The solution structures of three related peptides (A1, A2, and A9) corresponding to the Thr 671 -Leu 690 region of the skeletal muscle dihydropyridine receptor II-III loop have been investigated using nuclear magnetic resonance spectroscopy. Peptide A1, the native sequence, is less effective in activating ryanodine receptor calcium release channels than A2 (Ser 687 to Ala substitution and Asp 1112 has also been reported (8). The peptide A1 region may be necessary for the DHPR II-III loop to bind to the RyR, whereas other regions of the loop are necessary for the transmission of the ECC signal from the S4 part of the DHPR to the II-III loop/RyR binding region (7). Evidence for this hypothesis is that the cardiac DHPR can support skeletal type ECC when the peptide A region contains the cardiac sequence, provided that residues 725-742 contain the skeletal sequence (9), suggesting that (a) both cardiac and skeletal peptide A1 regions bind to the RyR, and (b) residues 725-742 are essential for transmission of the ECC (7). If this hypothesis is correct, activation of RyR channels by peptide A1 or by the II-III loop may reflect binding to the RyR, rather than activation equivalent to ECC. The hypothesis is supported by the fact that the full cardiac II-III loop activated skeletal RyRs (3, 4).Two regions of peptide A1 are important in its interaction with RyRs. The highly charged 681 RKRRK 685 sequence is essential for RyR activation by peptide A1 (6, 10), and Ser 687 is important, although its precise role is not clear. The II-III loop does not activate RyRs when Ser 687 is either phosphorylated or replaced by alanine (4). In contrast, replacement of Ser 687 with Ala increases the ability of peptide A1 to activate RyRs (7), whereas a similar replacement in a 25-amino acid peptide (Glu 666 -Pro 690 ) reduces [ 3 H]ryanodine binding (11). The different effects of Ser 687 to Ala substitution are likely to reflect structural differences between peptides of different lengths. The structure of the protein-protein interaction sites on the II-III loop and on the RyR must be understood in order to comprehend these functional observations and the structural changes that could occur during ECC. Neither structure has been determined.Here, we address the structural basis for Ca 2ϩ release from the SR by the native DHPR sequence from Thr 671 -Leu 690 (peptide A1) and the enhanced ability of peptide A2 (S687A substitution) to release Ca 2ϩ from skeletal SR and to activate skeletal RyRs. We determined the solution structures for three peptides using NMR spectroscopy. These peptides are a small, sevenresidue peptide encompassing the basic region essential for RyR activation (A9), peptide A1 (containing Ser 687 ), and peptide A2 (containing a S687A substitution). The results show that the smaller A9 peptide is unstructured, whereas both peptides A1 and A2 have a propensity to form helical structures at their N-terminal end. The C-terminal part of A1 is highly mobile, whereas the C-terminal part of A2 is more constrained. Because we find...

An alpha-helical II-III loop segment of the dihydropyridine receptor activates the ryanodine receptor calcium-release channel. We describe a novel manipulation in which this agonist's activity is increased by modifying its surface structure to resemble that of a toxin molecule. In a unique system, native beta-sheet scorpion toxins have been reported to activate skeletal muscle ryanodine receptor calcium channels with high affinity by binding to the same site as the lower-affinity alpha-helical dihydropyridine receptor segment. We increased the alignment of basic residues in the alpha-helical peptide to mimic the spatial orientation of active residues in the scorpion toxin, with a consequent 2-20-fold increase in the activity of the alpha-helical peptide. We hypothesized that, like the native peptide, the modified peptide and the scorpion toxin may bind to a common site. This was supported by (i) similar changes in ryanodine receptor channel gating induced by the native or modified alpha-helical peptide and the beta-sheet toxin, a 10-100-fold reduction in channel closed time, with a < or = 2-fold increase in open dwell time and (ii) a failure of the toxin to further activate channels activated by the peptides. These results suggest that diverse structural scaffolds can present similar conformational surface properties to target common receptor sites.

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 alpha-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.

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