Abstract
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.
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Acknowledgement
We thank Dr. Graham D. Lamb for his valuable comments on the paper. This work was supported by NH&MRC (grant 234420), an infrastructure grant from NSW Health through Hunter Medical Research Institute and National Institutes of Health grants AR 16922 from NIAMS and HL072841 from NHLBI. D. R. L. was supported by a Senior Brawn Fellowship from the University of Newcastle.
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Laver, D.R., Hamada, T., Fessenden, J.D. et al. The Ryanodine Receptor Pore Blocker Neomycin also Inhibits Channel Activity via a Previously Undescribed High-Affinity Ca2+ Binding Site. J Membrane Biol 220, 11–20 (2007). https://doi.org/10.1007/s00232-007-9067-3
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DOI: https://doi.org/10.1007/s00232-007-9067-3