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Biological Research

Print version ISSN 0716-9760

Biol. Res. vol.37 no.4 Santiago  2004 


Biol Res 37: 613-616, 2004


Peptide and protein modulation of local Ca2+ release events in permeabilized skeletal muscle fibers


Dept. of Biochemistry and Molecular Biology and Interdisciplinary Program in Muscle Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA

Dirección para Correspondencia


Local discrete elevations in myoplasmic Ca2+ (Ca2+ sparks) arise from the opening of a small group of RyRs. Summation of a large number of Ca2+ sparks gives rise to the whole cell Ca2+ transient necessary for muscle contraction. Unlike sarcoplasmic reticulum vesicle preparations and isolated single channels in artificial membranes, the study of Ca2+ sparks provides a means to understand the regulation of a small group of RyRs in the environment of a functionally intact triad and in the presence of endogenous regulatory proteins. To gain insight into the mechanisms that regulate the gating of RyRs we have utilized laser scanning confocal microscopy to measure Ca2+ sparks in permeabilized frog skeletal muscle fibers. This review summarizes our recent studies using both exogenous (ImperatoxinA and domain peptides) and endogenous (calmodulin) modulators of RyR to gain insight into the number of RyR Ca2+ release channels underlying a Ca2+ spark, how domain-domain interactions within RyR regulate the functional state of the channel as well as gating mechanisms of RyR in living muscle fibers.

Keywords: Excitation-Contraction Coupling, Ca2+ Sparks, IperatoxinA, DP4, Calmodulin.

In cardiac, skeletal and smooth muscle, the activity of small, functionally-coupled groups of ryanodine receptor (RyR) Ca2+ release channels gives rise to localized, discrete elevations in myoplasmic [Ca2+], termed Ca2+ sparks (Cheng et al., 1993; Tsugorka et al., 1995; Nelson et al., 1995; Klein et al., 1996). The simultaneous occurrence of large numbers of Ca2+ sparks appears to underlie the "macroscopic" Ca2+ transient that occurs during depolarization of frog skeletal muscle fibers (Lacampagne et al., 2000). Ca2+ sparks also occur "spontaneously," presumably initiated by ligand activation of RyR Ca2+ release channels independent of voltage sensor activation via Ca2+-induced calcium release (CICR, Klein et al., 1996; see also Rios and Zhou, this issue). These local Ca2+ release events can be studied in permeabilized muscle fibers, which provide a convenient preparation for investigating the modulation of RyR function by both physiological (Lacampagne et al., 1998) and experimental ligands, including peptides (Shtifman et al., 2000; Shtifman et al., 2001) and proteins (Rodney & Schneider, 2003) which can enter the permeabilized fiber.

Earlier work from this laboratory utilized the permeabilized fiber preparation to investigate very long-lasting (seconds duration) Ca2+ release events initiated by the peptide toxin ImperatoxinA (IpTxa) (Shtifman et al., 2000), which also produces similarly long duration sub-conductance openings of single RyR Ca2+ release channels in lipid bilayers. The long duration, low amplitude local elevations of [Ca2+] induced by IpTxa in muscle fibers provide insight into the number of channels underlying a Ca2+ spark, suggesting that there are 2 to 4 RyR channels simultaneously open during a Ca2+ spark. Our laboratory (Shtifman et al., 2001) has also previously characterized the effect of the RyR domain peptide DP4, corresponding to amino acids 2442-2477 of RyR1 (Yamamoto et al.,2000). Within this sequence lies a mutation (R2458C) that occurs in patients with the disease malignant hyperthermia. DP4, but not DP4 with the R2458C mutation (DP4mut), activates Ca2+ release via RyR Ca2+ release channels in SR vesicles (Yamamoto et al., 2000) . Addition of DP4 to permeabilized frog muscle resulted in an increase in Ca2+ spark frequency with minimal change in average spark properties (Shtifman et al., 2001). The mutant domain peptide DP4mut did not alter Ca2+ spark frequency. These data are consistent with a model in which an interdomain interaction involving this region stabilizes a closed state of the RyR Ca2+ release channel and that the mutation, which gives rise to malignant hyperthermia, decreases this interdomain interaction and thereby destabilizes the closed state (Ikemoto & Yamamoto, 2002).

In more recent studies we have used confocal line scan (xt) imaging of permeabilized frog skeletal muscle fibers exposed to fluo-3 containing internal solution to determine the effects of calmodulin (CaM) and various CaM mutants on the frequency and properties of Ca2+ sparks. CaM is a ubiquitous Ca2+ binding protein and an endogenous modulator of RyR function. Studies using isolated SR vesicles have shown that four CaM molecules bind non-cooperatively per RyR homotetramer at both nM and mM [Ca2+]. At nM [Ca2+] CaM is Ca2+-free and promotes activation of RyR. In contrast, an increase in [Ca2+] results in Ca2+ binding to CaM and a shift of CaM within the CaM binding site on RyR, which promotes RyR Ca2+ channel inactivation (Rodney et al., 2000; Rodney et al., 2001).

In permeabilized frog skeletal muscle fibers wild-type CaM (0.05-5.0 mM) caused a highly cooperative dose-dependent increase in Ca2+ spark frequency (Fig. 1, Rodney & Schneider, 2003). The values of relative spark frequency (f) as a function of [CaM] (x) were fit by the equation, f = fmin(R-1)(xn/(Kn+x n))+fmin, where f is the event frequency in the test solution normalized to the mean frequency in the same fibers prior to application of the test solution, R is the fractional maximal increase in normalized spark frequency (fmax/fmin), n is the Hill coefficient, and K is the concentration of CaM that elicits 50% of the increase in frequency (EC50). The fractional maximal increase (R) was 17.3 ± 5.5, the EC50 was 1.1 ± 0.1 mM and the Hill coefficient was 4.2 ± 1.1. Our finding that CaM potentiates the occurrence of Ca2+ sparks in frog skeletal muscle fibers in a cooperative manner differs from the non-cooperative binding and enhancement of [3H]-ryanodine binding observed in isolated SR vesicle preparations (Tripathy et al., 1995; Moore et al., 1999). This difference may be due in part to a disruption of an RyR macromolecular complex during SR vesicle preparations.

Figure 1. Dose-dependent effect of CaM on spontaneous Ca2+ spark frequency.

The spatial and temporal properties of calcium sparks were essentially unaffected by 1 mM CaM (Table 1). These observations indicate that CaM cooperatively potentiates either the opening rate of the channel(s) responsible for initiating a spark or increases the likelihood that an open channel can initiate a spark in permeabilized muscle fibers. In contrast, the constancy of spark properties indicates that CaM had negligible effect on the aggregate open time of the channels generating the sparks or the amount of Ca2+ released in a spark. Since added CaM localized to the triad in permeabilized fibers and saponin permeabilization did not appear to result in loss of a significant amount of native CaM from frog skeletal muscle fibers (Rodney and Schneider, 2003) our findings that exogenous, recombinant CaM potentiated the occurrence of Ca2+ sparks suggest that the recombinant CaM is modulating a population of RyR channels that is not regulated by endogenous CaM. This population of RyR channels in frog muscle could be either RyRa or RyRb, the homologues to the mammalian isoforms RyR1/RyR3, since amphibian skeletal muscle expresses equal proportions of RyR a and b isoforms. (N+3)CaM (2 mM), which binds RyR with a higher affinity than CaM but does not activate RyR at nM [Ca2+] in SR vesicles (Xiong et al., 2002), prevented the binding of fluorescently labeled CaM (1 mM) to permeabilized frog fibers and had no significant effect on spark frequency, serving as a negative control in these studies.


Effect of CaM on the spatiotemporal properties of spontaneous Ca2+ Sparks






(0.78,1.74)*, #

Rise Time











(1.35,1.85) *, #

Ca2+ spark properties were obtained from line scan images (2ms/line) as described in Materials and Methods. The values reported are the medians with 25th and 75th percentiles in parentheses. P < 0.05 versus Sham*, or CaM#.


To further elucidate the role of Ca2+ and CaM in regulating SR Ca2+ release, we assessed the effects of a mutant CaM that cannot bind Ca2+ (CaM1234) on the frequency, temporal and spatial properties of Ca2+ sparks. In-vitro studies have shown that CaM1234 activates RyR at [Ca2+] concentrations in which wild-type CaM inhibits RyR activity (Rodney et al., 2000), indicating that the Ca2+ binding to CaM converts CaM from an activator to an inhibitor of RyR. Since Ca2+-CaM is thought to be an effective inhibitor of RyR we predicted that if Ca2+ binding to CaM plays a role in the inactivation of SR Ca2+ release, that CaM1234 would prolong the release event. This would be best manifested as a prolongation in the rise time of the Ca2+ spark. Addition of recombinant CaM1234 resulted in an increase in Ca2+ spark frequency similar to wild-type CaM (R=11.8 ± 3.7, EC50=0.95 ± 0.06, n=3.7 ± 0.6) suggesting that potentiation of Ca2+ spark frequency in frog skeletal muscle fibers by CaM does not require Ca2+ binding to CaM. However, even though CaM1234 (1 mM) caused a marked increase in spark frequency, there was no significant alteration in the spatial or temporal properties of Ca2+ sparks (Table 1). Thus, the aggregate open time of RyR channels underlying the generation of a Ca2+ spark was the same for sparks promoted by either wild-type CaM or by the non-Ca2+ binding mutant CaM1234. Consequently, our data suggest that CaM acts to promote activation of RyR, but that termination of Ca2+ sparks activated by CaM may be through a mechanism that is independent of Ca2+ binding to CaM.

The global increase in myoplasmic Ca2+ that is required for myofilament activation and contraction of frog skeletal muscle is the summation of a large number of Ca2+ sparks. Ca2+ sparks provide an effective means to study the gating properties of RyR within a functioning muscle fiber. The frequency of occurrence of these events provides information on the opening rate of the SR Ca2+ release channel while the amplitude and rise time of the release event provides information on the amount of Ca2+ released and the effect open time of the channels underlying Ca2+ sparks. Here we have briefly reviewed our data using peptides and protein modulators in saponin permeabilized muscle fibers to gain further insight into the gating mechanisms of SR Ca2+ release channels in an intact, living skeletal muscle fiber.


Supported by R01-NS23346 to M.F.S. Dr. Rodney received fellowship support from National Institute of Health institutional training grant T32 NS007375 (Training Program in Cellular and Integrative Neuroscience), T32 AR07592 (Interdisciplinary Training Program in Muscle Biology) and an Individual National Research Service Award F32 NS44636.


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Corresponding author: Martin F. Schneider, Dept. of Biochemistry and Molecular Biology and Interdisciplinary Program in Muscle Biology, University of Maryland School of Medicine, 108 North Greene St., Rm 229, Baltimore, MD 21201, USA, Phone: 410 706-5787, Fax: 410 706-8297, E-mail:

Received: March 29, 2004. Accepted: May 3, 2004.


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