In this study, we present evidence for the mechanism of neomycin inhibition of skeletal ryanodine receptors (RyRs). In single-channel recordings, neomycin produced monophasic inhibition of RyR open probability and biphasic inhibition of [3H]ryanodine binding. The half maximal inhibitory concentration (IC₅0) for channel blockade by neomycin was dependent on membrane potential and cytoplasmic [Ca²⁺], suggesting that neomycin acts both as a pore plug and as a competitive antagonist at a cytoplasmic Ca²⁺ binding site that causes allosteric inhibition. This novel Ca²⁺/neomycin binding site had a neomycin affinity of 100 nM and a Ca²⁺ affinity of 35 nM, which is 30-fold higher than that of the well-described cytoplasmic Ca²⁺ activation site. Therefore, a new high affinity class of Ca²⁺ binding site(s) on the RyR exists that mediates neomycin inhibition. Neomycin plugging of the channel pore induced brief (1-2 ms) conductance substates at 30% of the fully open conductance, whereas allosteric inhibition caused complete channel closure with durations that depended on the neomycin concentration. We quantitatively account for these results using a dual inhibition model for neomycin that incorporates voltage-dependent pore plugging and Ca²⁺-dependent allosteric inhibition.
Journal of Membrane Biology Vol. 220, Issue 1-3, p. 11-20