Maurocalcine and Peptide A Stabilize Distinct Subconductance States of Ryanodine Receptor Type 1, Revealing a Proportional Gating Mechanism*

Maurocalcine (MCa) isolated fromScorpio maurus palmatus venom shares 82% sequence identity with imperatoxin A. Both scorpion toxins are putative mimics of the II-III loop peptide (termed peptide A (pA)) of α1s-dihydropyridine receptor and are thought to act at a common site on ryanodine receptor type 1 (RyR1) important for skeletal muscle EC coupling. The relationship between the actions of synthetic MCa (sMCa) and pA on RyR1 were examined. sMCa released Ca2+from SR vesicles (EC50 = 17.5 nm) in a manner inhibited by micromolar ryanodine or ruthenium red. pA (0.5–40 μm) failed to induce SR Ca2+ release. Rather, pA enhanced Ca2+ loading into SR and fully inhibited Ca2+-, caffeine-, and sMCa-induced Ca2+release. The two peptides modified single channel gating behavior in distinct ways. With Cs+-carrying current, 10 nmto 1 μm sMCa induced long lived subconductances having 48% of the characteristic full open state and occasional transitions to 29% at either positive or negative holding potentials. In contrast, pA stabilized long lived channel closures with occasional burst transitions to 65% (s1) and 86% (s2) of the full conductance. The actions of pA and sMCa were observed in tandem. sMCa stabilized additional subconductance states proportional to pA-induced subconductances (i.e. 43% of pA-modified s1 and s2 substates), revealing a proportional gating mechanism. [3H]Ryanodine binding and surface plasmon resonance analyses indicated that the peptides did not interact by simple competition for a single class of mutually exclusive sites on RyR1 to produce proportional gating. The actions of sMCa were also observed with ryanodine-modified channels and channels deficient in immunophilin 12-kDa FK506-binding protein. These results provide evidence that sMCa and pA stabilize distinct RyR1 channel states through distinct mechanisms that allosterically stabilize gating states having proportional conductance.

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