Identification of a key determinant of ryanodine receptor type 1 required for activation by 4-chloro-m-cresol.

4-Chloro-m-cresol (4-CmC) is a potent and specific activator of the intracellular Ca2+ release channel, the ryanodine receptor (RyR). We have previously shown that RyR1 expressed in dyspedic 1B5 myotubes is activated by 4-CmC, whereas RyR3 is not (Fessenden, J. D., Wang, Y., Moore, R. A., Chen, S. R. W., Allen, P. D., and Pessah, I. N. (2000) Biophys. J. 79, 2509-2525). To identify region(s) on RyR1 that are responsible for mediating activation by 4-CmC, we expressed RyR1-RyR3 chimeric proteins in dyspedic 1B5 myotubes and then measured 4-CmC-induced increases in intracellular Ca2+. Substitution of the C-terminal third of RyR1 into RyR3 imparted 4-CmC sensitivity to the resulting chimera, thus suggesting that determinants required for activation by 4-CmC are located in this region. We subdivided the C-terminal third of RyR1 into smaller segments and identified two overlapping regions of RyR1 (amino acids 3769-4180 and 4007-4382) that each imparted 4-CmC sensitivity to RyR3. Substitution of the 173 amino acids of RyR1 common to these two chimeras (amino acids 4007-4180) also weakly restored 4-CmC sensitivity in the resulting chimera. To confirm these findings, we created a complementary set of chimeras containing RyR3 substitutions in RyR1. Substitution of the RyR3 C terminus into RyR1 disrupted 4-CmC sensitivity in the resulting chimera. In addition, substitution of the corresponding RyR3 sequence into positions 4007-4180 of RyR1 disrupted 4-CmC sensitivity. Taken together, these results suggest that essential determinants required for activation of RyR1 by 4-CmC reside within a 173-amino acid region between residues 4007 and 4180.

Skeletal muscle contraction occurs in response to depolarization of the muscle cell plasma membrane (the sarcolemma) via excitation-contraction coupling. In this process, sarcolemmal depolarization is detected by voltage-gated L-type Ca 2ϩ channels, which, in turn, activate the intracellular Ca 2ϩ release channel known as the ryanodine receptor (RyR). 1 Stimulation of this protein releases stored Ca 2ϩ into the cytosol, which then activates the cellular contractile apparatus to initiate muscle contraction.
RyRs are modulated by numerous exogenous and endogenous compounds. The principal endogenous modulator of the RyR is Ca 2ϩ , which, at micromolar levels, can activate the RyR via Ca 2ϩ -induced Ca 2ϩ release. Moreover, studies of [ 3 H]ryanodine binding to sarcoplasmic reticulum membranes (2) and single RyRs fused into lipids bilayer (3) indicate that Ca 2ϩ 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 Ca 2ϩ release and sustained muscle contraction (10).
Recently, a new RyR activator has been discovered, 4-chlorom-cresol (4-CmC) (11)(12)(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 Ca 2ϩ (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 EC 50 of ϳ1.5 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 Ca 2ϩ 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.
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 g/ml streptomycin sulfate, and 2 mM L-glutamine at 37°C at 10% CO 2 . 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) heatinactivated horse serum and L-glutamine/antibiotics as described above at 37°C at 18% CO 2 . 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 Ca 2ϩ in 1B5 myotubes were measured using the Ca 2ϩ indicator dye Fluo-4. 1B5 myotubes were loaded in the presence of 5 M 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 MgSO 4 , 6 mM glucose, 2 mM CaCl 2 , and 25 mM HEPES, pH 7.4, with NaOH). After extensive washing with imaging solution, changes in intracellular Ca 2ϩ were monitored using an Olympus U-PlanApo/340 40ϫ 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 Ca 2ϩ transients induced by the RyR activators.
Quantification of Ca 2ϩ Transients-The relative amount of Ca 2ϩ 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 Ca 2ϩ 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 Ͻ 0.05.
Binding Assay Sample Preparation and Immunoblotting-Crude membrane preparations used for [ 3 H]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 Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , and 0.6 mM EDTA, pH 7.2) and pelleted at 250 ϫ 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 g/ml leupeptin, 0.7 g/ml pepstatin A, 5 g/ml aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride using a Polytron cell disrupter (Brinkmann Instruments). After clearing the homogenates at 1500 ϫ g for 30 min, the supernatant was centrifuged at 100,000 ϫ 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 N 2 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 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).

RESULTS
Two complementary approaches were utilized to locate regions of sequence required for 4-CmC activation of RyR1. First, we substituted segments of RyR3 into the corresponding regions of RyR1 and identified those substitutions that diminished responses to 4-CmC without altering responses to depolarization or caffeine. Chimeras exhibiting a selective loss of activation by 4-CmC were presumed to lack critical determinants required for stimulation. Second, to confirm the importance of regions identified using this loss-of-function approach, we created a parallel set of reverse chimeras that contained RyR1 substitutions in RyR3 and determined which RyR1 substitutions enhanced 4-CmC activation of RyR3. Ideally, these two approaches should independently identify regions in RyR1 that are essential for mediating activation by 4-CmC.
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 Ca 2ϩ 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 Ca 2ϩ release from myotubes transduced with these constructs was compared with that from myotubes expressing wtRyR1 (Fig. 1B) and wtRyR3 (Fig. 1C).

4-CmC Activation Domain of RyR1
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 Ca 2ϩ 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-CmCinduced 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.
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
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 Nterminal 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).
To determine whether the AB and/or CD wtRyR1 segment of chimera 1 could impart 4-CmC activation to wtRyR3, we cre-ated 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 Ca 2ϩ transients elicited by 4-CmC in chimera 1CDrev-expressing myotubes was 26.8% of that of the responses from chimera 1rev-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.
An independent analysis of the 4-CmC effector site was performed by measuring the ability of 4-CmC to enhance high affinity binding of [ 3 H]ryanodine to wtRyR1, wtRyR3, and chimeras 1 and 1rev (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. 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-CmCstimulated [ 3 H]ryanodine binding to these RyRs at two different Ca 2ϩ levels. In the presence of 0.1 M Ca 2ϩ (Fig. 6A)

4-CmC Activation Domain of RyR1
To determine the effect of Ca 2ϩ on 4-CmC-stimulated [ 3 H]ryanodine binding to these RyRs, we also conducted a parallel set of binding experiments in the presence of 1 M Ca 2ϩ (Fig. 6B). This 10-fold higher Ca 2ϩ level enhanced the level of [ 3 H]ryanodine binding for all constructs. As expected, 4-CmC significantly enhanced [ 3 H]ryanodine binding to both wtRyR1 (3.91fold stimulation) and chimera 1rev (2.35-fold stimulation). However, in the presence of 1 M Ca 2ϩ , 4-CmC also enhanced [ 3 H]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 [ 3 H]ryanodine binding to the RyR is partially dependent on Ca 2ϩ concentration, a finding that is consistent with published reports (15). DISCUSSION In our initial study of wtRyR1 and wtRyR3 expressed in 1B5 myotubes, several prominent functional differences between these isoforms were apparent (1). First, RyR1 (but not RyR3) could support depolarization-induced Ca 2ϩ release (excitationcontraction coupling). Second, RyR3 was much more sensitive to activation by caffeine compared with RyR1. Third, at the levels of 4-CmC tested, this compound could activate RyR1, but not RyR3. We took advantage of this isoform-specific difference in 4-CmC sensitivity to define regions of RyR1 required for 4-CmC activation. Using two complementary approaches, we have demonstrated that a principal 4-CmC effector site in the wtRyR1 primary sequence is located within a cluster of 173 amino acids located between positions 4007 and 4180.
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), Ca 2ϩ 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, and 1Mrev) more fully reconstitute 4-CmC activation compared with chi-

4-CmC Activation Domain of RyR1
mera 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 [ 3 H]ryanodine binding assays to provide an independent method to study the 4-CmC interaction site. Interestingly, binding studies conducted at 100 nM free Ca 2ϩ , a condition that approximates the resting intracellular free Ca 2ϩ 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 [ 3 H]ryanodine binding in the presence of 4-CmC. In addition, enhanced levels of 4-CmCstimulated ryanodine binding were observed for wtRyR1 and chimera 1rev, a finding that matches our intact cell data.
However, at 1 M free Ca 2ϩ , a condition that increases channel activity and [ 3 H]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 Ca 2ϩ are co-agonists of the RyR. Increased levels of Ca 2ϩ render the RyR more sensitive to pharmacological agents such as caffeine, halothane, and adenine nucleotides (4). In the case of RyR3 and chimera 1, Ca 2ϩ 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 EC 50 of ϳ1.5 mM (21). In our initial characterization of RyR1 and RyR3 expressed in 1B5 myotubes (1), we did not observe Ca 2ϩ transients in RyR3expressing 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 Ca 2ϩ 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 Ca 2ϩ (33)(34)(35). Since Ca 2ϩ 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 Ca 2ϩ rather than alterations to a putative 4-CmC-binding site in MH sensitivity region 2.
vates 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 [ 3 H]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 Ca 2ϩ permeability pore (37); and putative interaction sites for Ca 2ϩ (38,39), caffeine (29), ATP (40), and ryanodine (41)

4-CmC Activation Domain of RyR1
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.