Effects of peptide C corresponding to the Glu724–Pro760 region of the II–III loop of the DHP (dihydropyridine) receptor α1 subunit on the domain- switch-mediated activation of RyR1 (ryanodine receptor 1) Ca2+ channels

摘要

The Leu⁷²⁰–Leu⁷⁶⁴ region of the II–III loop of the dihydropyridine receptor is believed to be important for both orthograde and retrograde communications with the RyR (ryanodine receptor), but its actual role has not yet been resolved. Our recent studies suggest that voltage-dependent activation of the RyR channel is mediated by a pair of interacting N-terminal and central domains, designated as the ‘domain switch’. To investigate the effect of peptide C (a peptide corresponding to residues Glu⁷²⁴–Pro⁷⁶⁰) on domain- switch-mediated activation of the RyR, we measured Ca²⁺ release induced by DP (domain peptide) 1 or DP4 (which activates the RyR by mediation of the domain switch) and followed the Ca²⁺ release time course using a luminal Ca²⁺ probe (chlortetracycline) under Ca²⁺-clamped conditions. Peptide C produced a significant potentiation of the domain-switch-mediated Ca²⁺ release, provided that the Ca²⁺ concentration was sufficiently low (e.g. 0.1 μM) and the Ca²⁺ channel was only partially activated by the domain peptide. However, at micromolar Ca²⁺ concentrations, peptide C inhibits activation. Covalent cross-linking of fluorescently labelled peptide C to the RyR and screening of the fluorescently labelled tryptic fragments permitted us to localize the peptide-C-binding site to residues 450–1400, which may represent the primary region involved in physical coupling. Based on the above findings, we propose that the physiological role of residues Glu⁷²⁴–Pro⁷⁶⁰ is to facilitate depolarization-induced and domain-switch-mediated RyR activation at sub- or near-threshold concentrations of cytoplasmic Ca²⁺ and to suppress activation upon an increase of cytoplasmic Ca²⁺.