Mol093757 318..329

Ryanodine receptors (RyRs) are intracellular membrane channels playing key roles in many Ca signaling pathways and, as such, are emerging novel therapeutic and insecticidal targets. RyRs are so named because they bind the plant alkaloid ryanodine with high affinity and although it is established that ryanodine produces profound changes in all aspects of function, our understanding of the mechanisms underlying altered gating is minimal. We address this issue using detailed single-channel gating analysis, mathematical modeling, and energetic evaluation of state transitions establishing that, with ryanodine bound, the RyR pore adopts an extremely stable open conformation. We demonstrate that stability of this state is influenced by interaction of divalent cations with both activating and inhibitory cytosolic sites and, in the absence of activating Ca, trans-membrane voltage. Comparison of the conformational stability of ryanodineand Imperatoxin A-modified channels identifies significant differences in the mechanisms of action of these qualitatively similar ligands.