The ryanodine receptor pore blocker neomycin also inhibits channel activity via a previously undescribed high-affinity Ca2+ binding site

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 [H-3]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.