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Originally published In Press as doi:10.1074/jbc.M411136200 on October 6, 2004

J. Biol. Chem., Vol. 279, Issue 51, 53028-53035, December 17, 2004
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Mutational Analysis of Putative Calcium Binding Motifs within the Skeletal Ryanodine Receptor Isoform, RyR1*

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

From the {ddagger}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, Davis, California 95616

Received for publication, September 28, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The functional relevance of putative Ca2+ binding motifs previously identified with Ca2+ overlay binding analysis within the skeletal muscle ryanodine receptor isoform (RyR1) was examined using mutational analysis. EF hands between amino acid positions 4081 and 4092 (EF1) and 4116 and 4127 (EF2) were scrambled singly or in combination within the full-length rabbit RyR1 cDNA. These cDNAs were expressed in 1B5 RyR-deficient myotubes and channel function assessed using Ca2+-imaging techniques, [3H]ryanodine binding measurements, and single channel experiments. In intact myotubes, these mutations did not affect functional responses to either depolarization or RyR agonists (caffeine, 4-chloro-m-cresol) compared with wtRyR1. However, in [3H]ryanodine binding measurements, both Ca2+ activation and inhibition of the EF1 mutant was significantly altered compared with wtRyR1. No high affinity [3H]ryanodine binding was observed in membranes expressing the EF2 mutation, although in single channel measurements, the EF2-disrupted channel could be activated by micromolar Ca2+ concentrations. In addition, micromolar levels of ryanodine placed these channels into the classical half-conductance state, thus indicating that occupancy of high affinity ryanodine binding sites is not required for ryanodine-induced subconductance states in RyR1. Disruption of three additional putative RyR1 calcium binding motifs located between amino acid positions 4254 and 4265 (EF3), 4407 and 4418 (EF4), or 4490 and 4502 (EF5) either singly or in combination (EF3–5) did not affect functional responses in 1B5 myotubes except that the EC50 for caffeine activation for the EF3 construct was significantly increased compared with wtRyR1. However, in [3H]ryanodine binding experiments, the Ca2+-dependent activation and inactivation of mutated RyRs containing EF3, EF4, or EF5 was unaffected when compared with wtRyR1.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Calcium plays a critical role in skeletal muscle contraction. During excitation-contraction coupling, calcium is released from the sarcoplasmic reticulum (SR)1 through activation of the intracellular calcium release channel known as the type 1 ryanodine receptor (RyR1). Upon release, calcium binds to troponin C in the myosin head assembly to trigger muscle contraction.

However, calcium also regulates the contractile process by modulating activity of key proteins involved in EC coupling including the RyR itself. In fact, calcium has biphasic effects on RyR activity. At micromolar levels, calcium stimulates the channel in a process known as calcium-induced calcium release (CICR) whereas at millimolar levels, calcium inhibits RyR activity. This biphasic action is thought to occur through the binding of calcium to two classes of regulatory sites on the RyR, a stimulatory high affinity site and an inhibitory low affinity site (1).

Several potential calcium regulatory sites have been identified within the primary sequence of the RyR. A potential "calcium sensor" (i.e. a region of the RyR required for calcium activation of the channel) was identified at position 3885 of the RyR3 isoform. Mutation of this highly conserved glutamate to alanine resulted in a channel with drastically altered calcium-sensing properties in single channel studies (2). A subsequent report mutating the analogous residue in RyR2 yielded similar findings (3). However, the functional effects of the analogous mutation in RyR1 (E4032A) could be reversed using high concentrations of ryanodine (4). Whether this residue is involved in translating calcium binding to channel activation is still open to debate.

In addition, two EF hand calcium binding protein motifs (5) have been identified within the RyR primary sequence using sequence comparisons between rabbit skeletal muscle (RyR1), cardiac (RyR2), and the analogous skeletal muscle RyR isoform from lobster (6). The lobster isoform contains an EF hand motif that, when expressed as a peptide fragment, strongly bound 45Ca2+ in overlay and equilibrium binding studies. However much weaker 45Ca2+ binding was observed when the corresponding EF hand motif peptides from mammalian RyR1 and RyR2 were tested. Similarly, peptide fragments derived from the RyR homologue in Caenorhabditis elegans containing these EF hand motif bound 45Ca2+ in overlay experiments (7).

In addition, three highly negatively charged EF hand-like sequences in the C-terminal portion of the RyR were predicted after the initial cloning of RyR1 (8). RyR peptides containing these sequences bound 45Ca2+ in gel overlay assays (9) and an antibody raised against positions 4478–4512 of RyR1 (a region containing the third putative calcium binding sequence) abolished calcium sensing in single channel studies of the RyR (10). These findings strongly suggested that at least one of the putative sites in this region was the high affinity calcium binding site on RyR1 that mediates calcium activation of the channel.

To determine the functional significance of these predicted calcium binding sequences on RyR1 function, we have used mutational analysis to disrupt these regions in the rabbit RyR1 cDNA and then expressed these mutated RyRs in a dyspedic skeletal muscle cell line (1B5). We then examined the caffeine sensitivity of these mutated RyRs in intact cells as well as the ability of Ca2+ to regulate these proteins using [3H]ryanodine binding techniques and single channel analysis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning—The complementary DNA encoding the rabbit skeletal muscle RyR isoform (RyR1) has previously been cloned into pHSVpr-PUC thus enabling the generation of HSV amplicons required for transduction of 1B5 myotubes (11). Putative EF hand regions were mutated to the sequences described in Fig. 1 using primer extension-driven site-directed mutagenesis. The primers used to create these EF hand mutations are indicated below. (Note: mutated sequence is indicated in lowercase letters.)



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  		FIG. 1.
A, five putative calcium binding motifs lie between amino acids 4000–4500 of the wtRyR1 primary sequence (black bar); EF1, amino acids 4081–4092; EF2, 4116–4127; EF3, 4251–4266; EF4, 4402–4419; EF5, 4488–4499. B, EF1 and EF2 are each arranged as a helix-loop-helix motif. The putative amino acids that provide calcium chelation are located at the X, Y, Z, –X, and –Z positions. The scrambled sequence used to disrupt the calcium binding loops is indicated in bold. C–E, for EF3, -4, and -5, calcium binding sequences were disrupted by replacing all negatively charged amino acids with their neutral counterparts as indicated in bold.

 
EF1 (positions 12235–12276 of rabbit wtRyR1 cDNA sequence): forward 5'-GGCCTTCCAGgactacggcgacaaaccctctaagcttATCTCCAAAAAGGAC-3', reverse 5'GTCCTTTTTGGAGATaagcttagagggtttgtcgccGTAGTCCTGGAAGGCC-3'. EF2 (12346–12381): forward 5'-ttcgaagatgaagccaacgagaagTTCGCCAACCGCTTCC-3', reverse 5'-ttcgaagttaatttccatGGAGCACGAGAGCAGAAACTGG-3'. EF3 (12757–12798): forward 5'-CCCGgGGCCaACcAGaACcAGGGCATGGGCGAG-3', reverse 5'-CcCGGGCTgGCCCTgGGGCTgGGAGATCTGCG-3'. EF4 (13213–13248): forward 5'-tGGCcAGGGCaACGCGGCacAGGGTaACGGCGATG-3', reverse 5'-tgGCcaTtGCCCTgtCCCGCGCCGTtCGCGTCGCC-3'. EF5 (13458–13497): forward 5'-CCCGgGCCCcAGCCTcAGCCTcAGAAAGCCG-3', reverse 5'-CcCGGGCTgCGGTTgTGGAACCAGCTCCTCC-3'. PCR products generated using Pfu DNA polymerase (Promega, Madison, WI) were cloned into TOPO-TA vectors (Invitrogen, Carlsbad, CA) and confirmed using bi-directional DNA sequencing.

Cell Culture—1B5 myotubes were prepared as described previously (12, 13). After 5 days, differentiated myotubes were transduced with the RyR constructs using equivalent amounts of HSV virions (11) that contained the cDNAs encoding the RyRs. Myotubes were then 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 as described (14). 1B5 myotubes were loaded with 5 µM Fluo-4/AM (Molecular Probes) for 30 min at 37 °C, and changes in intracellular Ca2+ were monitored at x40 magnification (UPlanApo/340, Olympus; Melville, NY) using a Nikon Diaphot 300 microscope (Nikon; Melville, NY). Fluo-4 was excited at 495 nm using a DeltaRam lightsource (Photon Technology, Inc.; Monmouth Junction, NJ) and emitted fluorescence from the 1B5 myotubes was measured using an XR/Mega-12 ICCD camera (Stanford Photonics Inc.; Palo Alto, CA). Resultant images were acquired at the rate of 12 images per second and record traces representing changes in fluorescence of individual cells were analyzed using Microsoft Excel. Myotubes were challenged with either successive additions of 80 mM KCl, 40 mM caffeine, and 0.5 mM 4-chloro-m-cresol (4-CmC), or a graded series of caffeine concentrations dissolved in imaging solution.

Quantification of Ca2+ Transients and Determination of Caffeine EC50—Calcium transients were quantified as described previously (14). In caffeine dose response experiments, EC50 values for activation by caffeine were obtained for each RyR-expressing myotube by fitting a sigmoidal dose-response curve with variable slope using Prism V. 3.0 (Graphpad Software, San Diego, CA). The EC50 values reported for each construct were then obtained by averaging the EC50 values of all individual myotubes expressing a particular RyR. EC50 values for each mutated RyR were then compared with wtRyR1 EC50 values obtained from myotubes imaged on the same experimental day. Comparison of EC50 values between wild-type and mutant RyRs was made using two-way analysis of variance (ANOVA) with a 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 and single channel studies were prepared and characterized using Western blot techniques as described previously (13, 14). Western blot analysis revealed that each mutant construct was of identical size to wtRyR1 (data not shown).

[3H]Ryanodine Binding Assays—[3H]Ryanodine (specific activity 56 Ci/mmol) was purchased from PerkinElmer Life Sciences (Boston, MA). Specific binding of [3H]ryanodine to SR preparations was performed as described previously (14). The ability of crude SR preparations containing wild type or mutant RyR1 channels to bind 5 nM [3H]ryanodine was determined in 0.5-ml assays by incubating 35 µg of SR vesicles/assay in buffer consisting of 250 mM KCl, 20 mM Hepes, pH 7.1 (KOH), 100 µM EGTA, and variable amounts of CaCl2. Final free Ca2+ concentrations were calculated using the WebMaxChelator program. Binding reactions conducted at 37 °C for 3 h were terminated by rapid filtration onto GF/B glass fiber filters and then rinsed with 3 x 2 ml of ice cold 20 mM Tris-HCl, pH 7.4. The amount of [3H]ryanodine bound to each filter was determined by liquid scintillation counting.

EC50 values were determined by fitting the data points obtained with 0–100 µM [Ca2+]f to a sigmoidal dose-response curve with variable slope. IC50 values were determined by fitting data points obtained with 0.1–30 mM [Ca2+]f to a one-site competition model with variable slope. Both analyses were performed using Prism V. 3.0 software. Individual EC50 and IC50 values from separate dose response experiments were averaged together and compared using 2-way ANOVA.

Single Channel Measurements—Single channel behavior was measured by fusing membrane vesicles isolated from 1B5 myotubes containing the EF2 mutation into artificial planar lipid bilayers (5:2 phosphatidylethanolamine/phosphatidylcholine, 50 mg/ml in decane). The membrane preparation was introduced to the cis chamber as described previously (15). This chamber contained 0.7 ml of 500 mM CsCl, varied CaCl2 (10 nM to 10 µM) and 20 mM HEPES (pH7.4), whereas the trans side contained 50 mM CsCl, and 20 mM HEPES (pH7.4). Single channel activity of EF2 was measured at a holding potential of +40 mV (applied cis relative to the trans, i.e. ground side) using a patch clamp amplifier (Warner Instrument Corp, Hamden, CT). The data were filtered at 1 kHz and acquired at 10 kHz with a DigiData1320A (Axon Instruments, Union City, CA). The data were analyzed using pClamp 9 (Axon Instruments) without additional filtering.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Five putative calcium binding sequences have been proposed in the wtRyR1 primary sequence between amino acid positions 4000 and 4500 (Fig. 1A). EF hands 1 and 2, identified during the cloning of the lobster RyR1 cDNA (6) are each comprised of a classical helix-loop-helix motif that contains potential calcium-chelating amino acid residues located at the X, Y, Z, –X, and –Z positions of the calcium binding loop (Fig. 1B). To determine the potential functional significance of these putative EF hands, we scrambled these sequences, maintaining the overall amino acid composition but disrupting the spatial arrangement of the amino acids within the calcium binding loop. In particular, we ensured that the negatively charged amino acids at the –z position were not present in each of the scrambled Ca2+ binding loops since most EF hands contain either a glutamic or aspartic acid residue at this position. We also created a double mutant, EF1–2 that contained both scrambled sequences.

To determine the functional consequences of each mutation, we expressed these RyRs in 1B5 myotubes and examined calcium transients elicited by 80 mM KCl (to test for depolarization-induced calcium release (i.e. EC coupling)), 40 mM caffeine, and 0.5 mM 4-CmC. These latter two agonists are of especial interest since these compounds activate the RyR by increasing the apparent affinity of calcium for its activator site (16, 17).

1B5 myotubes expressing wtRyR1 (Fig. 2A) responded to all three test substances as has been described previously (13). RyR1 containing the disruption of EF1 (Fig. 2B), EF2 (Fig. 2C), or both (EF1–2; Fig. 2D) also responded to all three agonists. The percentage of myotubes expressing the EF mutants that responded to these stimuli was similar to wtRyR1 (Fig. 2E).



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  		FIG. 2.
Disruption of putative EF hands 1 and 2 was accomplished as described under "Experimental Procedures." 1B5 dyspedic myotubes transduced with HSV virions containing wild-type or mutant RyR1 cDNAs were challenged with consecutive 30-s additions of 80 mM KCl (black bar), 40 mM caffeine (clear bar), and 0.5 mM 4-chloro-m-cresol (stippled bar), and the resultant changes in myoplasmic calcium were monitored using the calcium-sensitive dye, Fluo-4. Calcium transients are shown for a single representative cell transduced with HSV virions containing either wtRyR1 (A) or mutant RyR1 cDNAs containing a disruption of EF1 (B), EF2 (C), or both (D). E, total number of 1B5 myotubes expressing either wild-type or mutant RyR1 responding to each of the three stimuli was determined. Values represent total number of cells that displayed a greater than 10% increase in Fluo-4 fluorescence over baseline (F/Fo) in response to each agonist.

 
Wild-type RyR1 (Fig. 3A), EF1 (Fig. 3B), EF2 (Fig. 3C), and EF1–2 (Fig. 3D) all had similar thresholds for activation by caffeine (~3 mM). The wtRyR1 EC50 value for activation by caffeine (2.83 mM) was not significantly different from the EC50 values for caffeine activation of the EF hand mutants (ranging from 2.37 mM for EF2 to 3.42 mM for EF1 (Fig. 3E)).



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  		FIG. 3.
The concentration dependence of caffeine activation of wild-type and mutated RyR1 proteins was determined as described under "Experimental Procedures." Caffeine-induced calcium transients from a representative myotube expressing wtRyR1 (A), EF1 (B), EF2 (C), or EF1–2 (D) are shown. E, caffeine dose response curves are shown for wild-type and mutant RyRs. Log EC values for each construct were determined as described under "Experimental Procedures." All curves are normalized to the amount of calcium 50released by 40 mM caffeine.

 
To more directly test if scrambling these putative EF hands altered Ca2+ regulation of RyR1, we examined the Ca2+ dependence of [3H]ryanodine binding to membrane vesicles isolated from 1B5 myotubes expressing either wtRyR1, EF1, EF2, or EF1–2 (Fig. 4). Although [3H]ryanodine binding to wtRyR1 and EF1 could be stimulated by either 1 µM Ca2+, 20 mM caffeine, or 0.5 mM 4-CmC, no specific high affinity [3H]ryanodine binding to EF2 or EF1–2 was detected (Fig. 4A) despite similar expression levels of the wild-type and mutated RyRs as determined in Western blot measurements (Fig. 4A, inset). For the EF1 construct, both Ca2+-dependent activation (Fig. 4B) and inhibition (Fig. 4C) were significantly altered compared with wtRyR1. The EC50 for Ca2+ activation of EF1 was decreased to 1.02 µM compared with wtRyR1 (2.8 µM) whereas the IC50 for calcium inhibition of EF1 was increased to 1.63 mM compared with wtRyR1 (0.85 mM) as summarized in Fig. 4D.



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  		FIG. 4.
A, [3H]ryanodine binding to membranes expressing wtRyR1, or the EF1, EF2 or EF1–2 mutant constructs was determined after stimulation with 1 µM calcium either alone (clear bars) or supplemented with 20 mM caffeine (filled bars) or 0.5 mM 4-CmC (cross-hatched bars). Data bars represent mean ± S.D. for four determinations. Inset, Western blot analysis for SR vesicles expressing wtRyR1, EF1, EF2, or EF1–2 mutant proteins (20 µg of total protein/lane) was performed as described under "Experimental Procedures." B and C, concentration dependence of Ca2+ activation (B) or inhibition (C) of [3H]ryanodine binding to membranes expressing wtRyR1 or EF1 was determined as described under "Experimental Procedures." D, log EC50 and log IC50 values for calcium activation/inhibition of wtRyR1, and EF1 constructs were determined as described under "Experimental Procedures." For the EF2 and EF1–2 constructs, no EC50 or IC50 values were obtained because of the lack of Ca2+-stimulated [3H]ryanodine binding for these mutants. *, p < 0.001 as determined using Student's t test.

 
To determine if the lack of high affinity [3H]ryanodine binding to membranes expressing EF2 and EF1–2 was due to loss of activity of these channels during isolation, we measured the activity of single EF2 channels from the same membrane preparations used for [3H]ryanodine binding measurements (Fig. 5). The open probability of single EF2 channels increased with defined cis Ca2+ concentrations ranging from 10 nM to 10 µM (Fig. 5, A–C). In addition, EF2 channel gating could be locked into a half conductance state by 10 µM ryanodine and fully inhibited by 10 µM ruthenium red (Fig. 5B). Finally, Ca2+-stimulated EF2 activity could be further enhanced by the addition of 2 mM caffeine (Fig. 5D). These findings indicate that the EF2 protein retains Ca2+ channel activity after isolation.



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  		FIG. 5.
Single channel activity of the EF2 mutant (n = 11) was determined in the presence of defined free [Ca2+], ranging from 10 nM-10 µM (A–C) as described under "Experimental Procedures." The defined free [Ca2+] was obtained by adding EGTA as calculated according to Bound and Determined software (30). Open current fluctuations are indicated as upward traces. RyR modulators such as ryanodine and ruthenium red (B) and caffeine (D) were added to the cis chamber. In B, the arrows with O, S, and C, indicate the current levels of fully open, subconductance, and closed states of the channel.

 
We also examined three additional potential calcium binding domains located in the C terminus of wtRyR1. These three sequences contain multiple negatively charged residues alternating between either proline or glycine residues (Fig. 1, C–E). We hypothesized that the structural basis of the potential interaction between these sequences and a calcium ion would reside in the negative charge of the glutamic or aspartic acid side chains. To disrupt this interaction, we replaced these negatively charged amino acids with their neutral counterparts (i.e. Glu-Gln or Asp-Asn substitution). We created separate mutated RyR1 constructs containing either mutations in a single motif (EF3, EF4, or EF5) or all three mutated sequences in a single construct (EF3–5).

As above, we first tested these mutated RyRs for their response to KCl, caffeine, and 4-CmC (Fig. 6). Similar to wtRyR1 (Fig. 6A), the mutated EF3 (Fig. 6B), EF4 (Fig. 6C), EF5 (Fig. 6D), and EF3–5 RyRs (Fig. 6E) all responded to these test substances. The relative proportion of myotubes responding to these agonists was unchanged compared with myotubes expressing wtRyR1 (Fig. 6F).



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  		FIG. 6.
Disruption of negatively charged sequences EF3, -4, and -5 was accomplished as described under "Experimental Procedures." 1B5 myotubes expressing wtRyR1 (A) or disruption of EF3 (B), EF4 (C), EF5 (D) or all three disruptions (EF3–5; E) were challenged with 80 mM KCl (black bar), 40 mM caffeine (clear bar), or 0.5 mM CmC (stippled bar). Representative calcium transients from each construct are shown. F, total number of myotubes expressing each construct that responded to the three stimuli are shown and expressed as a percentage of total myotubes examined.

 
Next, we examined the caffeine sensitivity of each mutated construct (Fig. 7). All 4 mutated constructs had similar thresholds for activation by caffeine (Fig. 7, B–E) compared with wtRyR1 (Fig. 7A). The EC50 values for activation by caffeine for these mutated RyRs ranged from 3.35 mM for EF3–5 to 6.99 mM for EF3 (Fig. 7F). Of these constructs, only the EC50 for EF3 was significantly different (increased) compared with wtRyR1.



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  		FIG. 7.
Caffeine dose response curves were obtained for 1B5 myotubes expressing wild-type or mutant RyRs. Caffeine-induced calcium transients from a representative myotube expressing EF3 (A), EF4 (B), EF5 (C), or EF3–5 (D) are shown. E, caffeine-induced calcium transients were quantified and normalized to the peak value at 40 mM caffeine. Normalized values are shown as dose response curves with each point representing mean ± S.E. for the number of measurements indicated. Log EC values were obtained as described under 50"Experimental Procedures." *, p < 0.05 as determined using ANOVA with a Dunnett's post-test.

 
However, Ca2+ had the expected biphasic effect on the level of [3H]ryanodine binding to EF3, EF4, and EF5 which were almost identical compared with wtRyR1 (Fig. 8). The EC50 for Ca2+ activation ranged from 1.8 for EF5 to 2.4 µM for EF3 and EF4 (Fig. 8A). None of these EC50 values were significantly changed compared with the EC50 for wtRyR1 (2.8 µM). Similarly, the IC50 values for calcium inactivation of these mutated constructs (which ranged from 1.16–1.27 mM) were also not significantly different from wtRyR1 (Fig. 8B).



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  		FIG. 8.
The calcium activation (A) and inhibition (B) of [3H]ryanodine binding to membranes expressing either wtRyR1, EF3, EF4, or EF5 were determined. Values normalized to the peak level of activation observed at 100 µM [Ca2+]f are depicted as dose response curves with each point representing mean ± S.D. for 5–13 measurements. Log EC50 and log IC50 values were determined as described under "Experimental Procedures." Statistical analyses using ANOVA yielded no significant differences for any condition tested.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Several lines of evidence suggest that calcium regulatory sequences in RyR1 reside within the C terminus of the protein. 1) A deletion mutant lacking the first 3660 amino acids of the RyR can still be activated by calcium (18). 2) Studies using RyR1-RyR2 chimeras suggest the presence of calcium inactivation sites at the C terminus of the protein (19, 20). 3) C-terminal RyR peptides bind calcium in 45Ca2+ overlay studies (9). 4) Analysis of the peptide sequence of the C terminus suggests the presence of calcium binding motifs either as large stretches of highly negatively charged sequences (8) or as EF hand Ca2+ binding motifs (6). In this report, we have used mutational analysis to determine the role of five putative C-terminal Ca2+ binding motifs in calcium regulation of RyR1.

EF Hand Ca2+ Binding Sites—Originally discovered in the crystal structure of parvalbumin (21), EF hands have since been identified in over 200 different proteins that are regulated by Ca2+ (22). Often found in pairs, EF hands are comprised of a central calcium binding loop flanked by two {alpha}-helices. Within the calcium binding loop, oxygen atoms in side chains of amino acids at the X, Y, Z, –X, and –Z positions (Fig. 1) coordinate the calcium ion with an additional coordination site provided by a peptide backbone carbonyl group. The –Z position is the most conserved position of the EF hand, containing either an aspartic or glutamic acid residue whose side chain chelates the calcium ion.

In the cDNA of the RyR homologue from lobster, a tandem set of EF hand motifs have been identified (6). The analogous regions from rabbit skeletal muscle RyR1 (EF1 and EF2 in this study) and cardiac RyR2 weakly bind 45Ca2+ (Kd~1–5 mM) in equilibrium binding studies, thus suggesting that these EF hands may play a role in calcium inactivation of mammalian RyRs.

We disrupted the EF1 and EF2 regions in the RyR1 cDNA by scrambling the amino acid sequence of the calcium binding loop. These mutations would be expected to disrupt the spatial geometry of the calcium binding loops while maintaining the overall amino acid composition. In intact myotubes, disruption of these EF hands did not affect any of the parameters of RyR1 function that we examined; namely, depolarization-induced calcium release and sensitivity to caffeine or 4-CmC. Since caffeine is hypothesized to activate the RyR by increasing the affinity of calcium for its activator site (16), we would predict that Ca2+-dependent regulation of these mutants should be unaffected compared with wtRyR1

However, for membranes expressing the EF1 mutant RyR, both Ca2+-dependent activation and inhibition of [3H]ryanodine binding were altered. We detected a 2-fold increase in the IC50 for EF1, compared with wtRyR1, which supports the hypothesis that this sequence may play a role in calcium (or Mg2+) inhibition of the channel. In addition, the EC50 for Ca2+ activation of the EF1 mutant was decreased by 2-fold compared with wtRyR1. These results support the hypothesis that the EF1 sequence is involved in Ca2+ regulation of RyR1. However, its physiologic role is probably minimal since the caffeine sensitivity of this mutant in intact myotubes was unaffected compared with wtRyR1. Most likely, other areas in the RyR1 primary sequence are also involved in Ca2+ sensing.

In contrast, RyRs containing the EF2 and EF1-EF2 mutations had no detectable high affinity [3H]ryanodine binding. We attribute this effect to the disruption of the EF2 region of RyR1 since this mutation is the common element in both of these constructs. This lack of high affinity [3H]ryanodine binding was not caused by poor channel expression since similar levels of the different EF mutant RyR constructs were detected using Western blot techniques. In addition, this lack of binding activity was not caused by nonspecific misfolding of the protein or subunit dissociation since EF2 channels were active both in intact cell studies and in broken cell preparations used for single channel measurements. In addition, the calcium activation site of these channels does not appear to be disrupted since increasing Ca2+ levels from nanomolar to micromolar levels activates single EF2 RyRs in lipid bilayers.

Thus, the most plausible explanation for the lack of [3H]ryanodine binding to the EF2 and EF1–2 constructs is that mutation of the EF2 sequence disrupts the high affinity [3H]ryanodine binding site. This phenomenon has also been observed in studies of RyR1-RyR2 chimeric proteins (23). Of 6 RyR1-RyR2 chimeras previously shown to be functional in intact myotubes (24), 3 did not have detectable levels of high affinity [3H]ryanodine binding. However, one of these chimeras (R4) was active in lipid bilayer experiments and could be locked into a half conductance state by 10 µM ryanodine (23). This phenotype is remarkably similar to the functional characteristics of the EF2-mutated RyRs examined in this study.

One possible explanation for the lack of [3H]ryanodine binding to EF2-disrupted RyRs is that the EF2 sequence represents the high affinity ryanodine binding site. However, this notion seems unlikely since the native EF2 sequence is present in RyR1-RyR2 chimeras that lack high affinity [3H]ryanodine binding (23). Also, previous biochemical studies have localized the high affinity ryanodine binding site to amino acid residues 4475–5037 in the C terminus of RyR1 (25). This site has been further localized to the putative Ca2+ pore-forming segment between residues 4820–4829 of the cardiac RyR isoform (26). Thus, the mechanism of how EF2 disruption impairs high affinity ryanodine binding remains to be determined although a more detailed mutational analysis of the EF2 region may provide additional insights into this phenomenon. However, it is clear from these and previous data on RyR1/RyR2 chimeras that the high affinity binding site is not required for ryanodine to place the channel into the classical half conductance state observed in single channel measurements.

Negatively Charged Putative Ca2+ Binding Regions—The earliest proposed calcium binding sequences in the RyR1 primary sequence were three highly negatively charged regions identified after the initial cloning of RyR1 cDNA (8). In subsequent studies, the functional significance of these sequences was tested using 45Ca2+ overlay experiments performed on trpE-RyR1 fusion proteins (9). Calcium could bind to a large fragment containing amino acids 4014–4765 of RyR1 as well as smaller peptides each containing one of these three sequences (EF3, -4, and -5 in the present study). Remarkably, affinity-purified antibodies raised against the EF5 sequence could selectively inhibit calcium or caffeine activation of single RyR1 channels in lipid bilayers while not affecting activation by adenine nucleotides or sensitivity to ryanodine inhibition (10). These studies strongly implicated EF5 as a calcium activator site of the RyR.

However, our results do not support this hypothesis. Mutation of EF3, EF4, and EF5 singly or together as a triple mutant does not impair RyR1 activation in intact cells, and caffeine sensitivity of these mutants is largely unchanged compared with wtRyR1. Although removing the negatively charged amino acids from the EF3 region alone did result in a slight but significant increase in the EC50 for caffeine activation compared with wtRyR1, the functional significance of this observed change is questionable because the EC50 for caffeine activation of EF3–5 is not significantly different from wtRyR1. These findings lend strong support to the conclusion that these regions do not play a significant role in calcium regulation of RyR1 and question the validity of Ca2+ overlay studies for identifying Ca2+ regulatory regions in the RyR.

Other Potential Calcium Regulatory Sites—Several approaches have been utilized to identify calcium regulatory sites on the RyR. One of the most promising findings was the identification of a "calcium sensor" using site-directed mutagenesis (2, 3). This regulatory site is comprised of a single highly conserved glutamic acid (position 4032 in RyR1) within a putative transmembrane loop of the RyR. Mutation of this residue to alanine in RyR3 dramatically increased the EC50 for calcium activation of the RyR in single channel studies thus suggesting that the calcium activator site had been perturbed by the mutation. However, no direct tests of calcium binding to this region have been reported. Moreover, the observation that high concentrations of ryanodine can overcome the functional defects incurred by the mutation introduced into either RyR1 (4) or RyR2 (27) suggests that this conserved residue may not comprise the actual calcium binding site although the possibility still exists that it is intimately involved in the pathway linking calcium binding to channel activation.

In a separate approach using RyR1/RyR2 chimeric proteins, divergent region 1 (D1), between amino acid positions 4254 and 4631 of wtRyR1, was implicated as a potential calcium regulatory sequence. Overlapping chimeras containing the RyR2 D1 region substituted into RyR1 exhibited decreased calcium inhibition of [3H]ryanodine binding compared with RyR1 to levels similar to RyR2, thus suggesting that calcium inactivation sites are located in the D1 region (19). Substitution of the N-terminal portion of the RyR2 D1 sequence into RyR1 also partially decreased the IC50 for calcium inactivation, thus suggesting that the N-terminal portion of D1 contained calcium inactivation site(s) (20). These studies are promising although they do not provide any insights as to the location of the calcium activation sites on the RyR.

Determination of calcium binding sequences in RyR1 is and will continue to be a difficult task. Most techniques used to study calcium modulation of RyR activity (calcium imaging of intact cells, single channel measurements in lipid bilayers, [3H]ryanodine binding) rely on RyR activity as an end point. If mutation of a particular RyR sequence interferes with calcium activation or inhibition of the RyR, then this sequence may be involved in calcium binding to the RyR or it may simply be important in conducting the calcium binding signal to channel gating. Functional assays cannot distinguish between these two possibilities.

An additional complicating feature in identifying calcium binding sequences in all RyRs is the highly acidic nature of the protein. The pI for wtRyR1 is ~5.0, and the pI in the 4000–4500 amino acid region examined in this study is 4.2. Thus, all 45Ca2+ binding studies have been and will be complicated by this property of the RyR because many potential calcium binding fragments identified with this technique might be due purely to electrostatic interactions that have questionable relevance to channel regulation in vivo.

The identification of physiological calcium modulatory sites on the RyR may best be achieved through a multidisciplinary approach combining biophysical and molecular biological techniques. For example, the trivalent cation, terbium can bind to both classes of calcium modulatory sites on RyR1. The intrinsic fluorescence of terbium increases when it is bound to the calcium inhibitory site (28). This method may be particularly useful as these studies can be done on the full-length RyR rather than smaller fragments of the channel. In addition, calcium-induced changes in intrinsic tryptophan fluorescence have been used to identify calcium regulatory sites (29). Thus, a potential strategy to map calcium binding sites on the RyR would be to use these biophysical methods in conjunction with mutational analysis of potential calcium binding sequences to determine which possible interactions are physiologically relevant. The utility of this approach has already been demonstrated in identifying calcium regulatory sites on the IP3 receptor (29).


   FOOTNOTES
 
* 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

§ To whom correspondence should be addressed: Boston Biomedical Research Institute, 64 Grove St., Watertown, MA 02472. Tel.: 617-658-7886; Fax: 617-972-1761; E-mail: fessenden{at}bbri.org.

1 The abbreviations used are: SR, sarcoplasmic reticulum; RyR1, ryanodine receptor isoform 1; 4-CmC, 4-chloro-m-cresol; HSV, Herpes simplex virus; ANOVA, analysis of variance; wt, wild type. Back



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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