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

Abstract

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, μM, compared with an EC_50act of 1.3 μM for ryanodine.

Using a low concentration (0.5 nM) of a high specific activity, radioiodinated derivative of ryanodine, C₁₀−O_eq N-(4-azido-5-¹²⁵iodo salicyloyl) glycyl ryanodine (1400 Ci/mmol) as the radioligand in displacement binding affinity assays, two distinct, sequential ryanodine binding isotherms were demonstrated within the normal 0–300 nM ryanodine sigmoidal displacement curve. A high affinity site had an IC₅₀ of 0.5 nM (K_d=0.26±0.02 nM). Above this concentration, an apparent plateau occurred between 3 and 6 nM ryanodine, and at higher concentrations a lower affinity site, was revealed that demonstrated an IC₅₀ of about 25 nM (K_d=11.7±1.2, nM). Scatchard analysis from direct binding of C₁₀−O_eq N-(4-azido-5-¹²⁵iodo salicyloyl) glycyl ryanodine to junctional sarcoplasmic reticulum vesicles also suggests the presence of more than one class of binding sites within the nanomolar concentration range. The high affinity site, demonstrated a B_max of 3 pmol/mg protein. We were unable to saturate the lower affinity binding sites with this ligand.

To evaluate the functional effects occurring among sarcoplasmic reticulum calcium release channel monomers as a consequence of ryanodine's binding, we utilized a photo-activatable derivative of ryanodine, C₁₀−O_eq N-(4-azido salicyloyl) glycyl ryanodine that demonstrates channel modulating characteristics similar to ryanodine. Covalently labeling the sarcoplasmic reticulum calcium-release channels with this ligand, followed by measurements of rates of calcium efflux and SDS-PAGE of the labeled protein, revealed that deactivation of the sarcoplasmic reticulum calcium release channels of skeletal muscle by this ryanoid occurred at concentrations which apparently produce virtually irreversibly, interactions between receptor monomers. This ‘polymerization’ was indicated by the progressive appearance of two higher molecular weight protein bands on SDS-PAGE concomitant with progressive decreases in the ryanodine receptor monomer band that runs at an apparent molecular mass of 365 kDa.

In summary, we have prepared an utilized, novel C₁₀−O_eq ester derivatives of ryanodine and dehydroryanodine in studies aimed at better understanding the molecular basis for the complex biphasic actions of ryanodine on the sarcoplasmic reticulum calcium release channels from rabbit skeletal muscle cells. The described studies presage correlations that may be useful in furthering our understanding of structure-function relationship among ryanoids.