The Ryanodine Receptor Pore Blocker Neomycin also Inhibits Channel Activity via a Previously Undescribed High-Affinity Ca2+ Binding Site

The Ryanodine Receptor Pore Blocker Neomycin also Inhibits Channel Activity via a Previously... 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Membrane Biology Springer Journals

The Ryanodine Receptor Pore Blocker Neomycin also Inhibits Channel Activity via a Previously Undescribed High-Affinity Ca2+ Binding Site

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Publisher
Springer Journals
Copyright
Copyright © 2007 by Springer Science+Business Media, LLC
Subject
Life Sciences; Human Physiology ; Biochemistry, general
ISSN
0022-2631
eISSN
1432-1424
D.O.I.
10.1007/s00232-007-9067-3
Publisher site
See Article on Publisher 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 [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

The Journal of Membrane BiologySpringer Journals

Published: Sep 18, 2007

References

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