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Originally published In Press as doi:10.1074/jbc.M600670200 on May 31, 2006

J. Biol. Chem., Vol. 281, Issue 30, 21022-21031, July 28, 2006
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Amino Acid Residues Gln4020 and Lys4021 of the Ryanodine Receptor Type 1 Are Required for Activation by 4-Chloro-m-cresol*

James D. Fessenden{ddagger}1, Wei Feng§, Isaac N. Pessah§, and Paul D. Allen{ddagger}

From the {ddagger}Department of Anesthesia Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115 and the §Department of Veterinary Medicine-Molecular Biosciences, Center for Children's Environmental Health, Davis, California 95616

Received for publication, January 23, 2006 , and in revised form, May 30, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
The ryanodine receptor type 1 (RyR1) and type 2 (RyR2), but not type 3 (RyR3), are efficiently activated by 4-chloro-m-cresol (4-CmC). We previously showed that a 173-amino acid segment of RyR1 (residues 4007-4180) is required for channel activation by 4-CmC (Fessenden, J. D., Perez, C. F., Goth, S., Pessah, I. N., and Allen, P. D. (2003) J. Biol. Chem. 278, 28727-28735). In the present study, we used site-directed mutagenesis to identify individual amino acid(s) within this region that mediate 4-CmC activation. In RyR1, substitution of 11 amino acids conserved between RyR1 and RyR2, but divergent in RyR3, with their RyR3 counterparts reduced 4-CmC sensitivity to the same degree as substitution of the entire 173-amino acid segment. Further analysis of various RyR1 mutants containing successively smaller numbers of these mutations identified 2 amino acid residues (Gln4020 and Lys4021) that, when mutated to their RyR3 counterparts (Leu3873 and Gln3874), abolished 4-CmC activation of RyR1. Mutation of either of these residues alone did not abolish 4-CmC sensitivity, although Q4020L partially reduced 4-CmC-induced Ca2+ transients. In addition, mutation of the corresponding residues in RyR3 to their RyR1 counterparts (L3873Q/Q3874K) imparted 4-CmC sensitivity to RyR3. Recordings of single RyR1 channels indicated that 4-CmC applied to either the luminal or cytoplasmic side activated the channel with equal potency. Secondary structure modeling in the vicinity of the Gln4020-Lys4021 dipeptide suggests that the region contains a surface-exposed region adjacent to a hydrophobic segment, indicating that both hydrophilic and hydrophobic regions of RyR1 are necessary for 4-CmC binding to the channel and/or to translate allosteric 4-CmC binding into channel activation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Striated muscle contraction is triggered by Ca2+ release from intracellular stores through the ryanodine receptor (RyR),2 a large homotetrameric channel protein embedded in the membrane of the sarcoplasmic reticulum (SR). In skeletal muscle, RyR1 is functionally associated with the {alpha}1s subunit of the dihydropyridine receptor, a voltage-gated Ca2+ channel that activates RyR1 in response to muscle cell depolarization in a process known as excitation-contraction (EC) coupling. Distinct RyR isoforms expressed in either skeletal (RyR1) or cardiac muscles (RyR2) mediate EC coupling in these muscle types (1, 2). The third RyR isoform (RyR3) is expressed at low levels in many tissues but has not been shown to participate in EC coupling in skeletal or cardiac muscle (3, 4).

Many pharmacological compounds activate RyR1 including the methylxanthine derivative, caffeine (5), and the phenol-based compound, 4-chloro-m-cresol (6). Both of these compounds are thought to directly activate RyR1 by increasing the affinity of Ca2+ binding to its activator site but these compounds differ in that 4-CmC is 25 times more potent in activating RyR1 compared with caffeine. In addition, these two compounds have distinct RyR isoform-dependent activation profiles: caffeine is much more potent in activating RyR2 and RyR3 compared with RyR1 (3, 7), whereas 4-CmC is a much more effective activator of RyR1 and RyR2 compared with RyR3 (3, 8).

This isoform-specific sensitivity to activation by caffeine and 4-CmC has made it possible to localize the activation sites of these compounds using chimeric RyR proteins. Substitution of amino acids 4187-4628 of RyR1 with the corresponding sequence from RyR2 increases the caffeine sensitivity of the resulting chimera compared with wtRyR1 (7, 9), whereas substitution of amino acids 4007-4180 of RyR1 into RyR3 enhances 4-CmC sensitivity in RyR3 (10), thus suggesting that determinants for caffeine or 4-CmC activation of the channel reside in these respective regions. In addition, performing the reverse substitution by replacing amino acids 4007-4180 in RyR1 with the equivalent RyR3 sequence greatly diminishes the 4-CmC sensitivity of the resulting chimeric protein (10), further strengthening the hypothesis that amino acid determinants required for full activation of RyR1 by 4-CmC reside within this 173-amino acid segment.

The amino acid sequence within the 4-CmC sensitivity region is highly conserved among all three RyR isoforms with over 66% identity and 87% similarity. This observation suggests that the isoform-specific differences in 4-CmC sensitivity are mediated by only a few amino acids that are divergent between the isoforms. To identify these amino acids, we performed site-directed mutagenesis on 11 divergent amino acids within the 173 amino acid RyR1 4-CmC sensitivity region and tested the resulting constructs for 4-CmC sensitivity. Our results demonstrate that the differential sensitivity to 4-CmC between RyR1 and RyR3 can be attributed to 2 amino acid residues in RyR1, Gln4020 and Lys4021 and their RyR3 counterparts Leu3873 and Gln3874. These findings further suggest that these residues mediate 4-CmC activation of RyR1.


Figure 1
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  		FIGURE 1.
A, bars represent the amino acid sequence of RyR1 (black line), RyR2 (gray line), and RyR3 (thin line), respectively. Substitution of amino acids 3730-4136 of RyR2 (gray bar) for amino acids 3621-4033 of RyR3 (thin bar) was performed to generate chimera 1 reverse RyR2 (Ch.1 Rev RyR2). The analogous chimera containing RyR1 sequence substituted into RyR3 (chimera 1 Rev) was constructed as described previously (10). The scale indicates amino acid number. B-D, agonist-induced changes in intracellular Ca2+ measured using Fluo-4 were determined in 1B5 myotubes expressing wtRyR3 (B), chimera 1 reverse RyR2 (Ch.1 Rev RyR2)(C), or chimera 1 reverse (Ch.1 Rev)(D). Myotubes were challenged with 30-s applications of 80 mM KCl (black bar), 40 mM caffeine (white bar), and 0.5 mM 4-CmC (cross-hatched bar). Data depict changes in Fluo-4 fluorescence normalized to the average resting fluorescence of the cell from the first 10 s of the measurement (F/Fo). Calibration bar = 0.2 F/Fo arbitrary units versus 30 s.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Construction of Mutated RyR1 and RyR3 cDNAs—cDNAs encoding the rabbit isoforms of RyR1, RyR2, and RyR3 have previously been cloned into pHSVprPUC thus enabling the generation of HSV amplicons required for transduction of 1B5 myotubes (11). Site-directed mutagenesis was performed using the QuikChange multi-mutagenesis Kit (Stratagene Inc., La Jolla, CA) on a RyR1 subclone created via the introduction of RyR1 sequence extending from the natural XhoI-HincII sites, located at positions 12,018 and 12,666, respectively, into the pGEM 3zf cloning vector (Promega Inc; Madison, WI). A silent mutation at position 12543 was added resulting in a unique AvrII restriction site that aided in the construction of full-length RyR1 cDNAs containing the mutations. Primers for mutagenesis, along with their reverse complement primers (not shown), were synthesized with the following sequence (nucleotides resulting in point mutations are indicated in italics): Mut 1 (Q4020L), 5'-GGAGCTGCTGGACCTGCTGAAGGACATGGTGG-3'; Mut2 (K4021Q), 5'-GGAGCTGCTGGACCTGCAACAGGACATGGTGG-3'; Mut1,2 (Q4020L and K4021Q), 5'-GGAGCTGCTGGACCTGCTGCAGGACATGGTGG-3'; Mut3 (M4047T), 5'-GGCAGATGGTGGACACGCTGGTGGAGTCCTCG-3'; Mut4 (S4099G), 5'-CCAGAAGGCCATGGACGGCCAGAAGCAGTTCACTGG-3'; Mut 5,6 (E4126I, E4127D), 5'-GATGATCAACTTCATCGACTTCGCCAACCGCTTCCAG-3'; Mut7,8 (R4161K,N4162C), 5'-GCACGACCCGCGCCTGAAGTGCTTCCTGGAGCTGGC-3'; Mut9,10 (F4163L and L4166P): 5'-GCGCCTGAAGTGCTTGCTGGAGCCGGCTGAGAGCATCC-3'; Mut11 (R4175E), 5'-CCTTGAGTACTTCGAGCCCTACCTAGGCCGCATGG-3'.

The introduction of each mutation was confirmed using DNA sequencing analysis.

RyR3 mutagenesis was performed using the primer extension method on a subclone comprised of RyR3 sequence between a previously introduced (10) AscI site at position 10862 and a naturally occurring AvrII site at position 12,090. The mutagenesis primer had the following sequence: RyR3 Mut1,2 (L3873Q/Q3874K), 5'-CTGCAGAAAGACATGGTGGTGATGCTTCTGTCCC-3'.


Figure 2
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  		FIGURE 2.
A, diagram depicts the chimera 1 region of RyR1 (black bar: amino acids 3768-4180) and the 1 CD region (amino acids 4007-4180), which mediates 4-CmC sensitivity in RyR1. B, peptide sequence comparison of the 1 CD region from rabbit RyR1, RyR2, and RyR3. Boxed residues indicate the position of the point mutations created in this study. Gray bar depicts the location of putative membrane-spanning segment M2 (26).

 
Cell Culture—1B5 myotubes were prepared as described (3, 12). Briefly, 1B5 myoblasts were propagated in Dulbecco's modified Eagle's medium (Invitrogen Laboratories) supplemented with 20% fetal bovine serum (v/v) (Hyclone; Logan, UT), 100 units/ml penicillin-G, 100 µg/ml streptomycin sulfate, 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% heat-inactivated horse serum (v/v) and L-glutamine/antibiotics as above at 37 °C at 18% CO2. After 7 days, differentiated myotubes were transduced with the RyR constructs using equivalent amounts of HSV virions (11) that contained the cDNAs encoding the RyRs. After2hat37 °Cin antibiotic-free medium, virus was removed and the myotubes were incubated for 2 days to allow full expression of the RyRs before testing.

Calcium Imaging—RyR-expressing myotubes were challenged with successive additions of 80 mM KCl, 40 mM caffeine, and 0.5 mM 4-CmC dissolved in imaging solution (125 mM NaCl, 5 mM KCl, 1.2 mM MgSO4,6mM glucose, 2 mM CaCl2,25 mM HEPES, pH 7.4 adjusted with NaOH). Note: for the 80 mM KCl solution, the NaCl concentration was reduced to 45 mM to maintain osmolarity. Changes in intracellular Ca2+ in 1B5 myotubes were measured as described previously (10) using the Ca2+ indicator dye Fluo-4. Record traces from individual cells were stored and analyzed using Microsoft Excel as follows.

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 the addition of the agonist. Comparison of 4-CmC-elicited responses from cells transduced with various wt and mutant 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 mutant were then compared with the values obtained from the 4-CmC induced transients from wtRyR1 and wtRyR3-expressing cells. This comparison was made using 2-way analysis of variance (ANOVA) with a Dunnett's post test. A significant difference was inferred at p < 0.05.

Measurements of Single Channel Activity in Lipid Bilayer Membranes—Bilayers were composed of phosphatidylethanolamine:phosphatidylserine:phosphatidylcholine (5:3:2 w/w) dissolved in decane at a final concentration of 50 mg/ml across a 200 µm aperture on a polysulfone cup (Warner Instrument Corp. CT). The bilayer partitioned two chambers (cis and trans), which contained the following buffer solutions (in mM): cis, 500 CsCl, 0.9 free Ca2+, and 20 Hepes-Tris (pH 7.4); trans, 500 CsCl and 20 Hepes-Tris (pH 7.4). The free Ca2+ concentration was adjusted by addition of CaCl2 and EGTA based on calculations from the "Bound and Determined" software (13). The cis chamber was virtually grounded, whereas the trans chamber was connected to the head stage input of an amplifier (Bilayer Clamp BC 525C, Warner Instrument, Hamden, CT). Single channels were obtained by introducing rabbit skeletal muscle junctional SR vesicles to the cis chamber to induce fusion with the bilayer membrane. Immediately after incorporation of RyR1 single channels into the lipid bilayer, the SR vesicles were perfused out of the cis chamber to prevent multiple vesicle fusions. Single channel gating was monitored and recorded at a holding potential of -40 mV (applied to the trans side). The amplified current signals, filtered at 1 kHz (Low-Pass Bessel Filter 8 Pole, Warner Instrument) was digitized and acquired at a sampling rate of 10 kHz (Digidata 1320A, Axon-Molecular Devices, Union City, CA). Most of the recordings were made for at least 3 min before the contents of the cis and/or trans chambers were changed. The channel open probability (Po), as well as the open and closed dwell times were calculated using pClamp software 9.0 (Axon-Molecular Devices). Junctional SR vesicles used in this work for single channel studies were from three different preparations.


Figure 3
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  		FIGURE 3.
A, mutation of all 11 amino acid residues in RyR1 described in Fig. 2B (white vertical bars) to their RyR3 counterparts was accomplished to create construct Mut1-11. B and C, agonist-induced changes in intracellular calcium for wtRyR1 (B) and Mut1-11 (C) were determined as described under "Experimental Procedures." Calibration bar = 0.5 F/Fo arbitrary units versus 30 s. D, for each RyR construct, the average size of the 4-CmC induced transients relative to the size of the caffeine-induced transients (taken as 100%) was determined. The numbers of myotubes analyzed for each construct were: RyR1 (n = 96 myotubes), RyR3 (n = 55), Mut 1-11 (n = 83). The asterisk indicates a statistically significant difference (p < 0.01) compared with wtRyR1 as determined using ANOVA with a Dunnett's post test.

 
Secondary Structure Prediction—The secondary structure predictions were performed using the Chou-Fasman method for {alpha}-helical predictions (14), the Kyte-Doolittle method for hydropathy predictions (15), and the method of Emini et al. (16) for surface probability prediction.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 CONCLUSION
 REFERENCES
 
Although 4-CmC can activate all three RyR isoforms (6, 8, 17), it activates RyR1 and RyR2 much more effectively compared with RyR3. A primary determinant on RyR1 that mediates 4-CmC sensitivity resides between amino acid residues 3768 and 4180 (10). Substitution of this region into the corresponding region of RyR3 results in a RyR1-RyR3 chimera (chimera 1 rev) with higher sensitivity to 4-CmC activation relative to wtRyR3. To determine whether 4-CmC acts on the analogous region of RyR2 (amino acids 3730-4136), this segment was substituted into RyR3 to create chimera 1 rev RyR2 (Fig. 1A). When intracellular Ca2+ release experiments were used to compare the ability of these constructs to respond to 80 mM KCl (to test for depolarization-induced calcium release), 40 mM caffeine and 0.5 mM 4-CmC, wtRyR3 (Fig. 1B) responded to caffeine but not to KCl or 4-CmC, whereas chimera 1 rev RyR2 (Fig. 1C) and chimera 1 rev (Fig. 1D) both responded to caffeine and 4-CmC but not to KCl depolarization. This finding suggests that determinants required for 4-CmC activation of RyR1 and RyR2 are identical.

The amino acid sequences of the three RyR isoforms were compared within the 173-amino acid 1 CD region (Fig. 2A), a subregion of chimera 1 that is the smallest determinant of 4-CmC sensitivity we have previously defined in wtRyR1 (10). Between the three isoforms, 66% of the amino acids in this region are identical and 87% are highly homologous (Fig. 2B). We reasoned that amino acid residues involved in 4-CmC activation of RyR1 and RyR2 should be (a) either identical or highly homologous between these isoforms and (b) divergent in RyR3. Eight amino acid residues were identified that were identical between RyR1 and RyR2 but not RyR3 (positions 1-6, 9, and 10). In addition, 3 residues were identified that were highly divergent between all three isoforms (positions 7, 8, and 11). Amino acid positions that were either identical or highly homologous between RyR3 and either RyR1 or RyR2 were not examined.


Figure 4
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  		FIGURE 4.
A, the following mutant RyRs were created containing mutations at positions 1, 2, 5-11 (Mut1,2,5-11), 1, 2, 7, 8 (Mut 1,2,7,8), 1, 2, 5, 6 (Mut1,2,5,6) as indicated by the white vertical bars. B-E, agonist-induced changes in intracellular Ca2+ from a representative myotube expressing Mut 1, 2, 5-11 (B), Mut 1, 2, 7, 8 (C), and Mut 1, 2, 5, 6 (D) are indicated. Calibration bar = 0.5 F/Fo arbitrary units versus 30 s. E, responses to 4-CmC were normalized to the response to caffeine as described under "Experimental Procedures" for the following numbers of myotubes: Mut1, 2, 5-11 (n = 39 myotubes), Mut 1, 2, 7, 8 (n = 69), Mut 1, 2, 5, 6 (n = 63). The asterisk indicates a statistically significant difference (p < 0.01) compared with wtRyR1 as determined using ANOVA with a Dunnett's post test.

 
A RyR1 construct containing all 11 mutations (Mut1-11) was tested for activation by KCl, caffeine, and 4-CmC (Fig. 3A). Compared with wtRyR1 (Fig. 3B), responses to chemical depolarization and caffeine activation of the Mut1-11 construct were unaffected. However, activation by 4-CmC was significantly reduced (Fig. 3, C and D).


Figure 5
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  		FIGURE 5.
A, RyR1 constructs containing mutations at position 1,2 (Mut1,2) or 3-11 (Mut3-11) were created. B and C, agonist-induced changes in intracellular Ca2+ from a representative myotube expressing Mut1,2 (B) or Mut3-11 (C) are indicated. Calibration bar = 0.5 F/Fo arbitrary units versus 30 s. D, responses to 4-CmC were normalized to the response to caffeine as described under "Experimental Procedures" for the following numbers of myotubes per construct: Mut1,2 (n = 32 myotubes) and Mut3-11 (n = 26). The asterisk indicates a statistically significant difference (p < 0.01) compared with wtRyR1 as determined using ANOVA with a Dunnett's post test.

 


Figure 6
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  		FIGURE 6.
A, RyR1 constructs containing mutations at position 1 (Mut1) or 2 (Mut2) were created. B and C, agonist-induced changes in intracellular Ca2+ from a representative myotube expressing Mut1 (B) or Mut2 (C) are indicated. Calibration bar = 0.5 F/Fo arbitrary units versus 30 s. D, responses to 4-CmC were normalized to the response to caffeine as described under "Experimental Procedures" for the following numbers of myotubes per construct: Mut1 (n = 60 myotubes) and Mut2 (n = 56). Asterisk indicates a statistically significant difference (p < 0.01) compared with wtRyR1 as determined using ANOVA with a Dunnett's post-test.

 
To identify which of these 11 mutated residues are responsible for the reduced 4-CmC sensitivity of the Mut1-11 construct, additional full-length RyR1 constructs containing smaller numbers of these mutations were tested (Fig. 4A). RyR1 constructs containing mutations at positions 1, 2, 5-11 (Fig. 4B), 1, 2, 7, 8 (Fig. 4C), or 1, 2, 5, 6 (Fig. 4D) all had significantly reduced 4-CmC-induced Ca2+ transients compared with wtRyR1 (Fig. 4E), whereas their responses to KCl and caffeine were unaffected.

Since all mutated constructs with reduced sensitivity to 4-CmC contain mutations at positions 1 and 2, a RyR1 channel containing mutations of only these two amino acids was tested (Mut1,2; Fig. 5A). This construct also had significantly reduced 4-CmC-induced Ca2+ transients compared with wtRyR1 (Fig. 5B). To determine whether positions 3-11 contribute to 4-CmC sensitivity, a RyR1 construct containing only mutations at these positions was tested (Mut3-11; Fig. 5A). The Mut3-11 construct could be activated by 4-CmC, although the 4-CmC-induced calcium transients were slightly reduced compared with wtRyR1 (Fig. 5, C and D). This finding leaves open the possibility that other residues in addition to positions 1 and 2 may be involved in 4-CmC activation of wtRyR1. However, the finding that mutations at positions 1 and 2 have a much more dramatic effect on 4-CmC sensitivity compared with mutations at positions 3-11 suggests that Gln4020 and Lys4021 constitute the primary determinant for activation of RyR1 by 4-CmC.

Full-length RyR1 constructs containing single mutations at either position 1 or position 2 were also tested (Fig. 6A). Activation of the Mut1 channel (Fig. 6B) with either caffeine or 4-CmC but not KCl resulted in repetitive oscillations in intracellular Ca2+ in 55% of myotubes tested. These oscillations were not seen in myotubes expressing Mut2 (Fig. 6C), which responded to all three methods of activation similarly to wtRyR1. Furthermore, the normalized 4-CmC responses of myotubes expressing Mut1 were significantly reduced relative to wtRyR1 (Fig. 6D).

Substitution of RyR1 segments required for 4-CmC activation into RyR3 can restore 4-CmC sensitivity of this isoform (Fig. 1D). To test the hypothesis that positions 1 and 2 mediate RyR1 sensitivity to 4-CmC, we mutated these residues in RyR3 to their RyR1 counterparts (L3873Q/Q3874K, Fig. 7A). This construct (RyR1 Mut1,2) responded to 4-CmC (Fig. 7B) with Ca2+ transients that were significantly larger than 4-CmC induced Ca2+ transients in RyR3 (Fig. 7C). These results further strengthen the hypothesis that these amino acid residues are the primary determinant of 4-CmC sensitivity in RyR1.

To determine whether 4-CmC preferentially activates either the cytoplasmic or luminal face of RyR1, single channel measurements were performed (Fig. 8). Consecutive additions of 4-CmC to the cytoplasmic (cis) face of RyR1 increased the Po from its resting level of 0.073 to 0.341 (25 µM 4-CmC) and 0.549 (50 µM 4-CmC; Fig. 8A). When applied to the luminal (trans) side of the channel, 4-CmC increased channel Po from a resting level of 0.106 to 0.431 (25 µM 4-CmC) and 0.636 (50 µM 4-CmC; Fig. 8B). Thus, under the experimental conditions used here, 4-CmC was equally potent and efficacious toward activating RyR1 channel activity from both cytoplasmic and luminal sides of the lipid bilayer. Moreover, addition of 4-CmC simultaneously to both cytoplasmic and luminal sides of the lipid bilayer produced an additive effect on channel activity, increasing a resting Po of 0.084-0.892 (25 µM 4-CmC each side) and 0.911 (50 µM 4-CmC; Fig. 8C). The dependence of RyR1 open probability on the concentration of 4-CmC applied to the cis, trans, or cis + trans sides of the bilayer is indicated in Fig. 9.


Figure 7
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  		FIGURE 7.
A, a RyR3 construct containing the mutations L3873Q/Q3874K (RyR3 Mut 1,2) was created. B, Agonist-induced changes in intracellular Ca2+ from a representative myotube expressing RyR3 Mut1,2 are indicated. Calibration bar = 0.2 F/Fo arbitrary units versus 30 s. C, Responses to 4-CmC were normalized to the response to caffeine as described in "Experimental Procedures" for n = 31 myotubes. The asterisk indicates a statistically significant difference (p < 0.01) compared with wtRyR3 as determined using ANOVA with a Dunnett's post test.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
CONCLUSION
 REFERENCES
 
In this study, we have defined 2 amino acid residues of rabbit RyR1 (Gln4020 and Lys4021) that are required for 4-CmC activation. Mutation of these residues to their RyR3 counterparts selectively reduces 4-CmC activation of the mutant channel compared with wtRyR1. Conversely, mutation of these residues in RyR3 to their RyR1 counterparts imparts 4-CmC sensitivity to RyR3. Of these 2 residues, Gln4020 appears to be more important in channel function since mutation of this residue in RyR1 results in repetitive Ca2+ oscillations evoked by caffeine or 4-CmC.


Figure 8
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  		FIGURE 8.
RyR1 single channel activity in the absence and presence of 4-CmC was measured as described under "Experiment Procedures." RyR1 activity in the presence of 0.9 µM cis free Ca2+ but in the absence of 4-CmC was used as a control (top traces). As indicated, consecutive additions of 25 and 50 µM 4-CmC were perfused into either the cis (A), trans (B), or both cis and trans sides of the lipid bilayer (C). Representative current traces were taken from original recordings of 1.5-5 min in duration. Channel gating kinetics were measured as described under "Experimental Procedures."

 
Does 4-CmC Bind to the Gln4020-Lys4021 Dipeptide?—The 4-CmC binding site most likely is located between amino acids 3759-4544 of RyR1 since N-terminal deletion mutants of RyR2 that contain the corresponding sequence (3722-4485) can be activated by 4-CmC, whereas deletion mutants lacking this sequence cannot (18). Given that the Gln4020-Lys4021 dipeptide is located within this region, this segment can be considered as a potential candidate for 4-CmC binding.

This hypothesis is strengthened by findings from our recent study on the structural determinants of the 4-CmC molecule that are required for activation of RyR1 (19). We found that the 1-OH of 4-CmC is critical for the ability of the compound to activate RyR1. Moreover, the phenolic proton appears to be shared between 4-CmC and a general base on RyR1, which has a pH profile consistent with it being either a histidine or a lysine residue. This residue could be the Lys4021 defined in this study.

A second finding in our recent work is that hydrophobic groups at the 3- and 4-positions of 4-CmC are required for activation of RyR1. This result is consistent with our findings in the present study that indicate the proximity of a hydrophobic segment, possibly M2, to the QK dipeptide. One model consistent with both sets of results predicts that the QK dipeptide stabilizes the 1-OH moiety of 4-CmC, whereas the M2 segment stabilizes the 3- and 4-hydrophobic groups. This model provides a framework for additional experiments aimed at understanding the molecular mechanisms by which RyR1 responds to 4-CmC.

A separate but related question is whether the 4-CmC binding site resides on the luminal or cytoplasmic face of RyR1. Single channel studies (20) originally suggested a luminal binding site for 4-CmC since this compound preferentially activated RyR1 when it was applied to the luminal face of the channel. However, in our attempts to reproduce this data, we found that 4-CmC activates RyR1 with roughly equal potency when applied to either the cytoplasmic or luminal side. These results clearly indicate that elements of RyR1 structure essential for 4-CmC activation can be equally accessed from either the cytoplasmic or luminal sides of the lipid bilayer. The reason for the discrepancy between our results and those of Herrmann-Frank and co-workers (20) is not clear considering that the experimental protocols were nearly identical in the two studies. Nevertheless, a new finding in the present study indicates that when 4-CmC is applied simultaneously to both sides of the lipid bilayer, its effect on channel activity is essentially additive. These new data support a hypothesis in which the 4-CmC binding site most likely resides in close proximity to the lipid bilayer or even at the protein/lipid interface. This hypothesis is supported by studies on structural analogs of 4-CmC such as 4-alkyl phenols whose activity is directly proportional to the length of the 4-alkyl chain (21). These alkyl chains could anchor the compound to the membrane allowing the compound to diffuse to a binding site at the protein/lipid interface of RyR1. Presumably, 4-CmC acts similarly, which could explain the lack of preferential activation we observe in our single channel measurements.

Location of the Gln4020-Lys4021 Dipeptide within the RyR1 Primary Structure—The Gln4020-Lys4021 dipeptide is located proximally to a number of important functional elements in the RyR1 primary structure. RyR1 is thought to be comprised of two overall structural domains, a massive N-terminal cytoplasmic domain and a smaller C-terminal transmembrane domain that anchors the channel to the membrane and also contains the Ca2+ permeation pore (22). The Gln4020-Lys4021 dipeptide is located at the beginning of this C-terminal channel domain. Directly adjacent to the dipeptide is the putative M2 transmembrane segment, which contains a highly conserved glutamate residue (Glu4032) that has been implicated as a "Ca2+ sensor"; i.e. a residue required to translate Ca2+ binding to the channel into RyR activation (23). In addition, putative binding sites for calmodulin (amino acids 3614-3643 (22, 24)) and Ca2+ (amino acids 4079-4090 and 4116-4127 (25)) are located near the Gln4020-Lys4021 dipeptide. Taken together, these observations suggest that the 4-CmC activation site and Ca2+ regulatory sites are proximal to each other. This arrangement could provide an attractive explanation for how localized conformational changes in RyR1 structure induced by 4-CmC binding could be conveyed to nearby Ca2+ regulatory sites, thus increasing the sensitivity of RyR1 to Ca2+ activation as has been reported (20).


Figure 9
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  		FIGURE 9.
RyR1 channel Po values in the presence of different (4-CmC) but constant recording conditions (cis and trans buffers, free [Ca2+], holding potential, sample rating) are indicated. The data were obtained from three different JSR membrane preparations and a total number of 34 independent single channel measurements.

 
Protein Modeling—To understand how the Q4020L and K4021Q mutations might alter the local structure of RyR1, we modeled the secondary structure between amino acids 4007 and 4037 and compared this predicted structure to the equivalent regions from RyR2 and RyR3 (Fig. 10, A-C). For all three isoforms, the predicted overall secondary structure is {alpha}-helical. In wtRyR1 and wtRyR2, the Gln4020-Lys4021 dipeptide is adjacent to a highly hydrophobic segment (residues 4023-4041 in RyR1) originally predicted as the M2 transmembrane segment (26). In RyR3, this hydrophobic region is extended to include the RyR3 equivalent of the 4-CmC activation dipeptide, Leu3873-Gln3874. When the Q4020L and K4021Q mutations are introduced into RyR1 (Fig. 10D), this hydrophobic segment becomes extended as well, producing a similar hydropathy profile to that of wtRyR3. These observations suggest that extending this hydrophobic segment to include the Gln4020-Lys4021 dipeptide reduces 4-CmC sensitivity of RyR1.

A second major difference between RyR1/RyR2 and RyR3 in the predicted secondary structure is the presence of a putative surface-exposed region in RyR1 and RyR2 that is not present in RyR3 (Fig. 10, A-C). This surface-exposed region lies just N-terminal to the Gln4020-Lys4021 dipeptide of RyR1 and to its equivalent in RyR2. Thus, a hypothesis consistent with our experimental data and also the modeled structure of this area is that this surface-exposed region is required for 4-CmC activation of RyR1 and RyR2. This hypothesis is strengthened by the observation that the Q4020L/K4021Q mutant also lacks the predicted surface exposed region (Fig 10D). Indeed, the presence of this putative surface-exposed region is correlated well with 4-CmC sensitivity of the RyR. Mutation of Lys4021 alone (K4021Q) affects neither 4-CmC sensitivity nor the predicted surface exposed region (Fig. 10F), whereas mutation of Gln4020 alone (Q4020L) diminishes the probability of the surface exposed sequence (Fig. 10E) and also diminishes 4-CmC-induced Ca2+ transients.


Figure 10
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  		FIGURE 10.
Diagrams indicate secondary structure analysis of wtRyR1 (A), wtRyR2 (B), wtRyR3 (C), Mut1,2 (D), Mut1 (E), and Mut2 (F) constructs within a 30-amino acid segment corresponding to residues 4007-4036 in wtRyR1 (top row of each panel). Middle row, hydropathy analysis was conducted using the Kyte-Doolittle method as described under "Experimental Procedures." Lower row, surface probability prediction was conducted according to the method of Emini et al. (16) as described under "Experimental Procedures." The Gln4020 and Lys4021 residues in RyR1 and the equivalent residues in the other constructs are bounded by dotted lines. The arrows indicate the presence of potential surface-exposed residues.

 

   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSION
REFERENCES
 
In this report, we have defined a critical dipeptide segment of RyR1, Gln4020-Lys4021, that is required for 4-CmC activation of the channel. Future work will be needed to understand the mechanism of channel activation by 4-CmC and how these residues are involved in this process.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants R01-AR43640 and 1F32 HL67572-01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Boston Biomedical Research Institute, 64 Grove St., Watertown, MA 02472. Tel.: 617-658-7886; Fax: 617-971-1761; E-mail: fessenden{at}bbri.org.

2 The abbreviations used are: RyR, ryanodine receptor; 4-CmC, 4-chloro-m-cresol; SR, sarcoplasmic reticulum; HSV, herpes simplex virus; wt, wild-type; ANOVA, analysis of variance. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Ronit Hirsch for expert technical assistance in preparing the HSV-1 amplicons for infection of 1B5 myotubes and Dr. Margarita M. Cala for preparing 1B5 myotubes for some imaging experiments.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
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 RESULTS
 DISCUSSION
 CONCLUSION
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