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Interdomain interactions within ryanodine receptors regulate Ca2+ spark frequency in skeletal muscle.

Shtifman A, Ward CW, Yamamoto T, Wang J, Olbinski B, Valdivia HH, Ikemoto N, Schneider MF - J. Gen. Physiol. (2002)

Bottom Line: Biol.DP4 caused a significant increase in Ca(2)+ spark frequency in muscle fibers.The same peptide with an Arg(17) to Cys(17) replacement (DP4mut), which corresponds to the Arg(2458)-to-Cys(2458) mutation in MH, did not produce a significant effect on RyR activation in muscle fibers, bilayers, or SR vesicles.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene Street, Baltimore, MD 21201, USA.

ABSTRACT
DP4 is a 36-residue synthetic peptide that corresponds to the Leu(2442)-Pro(2477) region of RyR1 that contains the reported malignant hyperthermia (MH) mutation site. It has been proposed that DP4 disrupts the normal interdomain interactions that stabilize the closed state of the Ca(2)+ release channel (Yamamoto, T., R. El-Hayek, and N. Ikemoto. 2000. J. Biol. Chem. 275:11618-11625). We have investigated the effects of DP4 on local SR Ca(2)+ release events (Ca(2)+ sparks) in saponin-permeabilized frog skeletal muscle fibers using laser scanning confocal microscopy (line-scan mode, 2 ms/line), as well as the effects of DP4 on frog SR vesicles and frog single RyR Ca(2)+ release channels reconstituted in planar lipid bilayers. DP4 caused a significant increase in Ca(2)+ spark frequency in muscle fibers. However, the mean values of the amplitude, rise time, spatial half width, and temporal half duration of the Ca(2)+ sparks, as well as the distribution of these parameters, remained essentially unchanged in the presence of DP4. Thus, DP4 increased the opening rate, but not the open time of the RyR Ca(2)+ release channel(s) generating the sparks. DP4 also increased [(3)H]ryanodine binding to SR vesicles isolated from frog and mammalian skeletal muscle, and increased the open probability of frog RyR Ca(2)+ release channels reconstituted in bilayers, without changing the amplitude of the current through those channels. However, unlike in Ca(2)+ spark experiments, DP4 produced a pronounced increase in the open time of channels in bilayers. The same peptide with an Arg(17) to Cys(17) replacement (DP4mut), which corresponds to the Arg(2458)-to-Cys(2458) mutation in MH, did not produce a significant effect on RyR activation in muscle fibers, bilayers, or SR vesicles. Mg(2)+ dependence experiments conducted with permeabilized muscle fibers indicate that DP4 preferentially binds to partially Mg(2)+-free RyR(s), thus promoting channel opening and production of Ca(2)+ sparks.

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DP4 increases Ca2+ spark frequency in frog skeletal muscle fibers. Ca2+ spark frequency detected in the fibers described in Fig. 1. Bars represent the mean ± SEM of six experiments performed in each condition.
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Figure 2: DP4 increases Ca2+ spark frequency in frog skeletal muscle fibers. Ca2+ spark frequency detected in the fibers described in Fig. 1. Bars represent the mean ± SEM of six experiments performed in each condition.

Mentions: Fig. 1 shows representative line-scan fluorescence (ΔF/F) images of permeabilized frog muscle fibers in control and after addition of either 50 μM DP4 or 50 μM DP4mut to the bathing solution. Each image was obtained from a set of five 1-s duration line-scan images recorded with ∼1 s separation between successive images. The distance along the fiber (x) is represented vertically and the time (t) is represented horizontally to give the x versus t image in each panel. Multiple successive runs of images were recorded in each condition. To avoid laser damage, the scan line was moved 1.8 μm perpendicular to the long axis of the fiber after each run. Each localized increase in [Ca2+] (Ca2+ spark) is characterized by a brief and localized increase in fluorescence (Klein et al. 1996; Schneider and Klein 1996). When added to the permeabilized muscle fibers, DP4 appeared to modulate SR Ca2+ release by producing a large increase in the frequency of Ca2+ sparks (Fig. 1 and Fig. 2). Contrary to the effects of DP4, addition of DP4mut did not produce an appreciable increase in Ca2+ spark frequency (Fig. 1 and Fig. 2), suggesting that Arg2458 of RyR (Arg17 of DP4) plays a critical role in stabilizing interdomain interaction within the channel.


Interdomain interactions within ryanodine receptors regulate Ca2+ spark frequency in skeletal muscle.

Shtifman A, Ward CW, Yamamoto T, Wang J, Olbinski B, Valdivia HH, Ikemoto N, Schneider MF - J. Gen. Physiol. (2002)

DP4 increases Ca2+ spark frequency in frog skeletal muscle fibers. Ca2+ spark frequency detected in the fibers described in Fig. 1. Bars represent the mean ± SEM of six experiments performed in each condition.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233852&req=5

Figure 2: DP4 increases Ca2+ spark frequency in frog skeletal muscle fibers. Ca2+ spark frequency detected in the fibers described in Fig. 1. Bars represent the mean ± SEM of six experiments performed in each condition.
Mentions: Fig. 1 shows representative line-scan fluorescence (ΔF/F) images of permeabilized frog muscle fibers in control and after addition of either 50 μM DP4 or 50 μM DP4mut to the bathing solution. Each image was obtained from a set of five 1-s duration line-scan images recorded with ∼1 s separation between successive images. The distance along the fiber (x) is represented vertically and the time (t) is represented horizontally to give the x versus t image in each panel. Multiple successive runs of images were recorded in each condition. To avoid laser damage, the scan line was moved 1.8 μm perpendicular to the long axis of the fiber after each run. Each localized increase in [Ca2+] (Ca2+ spark) is characterized by a brief and localized increase in fluorescence (Klein et al. 1996; Schneider and Klein 1996). When added to the permeabilized muscle fibers, DP4 appeared to modulate SR Ca2+ release by producing a large increase in the frequency of Ca2+ sparks (Fig. 1 and Fig. 2). Contrary to the effects of DP4, addition of DP4mut did not produce an appreciable increase in Ca2+ spark frequency (Fig. 1 and Fig. 2), suggesting that Arg2458 of RyR (Arg17 of DP4) plays a critical role in stabilizing interdomain interaction within the channel.

Bottom Line: Biol.DP4 caused a significant increase in Ca(2)+ spark frequency in muscle fibers.The same peptide with an Arg(17) to Cys(17) replacement (DP4mut), which corresponds to the Arg(2458)-to-Cys(2458) mutation in MH, did not produce a significant effect on RyR activation in muscle fibers, bilayers, or SR vesicles.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene Street, Baltimore, MD 21201, USA.

ABSTRACT
DP4 is a 36-residue synthetic peptide that corresponds to the Leu(2442)-Pro(2477) region of RyR1 that contains the reported malignant hyperthermia (MH) mutation site. It has been proposed that DP4 disrupts the normal interdomain interactions that stabilize the closed state of the Ca(2)+ release channel (Yamamoto, T., R. El-Hayek, and N. Ikemoto. 2000. J. Biol. Chem. 275:11618-11625). We have investigated the effects of DP4 on local SR Ca(2)+ release events (Ca(2)+ sparks) in saponin-permeabilized frog skeletal muscle fibers using laser scanning confocal microscopy (line-scan mode, 2 ms/line), as well as the effects of DP4 on frog SR vesicles and frog single RyR Ca(2)+ release channels reconstituted in planar lipid bilayers. DP4 caused a significant increase in Ca(2)+ spark frequency in muscle fibers. However, the mean values of the amplitude, rise time, spatial half width, and temporal half duration of the Ca(2)+ sparks, as well as the distribution of these parameters, remained essentially unchanged in the presence of DP4. Thus, DP4 increased the opening rate, but not the open time of the RyR Ca(2)+ release channel(s) generating the sparks. DP4 also increased [(3)H]ryanodine binding to SR vesicles isolated from frog and mammalian skeletal muscle, and increased the open probability of frog RyR Ca(2)+ release channels reconstituted in bilayers, without changing the amplitude of the current through those channels. However, unlike in Ca(2)+ spark experiments, DP4 produced a pronounced increase in the open time of channels in bilayers. The same peptide with an Arg(17) to Cys(17) replacement (DP4mut), which corresponds to the Arg(2458)-to-Cys(2458) mutation in MH, did not produce a significant effect on RyR activation in muscle fibers, bilayers, or SR vesicles. Mg(2)+ dependence experiments conducted with permeabilized muscle fibers indicate that DP4 preferentially binds to partially Mg(2)+-free RyR(s), thus promoting channel opening and production of Ca(2)+ sparks.

Show MeSH
Related in: MedlinePlus