Abstract
Localized distribution of malignant hyperthermia (MH) and central core disease (CCD) mutations in N-terminal and central domains of the ryanodine receptor suggests that the interaction between these domains may be involved in Ca(2+) channel regulation. To test this hypothesis, we investigated the effects of a new synthetic domain peptide DP4 corresponding to the Leu(2442)-Pro(2477) region of the central domain. DP4 enhanced ryanodine binding and induced a rapid Ca(2+) release. The concentration for half-maximal activation by agonists was considerably reduced in the presence of DP4. These effects of DP4 are analogous to the functional modifications of the ryanodine receptor caused by MH/CCD mutations (viz. hyperactivation of the channel and hypersensitization of the channel to agonists). Replacement of Arg of DP4 with Cys, mimicking the in vivo Arg(2458)-to-Cys(2458) mutation, abolished the activating effects of DP4. An N-terminal domain peptide DP1 (El-Hayek, R., Saiki, Y., Yamamoto, T., and Ikemoto, N. (1999) J. Biol. Chem. 274, 33341-33347) shows similar activation/sensitization effects. The addition of both DP4 and DP1 produced mutual interference of their activating functions. We tentatively propose that contact between the two (N-terminal and central) domains closes the channel, whereas removal of the contact by these domain peptides or by MH/CCD mutations de-blocks the channel, resulting in hyperactivation/hyper-sensitization effects.
Highlights
Skeletal muscle-type excitation-contraction coupling is triggered presumably by the voltage-dependent binding of the dihydropyridine receptor ␣1 subunit II-III loop to the Ca2ϩ release channel protein of the SR, referred as ryanodine receptor (RyR)1 [1,2,3,4,5,6,7]
Domain Peptide DP4 Activates Both RyR1 and RyR2 in a Concentration-dependent Manner—Fig. 1 depicts the data of ryanodine binding to the SR vesicles isolated from skeletal and cardiac muscle in the presence of different concentrations of DP4
The RyR opens its Ca2ϩ channel in response to various types of stimuli acting on its cytoplasmic region
Summary
RyR, ryanodine receptor; AC50, concentration for half maximal activation; CCD, central core disease; IC50, concentration for half maximal inhibition; MES, 2-(N-morpholino)ethanesulfonic acid; MH, malignant hyperthermia; MOPS, 3-(N-morpholino)propanesulfonic acid; SR, sarcoplasmic reticulum; DP, domain peptide. Replacement of Arg of DP4 with Cys, in the same way as it happens in the MH/CCD mutation (i.e. Arg2458-to-Cys2458 mutation [37]), abolished the activating function of DP4 These findings suggest that the Leu2442–Pro2477 region of the RyR plays a critical role in the Ca2ϩ channel regulation and that a single amino acid residue (Arg2458) mutation produces crucial effects on the function of this domain. Another central domain peptide DP5 produced no appreciable effect on the RyR if added alone. These findings, together with other pieces of evidence shown here, suggest that the interdomain interaction specified in the above hypothesis is involved in the channel regulation mechanism, and changes in this mechanism will result in the abnormal channel regulation
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