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Originally published In Press as doi:10.1074/jbc.M408189200 on January 6, 2005

J. Biol. Chem., Vol. 280, Issue 12, 11713-11722, March 25, 2005
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A Calmodulin Binding Domain of RyR Increases Activation of Spontaneous Ca2+ Sparks in Frog Skeletal Muscle*

George G. Rodney{ddagger}, Gerald M. Wilson{ddagger}§, and Martin F. Schneider{ddagger}

From the {ddagger}Department of Biochemistry and Molecular Biology and §Center for Fluorescence Spectroscopy, University of Maryland School of Medicine, Baltimore, Maryland 21201

Received for publication, July 20, 2004 , and in revised form, January 6, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The calmodulin C lobe binding region (residues 3614–3643) on the sarcoplasmic reticulum Ca2+ release channel (RyR1) is thought to be a region of contact between subunits within RyR1 homotetramer Ca2+ release channels. To determine whether the 3614–3643 region is a regulatory site/interaction domain within RyR in muscle fibers, we have investigated the effect of a synthetic peptide corresponding to this region (R3614–3643) on Ca2+ sparks in frog skeletal muscle fibers. R3614–3643 (0.2–3.0 µM) promoted the occurrence of Ca2+ sparks in a highly cooperative dose-dependent manner, with a half-maximal activation at 0.47 µM and a maximal increase in frequency of ~5-fold. A peptide with a single amino acid substitution within R3614–3643 (L3624D) retained the ability to bind Ca2+-free calmodulin but did not increase Ca2+ spark frequency, suggesting that R3614–3643 does not modulate Ca2+ sparks by removal of endogenous calmodulin. Our data support a model in which the calmodulin binding domain of RyR1 modulates channel activity by at least two mechanisms: direct binding of calmodulin as well as interactions with other regions of RyR.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The sarcoplasmic reticulum (SR)1 Ca2+ release channel (ryanodine receptor, RyR) is a homotetramer with a subunit molecular mass of ~560 kDa. In mammalian tissue three isoforms have been identified, skeletal muscle (RyR1), cardiac muscle (RyR2), and brain (RyR3) ryanodine receptors. All three isoforms are structurally and functionally related. The majority of the protein (~4/5) resides on the myoplasmic side of the SR where binding sites for endogenous modulators (e.g. Ca2+, Mg2+, FK506-binding protein, and calmodulin) are located (for review see Ref. 1). Recently, much attention has been focused on elucidating the role of calmodulin (CaM) in regulation of RyR function.

CaM is a ubiquitous Ca2+-binding protein that plays an important role in Ca2+ signaling in many cell types by modulating the activity of numerous proteins, including ion channels. Each RyR1 subunit binds one molecule of CaM or four CaM molecules per RyR1 homotetramer, regardless of the cytosolic [Ca2+] (2). In SR vesicle preparations CaM displays Ca2+ dependence in its functional effects on RyR1; at nanomolar [Ca2+] Ca2+-free CaM activates RyR1, whereas at millimolar [Ca2+] Ca2+-CaM inhibits RyR1 (3, 4). This Ca2+-dependent bi-functional regulation of RyR1 function was found to require Ca2+ binding to CaM (3), in particular at the two C-terminal Ca2+ binding sites (3, 5).

Further characterization of the interaction of CaM with RyR1 has led to the localization of a single binding site for Ca2+-free CaM or Ca2+-CaM binding to a region between amino acids 3614 and 3643 (2, 6), which is within the large cytoplasmic domain of the channel. Alkylation and tryptic cleavage studies identified cysteine 3635, which lies within the 3614–3643 region, as being important for inter-subunit cross-linking (7), suggesting that the CaM binding region also lies at an inter-subunit contact site within RyR1. More recently, Zhang et al. (8) have shown that Cys3635 forms an inter-subunit disulfide bond with a cysteine residue between 2000 and 2401 while the N-terminal lobe of CaM interacts with a region around amino acids 1975 and 1999 of RyR1, suggesting that CaM not only binds at a site of inter-subunit contact but may also span the two subunits.

These previous studies have been conducted on isolated SR vesicles and/or purified RyR1 channels, which removes the channel from the complex environment of the triad. In attempts to gain further understanding of the role of CaM in regulating SR Ca2+ release in a more fully constituted setting we have recently reported on the effect of CaM on spontaneous Ca2+ sparks in permeabilized frog skeletal muscle (9). Ca2+ sparks are local, discrete elevations in myoplasmic [Ca2+] due to the opening of RyR (10, 11). The measurement of Ca2+ sparks provides a convenient tool to assess the function and regulation of RyR in a more physiological setting within a living muscle cell. We found that exogenously applied CaM localized to the triad and caused a highly cooperative dose-dependent increase in Ca2+ spark frequency. Two possible mechanisms for these effects are that CaM promotes activation of RyR1 either by disrupting an inter-subunit interaction that stabilizes the closed state and/or by coordinating the movement of all four subunits within an RyR1 tetramer to the open state.

If the CaM binding region of RyR1 (amino acids 3614–3643) is indeed an inter-subunit interaction site then addition of an exogenous peptide corresponding to this sequence might disrupt a native interaction between RyR subunits and also possibly interfere with the interaction of CaM at this contact site, either of which might result in an alteration of RyR activity. Therefore, we tested the effects of a synthetic peptide corresponding to 3614–3643 of RyR1 (R3614–3643) on spontaneous Ca2+ sparks. We found that R3614–3643 increases Ca2+ spark occurrence in permeabilized frog skeletal muscle fibers in a highly cooperative dose-dependent manner. The maximum increase in Ca2+ spark frequency produced by R3614–3643 was about half that produced by exogenous recombinant CaM. A single amino acid mutation within R3614–3643 (L3624D) abolished the activating effect of the peptide. Both R3614–3643 and L3624D bind CaM with similar affinity. Thus, the spark-activating effect of R3614–3643 cannot be attributed to "stripping" endogenous CaM from the fiber. Interestingly, the maximum increase in Ca2+ spark frequency produced by exogenous recombinant CaM was the same in the presence or absence of R3614–3643, even though R3614–3643 binds to CaM. Our results support a model in which the CaM binding region of RyR1 is a site of inter-domain or inter-subunit contact within RyR that stabilizes the closed state of the channel. Addition of either exogenous R3614–3643 or exogenous CaM disrupts the native interaction, thereby destabilizing the closed state of the channel, with CaM having a stronger destabilizing effect than R3614–3643. Some of these data have been presented in abstract form (12, 13).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Synthesis and Purification of Peptides and Recombinant Calmodulin—Peptides were synthesized in the core facility at the University of Maryland School of Medicine (Baltimore, MD). CaM was expressed and purified as previously described (9).

Preparation of Skeletal Muscle Fibers—Frogs (Rana pipiens) were first placed in a cold-induced torpor (crushed ice-water slurry, 20 min) followed by rapid decapitation and spinal cord destruction according to protocols approved by the University of Maryland Institutional Animal Care and Use Committee. The ileofibularis muscle was removed and pinned in a dissecting chamber containing Ringer's solution (in mM): 115 NaCl, 2.5 KCl, 1.8 CaCl2, 1.0 MgCl2, 10 HEPES, pH 7.0. Small fiber segments (3–5 mm) were manually dissected in a relaxing solution containing (in mM): 120 potassium glutamate, 2 MgCl2, 1 EGTA, 5 Trizma (Tris base) maleate, pH 7.0. Cut fibers were mounted under stretching in a custom built experimental chamber (14). Solution equilibration into the myoplasm was realized by chemical permeabilization of fibers in a relaxing solution containing 0.01% saponin for 30–40 s. Immediately following the permeabilization procedure, the fiber was bathed in internal solution containing (in mM) 80 potassium glutamate, 5.5 MgCl2, 5 Na2ATP, 20 Tris maleate, 0.1 EGTA, 20 Na2-creatine phosphate, 5 glucose, 3 dithiothreitol, 0.05 Fluo-3 (pentapotassium salt) (Molecular Probes, Eugene, OR), pH 7.0. To avoid osmotic swelling observed with chemical permeabilization of fibers the internal solution was supplemented with 8% (41,000 Da) dextran (15, 16). Free [Ca2+] was estimated to be 0.1 µM using MaxChelator (17). Synthetic peptides and/or CaM were added to the internal solution from stock solutions. A complete change of the bathing solution occurred upon the addition of peptides and/or CaM, and fibers were allowed to equilibrate for 10 min prior to data collection. To control for buffer changes time-matched "sham" fibers were also examined.

Ca2+ Spark Measurements—Experimental protocols and data analysis were as previously described by our group (9). Briefly, fibers were imaged for spontaneous Ca2+ release events on an Olympus IX-70 inverted microscope (60x, 1.4 numerical aperture oil immersion objective) coupled to a Bio-Rad MRC 600 laser scanning confocal system (488 nm excitation). The confocal system was operated in linescan x-t mode (1024-ms acquisition time, 2 ms per line, 768 pixels per line, and 0.18 µm per pixel). Line scan images were processed to identify and store potential spark locations by an automated computer detection routine using a relative threshold algorithm as described by Cheng et al. (18) and analyzed as previously described (9). For each selected event, the peak amplitude ({Delta}F/F), 100% rise time, temporal half-duration (full duration at half-max, FDHM), and spatial half-width (full width at half-max, FWHM) were determined from the temporal and spatial fits. Events with a {Delta}F/F < 0.4 were excluded from data analysis post-hoc. The frequency of events per sarcomere was calculated from the number of sparks per image divided by the number of sarcomeres along the line and by the image duration (1.024 s).

To account for the variability in the starting Ca2+ spark frequency among fibers, each data point was normalized to the average Ca2+ spark frequency for the same group of fibers prior to the addition of exogenous protein. For the dose-dependent effect of R3614–3643 the data were fit to Equation 1,

(Eq. 1)
where f is the event frequency normalized to the average event frequency in the same group of fibers prior to R3614–3643 application, R is the fractional maximal increase in spark frequency (fmax/fmin), n is the Hill coefficient, and K is the concentration of R3614–3643 that elicits 50% of the increase in frequency (EC50).

Fluorescence Spectroscopy—The change in intrinsic tryptophan fluorescence of R3614–3643 or L3624D upon binding CaM was monitored using a Cary Eclipse fluorescence spectrophotometer (Varian Instruments). CaM and either R3614–3643 or L3624D were added to final concentrations of 2 and 1 µM, respectively. Tryptophan excitation was set at 295 nm, and emission spectra were recorded from 310 to 400 nm. Final fluorescence data were obtained by subtracting CaM and buffer effects from those of the CaM plus peptide and then normalized to the fluorescence for R3614–3643 or L3624D alone. Because CaM contains no tryptophan residues, the observed fluorescence is attributed to the single tryptophan residue in either R3614–3643 or L3624D.

To assess the relative affinity of R3614–3643 and L3624D for CaM the fluorescence emission spectra of Alexa Fluor® 488-labeled CaM (Alexa488-CaM, Molecular Probes, Eugene, OR) were collected by exciting at 485 nm and recording between 500 and 600 nm. Increasing amounts of either R3614–3643 or L3624D (0.25–200 nM) were titrated against 1 nM Alexa488-CaM in a buffer containing 80 mM potassium glutamate, 20 mM Tris maleate, 0.1 mM EGTA, 1 mM dithiothreitol, pH 7.0. Final fluorescence data were obtained by subtracting peptide and buffer effects from those of the Alexa488-CaM plus peptide and correcting for minor dilution during titration.

A variant of the Hill model (Equation 2) was used to resolve the change in Alexa Fluor® 488-conjugated CaM fluorescence as a function of peptide concentration ([P]), returning the Hill coefficient (n) and peptide concentration yielding half-maximal binding ([P]1/2). Nonlinear regression was performed using PRISM version 3.03 software (GraphPad).

(Eq. 2)
To estimate the fractional concentrations of each CaM species ([CaM]free, [CaM·P], and [CaM·P2]) for given concentrations of total calmodulin ([CaM]total) and peptide ([P]total), it was necessary to first estimate the macroscopic association constants (K1 and K2) describing each stage of CaM·P2 assembly. For a positively cooperative model, this is impossible without knowing the intrinsic fluorescence of the intermediate (CaM·P) species. However, inserting an approximation of this value into Equation 3 allowed estimation of K1 and K2 from regression of F518 versus [P] plots, assuming [CaM]total << 1/K1 or 1/K2.

(Eq. 3)
Using values of K1 and K2 estimated using Equation 3, equilibrium concentrations of all reaction species were resolved for any combination of [P]total and [CaM]total by the system of equations in Equations 4, 5, 6, 7.

(Eq. 4)

(Eq. 5)

(Eq. 6)

(Eq. 7)
All real solutions of ([P]free and [CaM]free) (>0 and >0) for this system of equations were determined for given values of [P]total, [CaM]total, K1, and K2 using Mathematica version 4.1 (Wolfram Research). Calculated values of [P]free and [CaM]free were then used to derive [CaM·P] and [CaM·P2] using Equations 6 and 7, which were then expressed as fractional concentrations of [CaM]total.

Data Analysis—Unless otherwise stated, results are reported as means ± S.E. Statistical analysis for comparison of means was performed using analysis of variance with a significance level of p < 0.05. The spatial and temporal properties of Ca2+ sparks (amplitude, rise time, FDHM, and FWHM) were not normally distributed; therefore a non-parametric analysis of variance was performed (Dunn's post hoc analysis). All statistical analysis was performed with SigmaStat (Jandel Scientific), and non-linear curve fitting was performed in SigmaPlot (Jandel Scientific), unless otherwise noted.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
R3614–3643 Increases the Frequency of Occurrence of Spontaneous Ca2+ Sparks—A sequence alignment of the CaM binding sequence (3614–3643) from rabbit RyR1 with that of RyR{alpha} and RyR{beta} from frog shows that this region is highly conserved (Fig. 1A). Therefore, we assessed the effects of a synthetic peptide representing amino acids 3614–3643 of the rabbit RyR1 sequence on RyR function in frog skeletal muscle. The frequency of occurrence of spontaneous Ca2+ sparks provides a measurement of the activation state of the RyR channels that give rise to the Ca2+ spark (19, 20). Application of R3614–3643 resulted in an increase in the frequency of spontaneous Ca2+ sparks, indicating an increase in the rate of activation of RyR. Representative {Delta}F/F linescan images from control conditions and after application of R3614–3643 (3.0 µM) are shown in Fig. 1B. The concentration dependence of the activation of Ca2+ sparks is shown in Fig. 1C. Addition of R3614–3643 (0.2–3.0 µM) resulted in a highly cooperative, dose-dependent increase in Ca2+ spark frequency. Fitting the data to Equation 1 resulted in a fractional maximal increase (R) of 5.3 ± 1.4, with a half-maximal activation (EC50) of 0.47 ± 0.02 µM and a Hill coefficient (n) of 8.2.



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  		FIG. 1.
R3614–3643 increases spontaneous Ca2+ spark frequency. A, sequence alignment of the CaM binding region (3614–3643) for rabbit RyR1, frog RyR{alpha}, and frog RyR{beta}. The underlined residues denote differences between isoforms in the primary structure. B, linescan images for Sham and 3 µM R3614–3643. Images display the fiber fluorescence as {Delta}F/F from fibers loaded with the Ca2+ indicator dye Fluo-3 (50 µM). Below each {Delta}F/F image is the temporal time course for the line indicated by the arrow. Addition of 3 µM R3614–3643 increased the number of spontaneous Ca2+ sparks when compared with Sham controls. C, dose-dependent effect of R3614–3643 on spontaneous Ca2+ spark frequency. The frequency of spontaneous Ca2+ sparks before and after the addition of various concentrations of R3614–3643 (0.2–3.0 µM) at a [Mg2+]free of 0.65 mM. To control for variability in the resting Ca2+ spark frequency, the frequency of each fiber in the presence of the indicated R3614–3643 was normalized to the average frequency for that group of fibers prior to addition of R3614–3643. The data for no added R3614–3643 are buffer and time controls (sham) as described under "Experimental Procedures." The solid line was obtained by fitting the data to Equation 1. The fractional maximal increase in spark frequency was 5.3 ± 1.4, the EC50 was 0.47 ± 0.02 µM, and the Hill coefficient was 8.2. All data points are presented as mean ± S.E. for at least three fibers at each R3614–3643 concentration.

 
The spatial and temporal properties of Ca2+ sparks reflect the underlying channel kinetics and resulting Ca2+ flux, which underlie the Ca2+ spark. To assess whether R3614–3643 alters the kinetics of the Ca2+ release event we measured the spatial and temporal properties of the Ca2+ sparks initiated by R3614–3643. The effect of R3614–3643 (3 µM) on the spatial and temporal properties of Ca2+ sparks is shown in Fig. 2. Ensemble averages and surface plots of identified Ca2+ spark events for Sham and 3 µM R3614–3643 are shown in Fig. 2A. Despite an approximate 5-fold increase in Ca2+ spark frequency only small differences in the spatial and temporal properties were observed (Fig. 2B). Median values of the population distribution for 3 µM R3614–3643 showed a small but statistically significant increase for FWHM and FDHM, no differences were observed for amplitude or rise time.



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  		FIG. 2.
Effect of R3614–3643 on the spatial and temporal properties of spontaneous Ca2+ sparks. A, average spark image and the spatial and temporal profiles through the peak {Delta}F/F for Sham (left, nevents = 107) and 3 µM R3614–3643 (right, nevents = 836). The surface plot for the average event is displayed below. B, distribution of the spatial (amplitude and FWHM) and temporal (rise time and FDHM) properties of Ca2+ sparks for Sham (open bars) and R3614–3643 (hatched bars). The box plots represent the 25th, 50th (median), 75th percentiles, whereas the error bar represents the 10th and 90th percentiles. p < 0.05 versus Sham (a).

 
A Single Amino Acid Substitution Abolishes the Activating Effect of R3614–3643—Given that the 3614–3643 region of RyR lies at an inter-subunit contact site (7, 8) and is also a binding site for CaM (21), it is conceivable that the increase in the rate of activation of RyR observed with R3614–3643 could be due to the peptide binding to either the inter-subunit contact site on RyR and/or binding to native CaM. In an effort to distinguish these possibilities we made the synthetic peptide R3614–3643 with a single amino acid mutation, L3624D. This mutation, when made in full-length RyR1, resulted in the loss of both CaM binding and functional regulation of RyR1 by CaM in SR vesicle preparations (6). Application of L3624D (2 µM and 10 µM) did not result in an increase in the frequency of occurrence of spontaneous Ca2+ sparks (Fig. 3). When added in combination with R3614–3643, excess L3624D (10 µM) attenuated the activation of Ca2+ sparks observed with R3614–3643 (2 µM, Fig. 3), suggesting competition for a similar binding site.



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  		FIG. 3.
The L3624D mutation within R3614–3643 does not increase Ca2+ spark frequency. Frequency was determined as the mean number of events identified in each fiber and normalized to the mean frequency of that group of fibers prior to the addition of either R3614–3643 (2 µM), L3624D (2 µM), L3624D (10 µM), or the combination of R3614–3643 (2 µM) plus L3624D (10 µM). Data for R3614–3643 are the same as presented in Fig. 1C. Bars represent mean ± S.E. p < 0.05 versus Sham (a), R3614–3643 (b), or L3624D (c).

 
R3614–3643 and L3624D Bind CaM with Similar Affinity—It is conceivable that the observed increase in Ca2+ spark frequency produced by R3614–3643 might be due to either: 1) a direct effect of R3614–3643 on RyR, 2) an indirect effect produced by R3614–3643 binding endogenous CaM and thereby removing CaM from the fiber, or 3) a combination of the two effects. In efforts to distinguish these mechanisms we assessed the binding of CaM to R3614–3643 and L3624D. Intrinsic tryptophan fluorescence of R3614–3643 and L3624D was monitored under saturation binding conditions (Fig. 4). CaM (2 µM) binding to R3614–3643 (1 µM) resulted in an increase and a 10 nm blue shift in the peak intrinsic tryptophan fluorescence of R3614–3643 (Fig. 4A). Upon binding CaM (2 µM), the peak intrinsic tryptophan fluorescence of L3624D (1 µM) also increased in a similar fashion to that for R3614–3643; however, there was little to no shift in the wavelength at which the peak tryptophan fluorescence occurred (Fig. 4B). These data indicate that CaM is able to bind both R3614–3643 and L3624D, but the lack of blue shift observed in the CaM-L3624D interaction suggests that there is some difference in the mechanism or structural consequence of the interaction of CaM with these two peptides.



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  		FIG. 4.
Changes in the intrinsic tryptophan fluorescence of R3614–3643 and L3624D upon CaM binding. Intrinsic tryptophan fluorescence was monitored in the absence (open circle) and presence (closed circle) of CaM (2 µM) for R3614–3643 (A, 1 µM) or L3624D (B, 1 µM) in internal solution used for Ca2+ sparks studies. Data were normalized to the peak fluorescence in the absence of CaM and are plotted as mean ± S.E.

 
We next assessed the relative affinity of both R3614–3643 and L3624D to bind CaM. Both R3614–3643 and L3624D (0.25–200 nM) quenched the fluorescence of Alexa488-CaM in a dose-dependent manner (Fig. 5, A and B, respectively). A plot of the fluorescence intensity at 518 nm as a function of the concentration of peptide added (Fig. 5C) shows that each peptide virtually fully quenched the Alex488-CaM fluorescence. The concentration of R3614–3643 for a half-maximal quenching of Alexa488-CaM fluorescence is 22 ± 2 nM, with a Hill coefficient of 1.6 ± 0.2. The concentration of L3624D needed for half-maximal quenching of Alexa488-CaM fluorescence is 33 ± 2 nM, with a Hill coefficient of 1.5 ± 0.1. The apparent affinity and Hill coefficients of R3614–3643 and L3624D for CaM are on the same order of magnitude as previously reported for R3614–3643 (21). Also, the apparent affinity of CaM for either R3614–3643 or L3624D is similar to the affinity of CaM for RyR1 (21). To ensure that the quenching of the Alexa488-CaM fluorescence was not due to nonspecific effects of the peptides we monitored the fluorescence of Alexa488-CaM in the presence of insulin {beta} chain (3.5 kDa), which has a molecular mass comparable to R3614–3643 and L3624D (3.7 kDa). Insulin {beta} chain (150 nM) resulted in less than a 5% quenching of the Alexa488-CaM fluorescence (data not shown). Our finding that the L3624D mutant does not increase the occurrence of spontaneous Ca2+ sparks cannot be attributed to a loss in CaM binding to the mutated peptide, suggesting that this single amino acid mutation does not abolish the spark-activating effect by disrupting the ability R3614–3643 to bind CaM. Instead the mutation likely disrupts the association of the R3614–3643 peptide with its complimentary contact site within RyR. Therefore, removal of endogenous CaM from RyR by R3614–3643 is unlikely to contribute to the increase in Ca2+ spark frequency.



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  		FIG. 5.
Changes in the Alexa488-CaM fluorescence intensity as a function of concentration of the peptides R3614–3643 or L3624D. The fluorescence emission spectra of Alexa488-CaM (1 nM) were recorded in the presence of increasing amounts of either R3614–3643 (A) or L3624D (B). The fluorescence intensity at 518 nm of each spectrum was plotted as a function of [peptide] (C) for R3614–3643 (closed circles) and L3624D (open circles). The solid lines were obtained by fitting the data to Equation 2. The peptide concentrations yielding half-maximal binding were 22 ± 2 nM and 33 ± 2 nM for R3614–3643 and L3624D, respectively. The Hill coefficients for R3614–3643 and L3624D were 1.6 ± 0.2 and 1.5 ± 0.1, respectively. All data points are presented as mean ± S.E. for three independent titrations.

 
The Effects of R3614–3643 and Recombinant CaM on Ca2+ Sparks Are Not Additive—We have previously shown that recombinant CaM increased the frequency of spontaneous Ca2+ sparks in frog skeletal muscle fibers (9). We next set out to determine whether the increase in Ca2+ spark frequency observed with either R3614–3643 alone (Fig. 1) or CaM alone (see Fig. 7 of Ref. 9) are additive. If R3614–3643 and CaM were activating RyR by different independent mechanisms, we might expect to observe some degree of additive effect upon the application of both ligands. However, in light of the fact that these two ligands bind each other in a cooperative manner (see Fig. 5C) it is also conceivable that the combination of R3614–3643 and CaM might have no effect on RyR channel activation. Addition of R3614–3643, at either 2 or 4 µM, in combination with recombinant wild type CaM (2 µM) resulted in an increase in Ca2+ spark frequency that was not different from CaM (2 µM) alone (Fig. 6A). One possible explanation of this finding might be that in the presence of bound exogenous CaM, R3614–3643 cannot bind to RyR. To test this possibility we made use of the dominant negative CaM ((N+3)CaM). Previous studies have demonstrated that (N+3)CaM binds to isolated RyR1 with ~5-fold higher affinity than wild type CaM, binds R3614–3643 similar to wild type CaM, prevents the localization of fluorescently labeled wild type CaM (Alexa488-CaM) in permeabilized muscle fibers, and prevents the increase in spark frequency due to exogenous wild type CaM in permeabilized muscle fibers, but it does not itself increase the activation rate of RyR (9, 22). Fig. 6B shows that in the presence of (N+3)CaM (2 µM), R3614–3643 (2 µM) still increased Ca2+ spark frequency to a level that is not different from R3614–3643 alone.



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  		FIG. 7.
Proposed mechanism for how R3614–3643 increases channel activity. The CaM binding region of RyR interacts with another subunit within RyR, stabilizing a closed state of the channel. Addition of R3614–3643 disrupts this native subunit-subunit interaction, promoting opening of the channel by destabilizing the closed state.

 



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  		FIG. 6.
R3614–3643 does not alter the CaM effect on Ca2+ spark frequency. Frequency was determined as the mean number of events identified in each fiber and normalized to the mean frequency of that group of fibers prior to the addition of either CaM plus R3614–3643 (A) or (N+3)CaM plus R3614–3643 (B). Ligand combinations were mixed prior to application to the muscle fiber. Data for R3614–3643 are the same as that presented in Fig. 1C. Bars represent mean ± S.E. p < 0.05 versus Sham (a), R3614–3643 (b), and (N+3)CaM (c).

 
To determine which ligand species might be leading to the activation of Ca2+ sparks during simultaneous application of CaM and R3614–3643, we used the binding data obtained in Fig. 5C and Equations 3, 4, 5, 6, 7 to simulate the fractional concentrations of each CaM species ([CaM]free, [CaM·P], and [CaM·P2]) for the given concentrations of total CaM ([CaM]total) and R3614–3643 ([P]total) used for the Ca2+ spark experiments described in Fig. 6. Because we do not know the intrinsic fluorescence of the intermediate (CaM·P) species, we performed simulations assuming 20, 50, and 80% quenching efficiency for the CaM·P species (Table I). At 2 µM of each ligand, CaM and R3614–3643, the [CaM]free was predicted to be at most 820 nM. This value is below the EC50 value (1.1 µM) we have previously reported for the CaM-dependent increase in Ca2+ spark frequency (9), and thus would not have resulted in the Ca2+ spark frequency observed in Fig. 6A. In fact, assuming that both the CaMfree and CaM·P species are the functional forms cannot predict the frequency response observed for the combination of CaM and R3614–3643 at 2 µM of each ligand. These observations are more evident in the simulations of 2 µM CaM plus 4 µM R3614–3643, which resulted in the same increase in Ca2+ spark frequency as 2 µM CaM plus 2 µM R3614–3643 as well as 2 µM CaM alone (Fig. 6) but negligible [CaM]free and [CaM·P] (Table I). Taken together, these data suggest that both R3614–3643 and CaM can simultaneously bind RyR, but that R3614–3643 binding does not increase or decrease the CaM-induced activation of RyR.


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  		TABLE I
Equilibrium simulations

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Monitoring spontaneous, discrete, local Ca2+ fluxes (i.e. Ca2+ sparks) can provide insight into the regulation of RyR in the complex anatomical and biochemical arrangement of the triad. In chronically depolarized permeabilized frog skeletal muscle Ca2+ sparks arise spontaneously in the absence of voltage sensor activation and are most likely due to ligand activation of SR Ca2+ release (20). Our recent finding that recombinant CaM increased spontaneous Ca2+ sparks (9) in permeabilized frog skeletal muscle suggested that a significant proportion of the CaM-induced events arise from RyR channels not associated with native CaM. Because the CaM binding site is thought to be at an inter-subunit contact site, then CaM may be sensitizing the channel to activation by disrupting this inter-subunit interaction. In the present experiments we addressed the possibility that the CaM binding domain is involved in an inter-subunit interaction by testing the effects of a synthetic peptide representing the CaM C-lobe binding region of RyR1 (R3614–3643). We found that R3614–3643 increased the occurrence of spontaneous Ca2+ sparks in a highly cooperative dose-dependent manner.

The CaM Binding Domain of RyR1 Promotes Activation of RyR—How might R3614–3643 regulate the functional state of RyR? If amino acids 3614–3643 of RyR1 are within an inter-subunit contact site as proposed by Zhang et al. (8), then addition of a synthetic peptide corresponding to this sequence could disrupt the native interaction. A model (Fig. 7) in which this synthetic peptide disrupts a native inter-domain or inter-subunit interaction, promoting activation of RyR by destabilizing a closed state of the channel, could explain our findings that R3614–3643 increased Ca2+ spark frequency. This model would be analogous to one proposed for domain peptide 4, which is thought to interact with another domain of RyR1, resulting in disruption of a native domain-domain interaction and destabilization of a closed state of the channel (23) and increased frequency of Ca2+ sparks (24).

A possible alternative mechanism by which R3614–3643 might modulate Ca2+ spark frequency is by altering the activation of a population of RyRs by removal of endogenous CaM while directly binding and activating another population of RyRs. This scenario seems unlikely for several reasons. First, the possible contribution of endogenous CaM to the resting spontaneous Ca2+ spark frequency appears to be small (<5%) compared with the maximal effect of added recombinant CaM (~1600%, see Fig. 7 of Ref. 9). The removal of endogenous CaM by the R3614–3643 peptide would, at best, underestimate the level of activation of Ca2+ sparks produced by R3614–3643 by 5%, a very small portion of the total activation observed (~450%, see Fig. 1). Second, if R3614–3643 altered the activation of some RyR channels by removal of CaM, then we might predict that L3624D would result in a similar change in Ca2+ spark frequency, because the L3624D mutant peptide was found to bind CaM with an affinity similar to R3614–3643. However, L3624D did not alter Ca2+ spark frequency. In addition, Zhu et al. (25) have recently shown that R3614–3643 increased [3H]ryanodine binding to purified RyR in SR vesicles, which do not contain CaM. Thus, it appears that removal of endogenous CaM from RyR does not contribute significantly to the modulation of Ca2+ sparks by R3614–3643.

We found that, upon application of CaM (2 µM) and R3614–3643 at molar ratios of 1:1 and 1:2 of CaM:R3614–3643, respectively, the increase in Ca2+ spark frequency was not different than that observed for addition of CaM alone (Fig. 6A). The amounts of [CaM]free under our conditions are modeled to be submicromolar (Table I), suggesting that CaM is able to bind to and activate RyR whether or not 1 or 2 mol of the R3614–3643 CaM-binding peptide is bound per mole of CaM. In addition, given that a dominant negative form of CaM, (N+3)CaM, binds R3614–3643 similar to wild type CaM (22) but does not prevent the R3614–3643-induced increase in Ca2+ spark frequency (Fig. 6B), it is likely that R3614–3643 is able to bind to and modulate RyR when bound to CaM. Taken together, these data support a model in which CaM and R3614–3643 simultaneously interact with RyR, but at different sites, and that with respect to the functional state of RyR this interaction is not additive. Future structural studies probing the conformational state of RyR in the presence of single and multiple ligands will provide valuable information for our understanding of the mechanisms of RyR channel regulation and thus regulation of SR Ca2+ release.

Valdivia and colleagues (25) have suggested that the activation of RyR1 by the CaM-binding peptide in their isolated SR vesicle experiments may be due to formation of a disulfide bond between the cysteine residue located at position 22 of the synthetic peptide, corresponding to Cys3635 of RyR1, and a highly reactive cysteine residue in another region within RyR1. This highly reactive cysteine within RyR could be either Cys3635 or one of the other cysteine residues thought to be involved in an inter-subunit disulfide bond formation within RyR1, located somewhere between amino acids 2000 and 2401 (8). In our experiments the internal solution used for dilution of stock peptide as well as to record Ca2+ sparks contains 3 mM dithiothreitol (see "Experimental Procedures"). This concentration is likely sufficient to maintain reducing conditions within the muscle fiber. Increasing the concentration of dithiothreitol up to 8 mM did not alter the ability of R3614–3643 to increase Ca2+ spark frequency (data not shown). Furthermore, the mutant peptide L3624D also contains the cysteine at position 22, and if disulfide bond formation between the synthetic peptide and a region of RyR was required for the activating effect of R3614–3643, then L3624D should have also increased Ca2+ spark frequency. However, L3624D had no effect on Ca2+ spark frequency. In addition, Meissner and colleagues (26) have suggested that Cys3635 of RyR1 does not significantly contribute to redox modulation of RyR1 by O2 tension or glutathione. Taken together, these data indicate that it is unlikely that disulfide bond formation is involved in the activation of RyR by R3614–3643.

Our finding that a single amino acid mutation (L3624D) within the synthetic peptide destroys the ability of R3614–3643 to increase Ca2+ spark frequency may provide some insight into the conformation of the inter-subunit interaction within RyR. The loss of a functional effect upon mutating a single amino acid within a putative domain-domain interaction site is analogous to studies conducted by Yamamoto et al. (23) who showed that a single amino acid mutation within domain peptide 4 prevented the peptide from disrupting the domain-domain interaction within RyR1 and thereby abolishing the activating effect of domain peptide 4. Interestingly, the mutation made in domain peptide 4 corresponds to a mutation occurring in RyR1 that is associated with malignant hyperthermia (23), providing some indications as to how single amino acids mutations within RyR1 can lead to altered SR Ca2+ release. Our findings that the L3624D peptide had no effect on the functional state of the channel would support a model in which the L3624D mutation in the full-length RyR would destabilize this region of RyR, leading to an altered inter-subunit interaction. Although there are no known mutations that have been identified within the 3614–3643 CaM binding region that lead to an altered RyR channel, our finding that a single residue change within this region (L3624D) removes the effectiveness of R3614–3643 to activate Ca2+ sparks provides information regarding the structural requirements within this region of RyR necessary for either subunit-subunit interactions or interactions with protein modulators. In full-length RyR the binding of CaM might require proper orientation between two subunits. Disruption of this proper arrangement in the tertiary structure of RyR might explain the loss of both Ca2+-free CaM and Ca2+-CaM binding as well as CaM-dependent regulation of RyR1 previously reported for RyR1 containing the L3624D mutation (6).

A region between 1393 and 1527 of the carboxyl-tail of the {alpha}1s subunit of the DHPR may also interact with the 3614–3643 region of RyR, independent of CaM (27). Hamilton and colleagues (28) have also previously shown that a synthetic peptide within this region (amino acids 1487–1506) inhibited [3H]ryanodine binding to SR vesicles and decreased single channel activity of RyR1 channels incorporated into planar lipid bilayers. Therefore, it is possible that an interaction between the 3614–3643 region of RyR and the 1487–1506 region of the DHPR stabilizes a closed state of RyR. Addition of R3614–3643 may disrupt this interaction, destabilizing a closed state of RyR and thereby increasing the frequency of Ca2+ sparks. At present, we cannot rule out this possibility, but future studies using these regions of the carboxyl-tail of the {alpha}1s subunit of the DHPR should help provide further insight into these possible mechanisms.

CaM Binding Domain of RyR1 Does Not Alter the Closing Rate of the Channel during a Spark—The spatial and temporal properties of Ca2+ sparks is determined by the behavior of the underlying RyR channels giving rise to those sparks and are a measure of the amount and extent of SR Ca2+ release (20). Several studies have shown that ligands, which alter the gating properties of RyR channels, incorporated into planar lipid bilayers also show alterations in the spatial and/or temporal properties of Ca2+ sparks (29–31). In the studies described here R3614–3643 resulted in only small increases in FDHM and FWHM, with no significant alterations of amplitude or rise time of Ca2+ sparks. These data indicate that the principal effect of R3614–3643 is restricted to an increase in the activation rate of RyR and does not alter either the closing rate of the channel or the amount of Ca2+ released from an open channel. This finding is supported by recent work by Zhu et al. (25) who demonstrated that, in isolated single RyR1 channels incorporated into planar lipid bilayers, the CaM binding domain peptide increased single channel activity without an alteration of the mean open time or single channel conductance.

In summary, we have shown that a synthetic peptide corresponding to the putative CaM C-lobe binding domain of RyR1 (R3614–3643) increases the frequency of spontaneous Ca2+ sparks in frog skeletal muscle, indicating an increase in the rate of activation of RyR, with no alteration of the closing rate or the amount of Ca2+ released during a spark. These results are consistent with a model (Fig. 7) in which the 3614–3643 region of RyR is involved in an inter-subunit interaction that stabilizes a closed state of the channel. The binding of peptide R3614–3643 to its corresponding contact site in RyR would disrupt this native interaction, destabilize the closed state of the channel, and lead to an increase in the rate of activation of RyR. A single amino acid mutation within this peptide (L3624D) prevents the R3614–3643 peptide from disrupting the inter-subunit interaction but does not appreciably alter the peptide binding to CaM. The results from this study provide further detail into intrinsic RyR interactions as well as RyR-ligand interactions and thus provide further insight into the basic mechanisms of SR Ca2+ release.


   FOOTNOTES
 
* This work was supported by an Individual National Research Service Award and by National Institutes of Health (NIH) Grants F32-NS44636 (to G. G. R.) and R01-NS23346 and T32-AR07592 (to M. F. S.). Additional support for the Center of Fluorescence Spectroscopy was provided by NIH Grant P41-RR08119. 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: Dept. of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 N. Greene St., Baltimore, MD 21201. Tel.: 410-706-7812; Fax: 410-706-8297; E-mail: mschneid{at}umaryland.edu.

1 The abbreviations used are: SR, sarcoplasmic reticulum; RyR, sarcoplasmic reticulum calcium release channel, ryanodine receptor; CaM, calmodulin; FDHM, temporal half-duration; FWHM, spatial half-width; Alexa488-CaM, Alexa Fluor® 488-labeled CaM; (N+3)CaM, dominant negative CaM; R3614–3643, synthetic peptide corresponding to the region 3614–3643. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Hector Valdivia for supplying the R3614–3643 peptide to initiate these studies.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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