci/mmol; 5 nM [3H]ryanodine + 5 nM unlabeled ryanodine) was determined by incubating 100 µg/ml (recombinant) or 50 µg/ml (rabbit skeletal muscle) sarcoplasmic reticulum vesicles in assay buffer consisting of 250 mM KCl, 20 mM HEPES/KOH, pH 7.4, and 100 µM CaCl2. Final free Ca2+ concentrations were achieved with EGTA buffering using the Bound and Determined software (26). Reaction mixtures were allowed to equilibrate at 37 °C for 3 h with constant shaking. Reactions were then quenched by rapid filtration on GF/B glass-fiber filters and quickly rinsed with 3 x 2 ml of ice-cold 20 mM HEPES and 100 µM CaCl2, pH 7.4. The amount of [3H]ryanodine bound to each filter was determined by liquid scintillation counting.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Wild-type and chimeric RyR cDNAs were introduced into 1B5 myotubes using HSV type 1 virions (23). These myotubes were then loaded with Fluo-4 and challenged with three different test substances. First, myotubes were challenged with 80 mM KCl, a treatment that triggers depolarization-induced Ca2+ release (excitation-contraction coupling) in 1B5 myotubes expressing RyR1 (1, 24). Since only RyR1 (but not RyR3) can support excitation-contraction coupling in 1B5 myotubes (1, 25), this chemical depolarization of the cells was used to verify the backbone of the chimeric receptor (RyR1 versus RyR3 background). Myotubes were then challenged with 40 mM caffeine to ensure that the expressed chimeric protein was functional. Finally, myotubes were challenged with 0.5 mM 4-CmC, a concentration that maximally activates RyR1, but has little effect on RyR3 (1).
First, we determined whether the 4-CmC effector site is located in the large cytosolic foot region or the putative transmembrane assembly of the channel. We constructed two chimeric RyRs (Fig. 1A): chimera R1R3, consisting of a RyR1 backbone with the C-terminal 1269 amino acids (containing the putative transmembrane assembly of the RyR) substituted with the equivalent RyR3 sequence, and the exact reverse of chimera R1R3, chimera R3R1, consisting of a RyR3 backbone containing the corresponding C-terminal amino acids from RyR1. RyR agonist-induced Ca2+ release from myotubes transduced with these constructs was compared with that from myotubes expressing wtRyR1 (Fig. 1B) and wtRyR3 (Fig. 1C). Myotubes expressing chimera R1R3 (Fig. 1D) responded to both KCl and caffeine, thus verifying that the chimeric receptor was functional and that it contained a RyR1 backbone. However, 4-CmC-induced Ca2+ transients in chimera R1R3-expressing myotubes were significantly reduced relative to either responses in these myotubes to 40 mM caffeine (which themselves were reduced by an unknown mechanism) or to 4-CmC-induced transients in wtRyR1-expressing 1B5 myotubes (Fig. 1F). We then tested chimera R3R1 (Fig. 1E). Myotubes expressing this construct did not respond to KCl depolarization (presumably due to the wtRyR3 backbone), although they did respond to caffeine. Interestingly, these myotubes responded to 4-CmC, and the magnitude of these responses was similar to that of the responses exhibited by myotubes expressing wtRyR1 (Fig. 1F). Taken together, these results suggest that the essential structural determinant(s) required for 4-CmC activation of the RyR reside in the C-terminal third of the protein.
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To further define the 4-CmC interaction site, we divided the C-terminal third of RyR1 into three sections of nearly equal length and then individually substituted the corresponding RyR3 sequences into these three sections to produce chimeras 1–3 (Fig. 2A). Although all chimeras displayed similar responses to KCl and caffeine compared with wtRyR1, chimera 1 (RyR1 amino acids 3768–4181 substituted with RyR3 sequence) (Fig. 2B) had responses to 4-CmC that were significantly reduced compared with wtRyR1 (Fig. 2F), whereas chimera 2 (RyR1 amino acids 4181–4595 substituted) (Fig. 2D) and chimera 3 (RyR1 amino acids 4596–5037 substituted) (Fig. 2E) did not. However, chimera 1M (RyR1 amino acids 4007–4383 substituted with RyR3 sequence) (Fig. 2C), containing a RyR3 substitution in an overlapping region between chimeras 1 and 2, also had significantly reduced responses to 4-CmC (Fig. 2F). Taken together, these studies indicate that structural determinants required for activation by 4-CmC reside between RyR1 amino acids 3768 and 4383 (i.e. the RyR1 region substituted with RyR3 sequence in chimeras 1 and 1M).
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To confirm these findings, we created a series of reverse chimeras in which the identical regions of RyR1 described above were substituted into RyR3 (Fig. 3A). Since substitution of RyR3 sequence into the C-terminal 441 amino acids of RyR1 (chimera 3) did not affect 4-CmC-induced Ca2+ transients (Fig. 2E), we tested the reverse of only chimeras 1, 1M, and 2 (Fig. 3A). Expression of chimera 1rev [PDB] (Fig. 3B) and chimera 1Mrev (Fig. 3C) restored responses to 4-CmC that were elevated significantly compared with 4-CmC responses in myotubes expressing wtRyR3 (Fig. 3E). This finding is significant since substitution of the corresponding RyR3 sequences into the chimera 1 and 1M regions of wtRyR1 abolished 4-CmC activation of RyR1 (Fig. 2, B and C). No significant difference in 4-CmC activation was observed between myotubes expressing chimera 2rev (Fig. 3D) and wtRyR3.
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To further define the 4-CmC effector site, chimera 1 was subdivided into four parts designated A–D, and different combinations of these sections from RyR3 were substituted into RyR1 (Fig. 4A). Substitution of RyR3 sequences into the N-terminal half of the RyR1 chimera 1 region (chimera 1AB, RyR1 amino acids 3768–4006 substituted) resulted in a chimeric RyR with responses to 4-CmC essentially identical to those of wtRyR1 (Fig. 4, B and F). However, substitution of RyR3 sequences into the C-terminal half of chimera 1 (chimera 1CD, RyR1 amino acids 4007–4180 substituted) selectively eliminated responses to 4-CmC without altering responses to KCl or caffeine (Fig. 4C). This finding is intriguing since the CD region of chimera 1 represents the overlapping portion between chimeras 1 and 1M, both of which do not respond to 4-CmC. Further subdivision of this region into chimera 1C (Fig. 4D) and chimera 1D (Fig. 4E) resulted in proteins with responses to 4-CmC that were not statistically different in magnitude compared with those of wtRyR1 (Fig. 4F).
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To determine whether the AB and/or CD wtRyR1 segment of chimera 1 could impart 4-CmC activation to wtRyR3, we created the reverse chimeras, 1ABrev and 1CDrev (Fig. 5A). Upon exposure to 0.5 mM 4-CmC, myotubes expressing chimera 1CDrev (Fig. 5C), but not chimera 1ABrev (Fig. 5B), gave responses to 4-CmC that were significantly greater than those of myotubes expressing wtRyR3 (Fig. 5D). The magnitude of Ca2+ transients elicited by 4-CmC in chimera 1CDrev-expressing myotubes was 26.8% of that of the responses from chimera 1rev [PDB] -expressing myotubes, thus suggesting that although essential structural determinants required for 4-CmC-induced activation of RyR1 channels reside in the chimera 1CD region (amino acids 4007–4180), flanking sequences are required to fully restore the 4-CmC effector site in RyR1.
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An independent analysis of the 4-CmC effector site was performed by measuring the ability of 4-CmC to enhance high affinity binding of [3H]ryanodine to wtRyR1, wtRyR3, and chimeras 1 and 1rev [PDB] (Fig. 6). We prepared heavy sarcoplasmic reticulum vesicles from either rabbit skeletal muscle (as a source of wtRyR1) or 1B5 myotubes expressing wtRyR3, chimera 1, or chimera 1rev [PDB] . The molecular weight and expression level of these proteins were confirmed using Western blot analysis (data not shown). We then measured the level of 4-CmC-stimulated [3H]ryanodine binding to these RyRs at two different Ca2+ levels. In the presence of 0.1 µM Ca2+ (Fig. 6A), 0.25 mM 4-CmC stimulated binding of [3H]ryanodine to wtRyR1 from 79.5 to 7480 fmol of [3H]ryanodine bound per mg of protein. Interestingly, 4-CmC failed to enhance [3H]ryanodine binding to vesicles isolated from myotubes expressing wtRyR3 or chimera 1, a finding consistent with our observations in intact cells. In addition, 4-CmC significantly enhanced binding of [3H]ryanodine to vesicles isolated from myotubes expressing chimera 1rev [PDB] from 0.27 to 253 fmol of [3H]ryanodine bound per mg of protein.
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To determine the effect of Ca2+ on 4-CmC-stimulated [3H]ryanodine binding to these RyRs, we also conducted a parallel set of binding experiments in the presence of 1 µM Ca2+ (Fig. 6B). This 10-fold higher Ca2+ level enhanced the level of [3H]ryanodine binding for all constructs. As expected, 4-CmC significantly enhanced [3H]ryanodine binding to both wtRyR1 (3.91-fold stimulation) and chimera 1rev [PDB] (2.35-fold stimulation). However, in the presence of 1 µM Ca2+, 4-CmC also enhanced [3H]ryanodine binding to membrane preparations containing wtRyR3 and chimera 1 (2.24- and 1.45-fold stimulation, respectively). These results suggest that the ability of 4-CmC to stimulate [3H]ryanodine binding to the RyR is partially dependent on Ca2+ concentration, a finding that is consistent with published reports (15).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The RyR1-RyR3 Chimeric Approach—Chimeric RyRs have proven useful in identifying areas within the RyR primary sequence involved in different functional phenotypes. This approach has been used to identify regions of RyR1 that mediate excitation-contraction coupling (25, 27, 28), Ca2+ sensitivity (29, 30), and caffeine sensitivity (31). A potential advantage to using chimeras is that substitution of homologous regions between proteins should ideally result in less severe perturbations of the overall protein conformation compared with large deletions, thus providing more specific and relevant information in structure-function studies. In addition, through the use of chimeric proteins, large areas can first be surveyed, followed by successively smaller substitutions. Ideally, if a relatively small region governs a specific functional difference between two isoforms, then its identification using this approach can serve as a starting point for site-directed mutagenesis to further define the site being studied.
Topography of the Interaction Site—Through the use of RyR1-RyR3 chimeric proteins, we have identified a 173-amino acid segment from RyR1 (chimera 1CD region) that partially restores 4-CmC activation of wtRyR3. However, flanking sequences are required since RyR3-based chimeras containing longer segments of RyR1 sequence (R3R1, 1rev [PDB] , and 1Mrev) more fully reconstitute 4-CmC activation compared with chimera 1CDrev. These longer stretches of RyR1 sequence are required presumably to maintain the correct structure for channel activation by 4-CmC. One assumption in the use of chimeric proteins is that an exogenous amino acid sequence maintains its original three-dimensional conformation when introduced into a new protein. This assumption is most likely valid when large, highly homologous protein segments are substituted. However, as smaller and smaller regions are examined, the likelihood increases that the substituted amino acid segment can adopt conformations different from its original conformation. The 173-amino acid region we have identified using chimeric RyRs may represent the resolution limit of this approach in this specific case.
Binding Studies and 4-CmC Activation Mechanism—We conducted [3H]ryanodine binding assays to provide an independent method to study the 4-CmC interaction site. Interestingly, binding studies conducted at 100 nM free Ca2+, a condition that approximates the resting intracellular free Ca2+ level in intact cells, yielded results that correlated well with our findings from intact cells. Namely, RyR3 and chimera 1, constructs for which 4-CmC stimulation was weak in intact cells, did not show enhancement of [3H]ryanodine binding in the presence of 4-CmC. In addition, enhanced levels of 4-CmC-stimulated ryanodine binding were observed for wtRyR1 and chimera 1rev [PDB] , a finding that matches our intact cell data.
However, at 1 µM free Ca2+, a condition that increases channel activity and [3H]ryanodine binding, 4-CmC-stimulated ryanodine binding was observed for all constructs tested, including wtRyR3 and chimera 1. This result is not surprising when one considers that 4-CmC and Ca2+ are co-agonists of the RyR. Increased levels of Ca2+ render the RyR more sensitive to pharmacological agents such as caffeine, halothane, and adenine nucleotides (4). In the case of RyR3 and chimera 1, Ca2+ may act similarly to sensitize these channels to activation by 4-CmC.
Thus, 4-CmC most likely can directly activate RyR3, albeit with reduced
efficacy and/or potency. This conclusion is supported by studies in primary
astrocyte cultures indicating that 4-CmC can activate RyR3 with an
EC50 of 1.5 mM
(21). In our initial
characterization of RyR1 and RyR3 expressed in 1B5 myotubes
(1), we did not observe
Ca2+ transients in RyR3-expressing cells induced by
4-CmC concentrations up to 0.5 mM. However, we did not test 4-CmC
concentrations higher than 0.5 mM because, at these levels, 4-CmC
was able to induce small Ca2+ transients in
non-transfected RyR-null 1B5 myotubes.
Other Potential 4-CmC Effector Sites—Point mutations in the MH sensitivity region 2 of wtRyR1 (residues 2129–2458) can either increase (11, 17, 18) or decrease (32) 4-CmC sensitivity, thus suggesting that this domain might also contain a potential 4-CmC effector site. However, these mutations also alter sensitivity to other RyR agonists such as caffeine and halothane, thus suggesting a more general effect on RyR activity. Indeed, myotubes expressing RyR1 channels containing MH or central core disease mutations have elevated levels of resting free Ca2+ (33–35). Since Ca2+ can alter the sensitivity of the RyR to 4-CmC, it seems likely that the differences in 4-CmC activation potential of these various MH mutants are due to this global change in intracellular Ca2+ rather than alterations to a putative 4-CmC-binding site in MH sensitivity region 2.
Mechanism—The precise mechanism by which 4-CmC activates the RyR remains to be determined. One possibility is that the 173-amino acid segment defined in this study contains the 4-CmC-binding site and that differences in amino acid composition between RyR1 and RyR3 result in differential affinity of 4-CmC binding to this site. A second possibility is that 4-CmC can bind to both RyR1 and RyR3 with equal affinity, but that this binding is translated more effectively into channel activation for RyR1. In this case, the chimera 1CD region might contain important structural determinants that translate 4-CmC binding to channel activation. A final possibility is that 4-CmC binds to an accessory protein on the RyR, which then activates the RyR.
At the present time, we cannot distinguish between these possibilities since there is no conclusive evidence that 4-CmC binds directly to the RyR or to one of its accessory proteins. Although 4-CmC can activate isolated RyR1 proteins in single channel studies and also activate [3H]ryanodine binding to RyR1 in lipid vesicles (15), the possibility still exists that 4-CmC could bind to a tightly associated accessory protein. If this is the case, however, then presumably the interaction site between this protein and the RyR involves the critical region defined in this study.
Topography of the C terminus—The C-terminal third of the RyR is thought to contain a number of important structural features, including the transmembrane complex (36); the Ca2+ permeability pore (37); and putative interaction sites for Ca2+ (38, 39), caffeine (29), ATP (40), and ryanodine (41), among others. In addition, this area contains central core disease/MH region 3 (which includes amino acids 4637–4898) (34) and also divergent area D1, the amino acid composition of which is poorly conserved between the three RyR isoforms.
Our work indicates that the 4-CmC effector site is also located in the C terminus. However, this site does not overlap with the D1 region, but instead lies adjacent to it on the N-terminal side. Interestingly, the chimera 1 region shares a high degree of homology between RyR1 and RyR3, with 84% amino acid similarity. Moreover, within the chimera 1CD region, the amino acid similarity is 81%, with only 23 dissimilar amino acids. It is intriguing that such a well conserved region should be implicated in a pharmacological phenotype that is so highly divergent between RyR1 and RyR3. However, this high degree of homology should be beneficial in identifying the individual amino acids that are required to mediate activation of the RyR by 4-CmC.
Conclusions—Through the use of RyR1-RyR3 chimeric proteins, we have identified a 173-amino acid segment of RyR1 that can impart 4-CmC sensitivity to RyR3. These findings suggest that a critical determinant on RyR1 required for activation by 4-CmC resides between amino acids 4007 and 4180.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Anesthesia Research,
Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. Tel.:
617-732-6881; Fax: 617-732-6927; E-mail:
fessenden{at}zeus.bwh.harvard.edu.
1 The abbreviations used are: RyR, ryanodine receptor; wtRyR, wild-type
ryanodine receptor; MH, malignant hyperthermia; 4-CmC,
4-chloro-m-cresol; HSV, herpes simplex virus.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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