Keshore R. Bidasee

Structure-Function Relationships among Ryanodine Derivatives PYRIDYL RYANODINE DEFINITIVELY SEPARATES ACTIVATION POTENCY FROM HIGH AFFINITY

Ryanodine derivatives are differentially effective on the two limbs of the ryanodine concentration-effect curve. This study comparing ryanodine, ryanodol, and pyridyl ryanodine and nine C10Oeq esters of them focuses on structure-function relations underlying their differential effectiveness. Ryanodol and pyridyl ryanodine had significantly lower affinities than ryanodine, but their EC50act values (concentration of ryanoid that induces one-half of full efficacy), potencies, and efficacies were not diminished in like fashion. Ryanodine and ryanodol were partial agonists, whereas pyridyl ryanodine was a full agonist, having a diminished deactivation potency. C10Oeq esterifications enhanced affinities and efficacies of the base ryanoids. The C10-Oeqester derivatives of ryanodine and pyridyl ryanodine, but not those of ryanodol, lost their capacity to deactivate RyR1s. Thus, affinity differences among ryanoids clearly do not predicate functional differences as regards activation of Ca2+ release channels. The pyrrole carboxylate on the C3 of ryanodine is dispensable to ryanoid activation of Ca2+ release channels. Ryanodol lacks this ring, but it nevertheless effects substantial activation. Moreover, its C10-Oeq esters display full efficacy. The increased ability of all the C10-Oeq derivatives to release Ca2+from the vesicles strengthens their role in directly impeding deactivation of RyR1, perhaps by interaction with some component within the transmembrane ionic flux pathway.

Effects of ryanodine on calcium sparks in cut twitch fibres of Rana temporaria.

1 Localized calcium release events (calcium sparks) were studied in voltage‐clamped cut twitch fibres of Rana temporaria. 2 A histogram of thousands of spontaneous sparks displayed a monotonically decreasing amplitude distribution from the low to the high limit of > 7 ΔF/F0 units. 3 Several effects of low micromolar concentrations of ryanodine (0.4‐2 μm) on spontaneous sparks, reproducing the agents effects on single ryanodine receptor channel current in bilayers, were observed collectively for the first time in live fibres, namely (a) increases in spark frequency followed by (b) conversions of sparks into steady glows lasting tens of seconds, (c) occasional interruptions of the glows by brief gaps of darkness, and (d) abolition of sparks at the locations of the glows. The glow could reflect the incessant Ca2+ flux through a single (or a few) calcium release channel locked in the semi‐open state, which was allowed to make occasional transitions to the closed state but not to the fully open state. 4 Higher concentrations of ryanodine (≥ 20 μm) suppressed the spontaneous sparks effectively and permanently, presumably by deactivating the ryanodine receptors. 5 Depolarization‐evoked sparks elicited with small pulses had higher frequencies and larger amplitudes than spontaneous sparks and were abolished by both concentrations of ryanodine. 6 With 1‐2 μm ryanodine, however, a uniform non‐sparking calcium release persisted during the pulse, with the globally averaged increase in fluorescence intensity being about half that of the control. A possible origin of this non‐sparking release may be related to the structural coupling between the voltage sensors and the ryanodine receptors that can exist only in live fibres but not in the bilayer preparation.

Activation and deactivation of sarcoplasmic reticulum calcium release channels: Molecular dissection of mechanisms via novel semi-synthetic ryanoids

The plant alkaloids ryanodine and dehydroryanodine are high affinity, biphasic modulators of the intracellularly located, calcium- regulated calcium release channels of a variety of cell types. To date, little is certain about the molecular basis of the interactions that prompt low concentrations of ryanodine (nanomolar to low micromolar) to activate (open) the channels and higher concentrations to deactivate (functionally close) the sarcoplasmic reticulum calcium release channel. In the present study, we approached this question using novel, semi-synthetic C10-Oeq ester derivatives of ryanodine and dehydroryanodine as molecular probes of the ryanodine binding sites on the calcium release channel.