Discrimination of multiple binding sites for antagonists of the calcium release channel complex of skeletal and cardiac sarcoplasmic reticulum.
The mechanisms by which ruthenium red (RR), neomycin and FLA 365 ([2,6-dichloro-4-aminophenyl]isopropylamine) inhibit calcium channels of skeletal and cardiac sarcoplasmic reticulum (SR) are characterized. Neomycin and FLA 365 inhibit ryanodine-enhanced calcium release from skeletal SR vesicles in a dose-dependent manner. The apparent affinity of [3H]ryanodine is reduced in a dose-dependent manner by each inhibitor indicative of competitive mechanisms. Displacement studies with skeletal and cardiac SR demonstrate that the order of inhibitory potency is RR greater than neomycin greater than FLA 365 and RR greater than FLA 365 greater than neomycin, respectively. Neomycin is 100-fold less potent in cardiac SR and inhibition of [3H]ryanodine binding is biphasic in both tissues. Neomycin induces a greater proportion of [3H]ryanodine binding states recalcitrant to inhibition in cardiac SR. The ability of neomycin to increase the apparent affinity of [3H]ryanodine for its binding sites is potentiated by RR and attenuated by FLA 365. Kinetic binding studies reveal that increasing neomycin concentrations decreases the association of [3H]ryanodine as predicted for competitive inhibition. However, high (much greater than Kn) neomycin increases [3H]ryanodine binding affinity by slowing dissociation of the radioligand demonstrating that, like micromolar ryanodine, neomycin induces allosterism. Studies with combinations of antagonists demonstrate the existence of two non-overlapping inhibitor recognition sites within the ryanoid site, one polycationic inhibitor site and one FLA 365 inhibitor site. These results suggest that aminoglycoside-induced muscle paralysis may be mediated by direct block of pre- and postsynaptic calcium release channels of endoplasmic reticulum.
Mack
W M
WM
Department of Veterinary Pharmacology and Toxicology, University of California, Davis.
Zimányi
I
I
Pessah
I N
IN
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000187:drug effects* / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects; D006321:Heart / Q000187:drug effects*; D007700:Kinetics; D008996:Monoamine Oxidase Inhibitors / Q000494:pharmacology*; D009132:Muscles / Q000187:drug effects / Q000378:metabolism; D009355:Neomycin / Q000494:pharmacology*; D010627:Phenethylamines / Q000494:pharmacology*; D051381:Rats; D012430:Ruthenium Red / Q000494:pharmacology*; D012433:Ryanodine / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000187:drug effects* / Q000378:metabolism
D015220:Calcium Channels; D008996:Monoamine Oxidase Inhibitors; D010627:Phenethylamines; D012430:Ruthenium Red; D012433:Ryanodine; C060909:FLA 365; D009355:Neomycin; D002118:Calcium