Biophys J, June 2001, p. 2715-2726, Vol. 80, No. 6

Structural Determinants for Activation or Inhibition of Ryanodine Receptors by Basic Residues in the Dihydropyridine Receptor II-III Loop

Marco G. Casarotto,^* Daniel Green,^* Suzi M. Pace,^* Suzanne M. Curtis,^* and Angela F. Dulhunty^*

 ^*Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research; and  Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia

The structures of peptide A, and six other 7-20 amino acid peptides corresponding to sequences in the A region (Thr⁶⁷¹- Leu⁶⁹⁰) of the skeletal muscle dihydropyridine receptor II-III loop have been examined, and are correlated with the ability of the peptides to activate or inhibit skeletal ryanodine receptor calcium release channels. The peptides adopted either random coil or nascent helix-like structures, which depended upon the polarity of the terminal residues as well as the presence and ionisation state of two glutamate residues. Enhanced activation of Ca²⁺ release from sarcoplasmic reticulum, and activation of current flow through single ryanodine receptor channels (at 40 mV), was seen with peptides containing the basic residues ⁶⁸¹Arg Lys Arg Arg Lys⁶⁸⁵, and was strongest when the residues were a part of an -helix. Inhibition of channels (at +40 mV) was also seen with peptides containing the five positively charged residues, but was not enhanced in helical peptides. These results confirm the hypothesis that activation of ryanodine receptor channels by the II-III loop peptides requires both the basic residues and their participation in helical structure, and show for the first time that inhibition requires the basic residues, but is not structure-dependent. These findings imply that activation and inhibition result from peptide binding to separate sites on the ryanodine receptor.

Biophys J, June 2001, p. 2715-2726, Vol. 80, No. 6
© 2001 by the Biophysical Society   0006-3495/01/06/2715/12  $2.00

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