Journal
JOURNAL OF GENERAL PHYSIOLOGY
Volume 119, Issue 1, Pages 15-31Publisher
ROCKEFELLER UNIV PRESS
DOI: 10.1085/jgp.119.1.15
Keywords
E-C coupling; Ca2+ release channel; domain peptides; Ca2+-induced; Ca2+ release; sarcoplasmic reticulum
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Funding
- NHLBI NIH HHS [P01-HL47053, P01 HL047053] Funding Source: Medline
- NIAMS NIH HHS [F32 AR008544, R01 AR016922, F32-AR08544, R01-AR16922] Funding Source: Medline
- NINDS NIH HHS [R01-NS23346, R01 NS023346] Funding Source: Medline
- NATIONAL HEART, LUNG, AND BLOOD INSTITUTE [P01HL047053] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES [R01AR016922, F32AR008544] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE [R01NS023346] Funding Source: NIH RePORTER
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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 Ca2+ release channel (Yamamoto, T., R. El-Hayek, and N. Ikemoto. 2000. J. Biol. Chem. 275:11618-11625). We have investigated the effects of DP4 oil local SR Ca2+ release events (Ca2+ 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 Ca2+ release channels reconstituted in planar lipid bilayers. DP4 caused a significant increase in Ca2+ spark frequency in muscle fibers. However, the mean values of the amplitude, rise time, spatial half width. and temporal half duration of the Ca2+ 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 Ca2+ release channel (s) generating the sparks. DP4 also increased [H-3] ryanodine binding to SR vesicles isolated from frog and mammalian skeletal muscle, and increased the open probability of frog RyR Ca2+ release channels reconstituted in bilayers, without changing the amplitude of the Current through those channels. However. unlike in Ca2+ 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(2158)-to-Cys(2458) mutation in MH, did not produce a significant effect on RyR activation in muscle fibers, bilayers, or SR vesicles. Mg2+ dependence experiments conducted with permeabilized muscle fibers indicate that DP4 preferentially binds to partially Mg2+-free RyR(s), thus promoting channel opening and production of Ca2+ sparks.
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