title: The ryanodine receptor pore blocker neomycin also inhibits channel activity via a previously undescribed high-affinity Ca(2+) binding site.
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 [(3)H]ryanodine binding. The half-maximal inhibitory concentration (IC(50)) for channel blockade by neomycin was dependent on membrane potential and cytoplasmic [Ca(2+)], suggesting that neomycin acts both as a pore plug and as a competitive antagonist at a cytoplasmic Ca(2+) binding site that causes allosteric inhibition. This novel Ca(2+)/neomycin binding site had a neomycin affinity of 100 nM: and a Ca(2+) affinity of 35 nM,: which is 30-fold higher than that of the well-described cytoplasmic Ca(2+) activation site. Therefore, a new high-affinity class of Ca(2+) 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 Ca(2+)-dependent allosteric inhibition.
authors: School of Biomedical Sciences, University of Newcastle and Hunter Medical Research Institute, Callaghan, NSW 2308, Australia. Derek.Laver@newcastle.edu.au
authors: Derek R
authors: DR
authors: Laver
authors: Tomoyo
authors: T
authors: Hamada
authors: James D
authors: JD
authors: Fessenden
authors: Noriaki
authors: N
authors: Ikemoto
mesh_terms: D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015640:Ion Channel Gating / Q000187:drug effects* / Q000502:physiology; D008051:Lipid Bilayers / Q000378:metabolism; D008390:Markov Chains; D018482:Muscle, Skeletal / Q000378:metabolism; D009355:Neomycin / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D011817:Rabbits; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism / Q000502:physiology*
chemical_list: D008051:Lipid Bilayers; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D009355:Neomycin; D002118:Calcium