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–O_eq ester derivatives of ryanodine and dehydroryanodine as molecular probes of the ryanodine binding sites on the calcium release channel.Binding affinities of these C₁₀–O_eq ester derivatives of ryanodine and dehydroryanodine with acidic, basic and neutral side chains (K_d values> 53.9 nM, K_d values 0.3–0.7 nM and K_d values 1.3–20.4 nM, compared with 2.3 and 2.8 nM for ryanodine and dehydroryanodine, respectively) were evaluated for their ability to modulate, the patency of the sarcoplasmic reticulum calcium release channel. With the exception of only two derivatives tested to date, all the semi-synthetic C₁₀–O_eq esters selectivelyactivate the Ca²⁺ release channel. That is, they produce no functional closure of the sarcoplasmic reticulum calcium release channels at the highest concentration, that could be tested. Half-maximal concentrations for activation (EC_50act , values) ranged from 0.87–4.2, M, compared with an EC_50act of 1.3 M for ryanodine.