Ryanodine Receptor Projects

The ryanodine receptor (RyR) releases calcium ions from the sarcoplasmic /endoplasmic reticulum (SR/ER) into the cytosol and thereby converts extracellular stimuli into intracellular calcium signals or amplifies and regulates the intracellular calcium concentration. RyRs are found in most cell types but they are concentrated in striated muscle; they are crucial components of the cascade leading to muscle contraction. RyRs are large transmembrane proteins of 565 kDa that form tetrameric Ca2+ channels. They show sequence similarity with inositol 1,4,5-trisphosphate (InsP3)-gated calcium channels of the SR/ER, but have distinct biophysical and pharmacological properties. As for the InsP3R, the 3D structure of the RyR will provide a foundation for understanding structure-function studies and for predicting regulatory sites. The structure, determined at 24 Å resolution, is consistent with the predicted topology for the RyR.

Caffeine is a very effective activator of the RyR. Ryanodine has dual effects on the channel activity. At low concentrations ryanodine locks the channel open in a subconducting state. At higher concentrations the channel is closed. An interesting feature of ryanodine’s action on its receptor is that it only acts on the open channel. Heparin is traditionally used as an inhibitor of the InsP3R. Because heparin is often added to cells to determine the contribution of InsP3 induced release in cells that contain both InsP3R and RyR, the effect of heparin on the RyR was tested. At the same concentration that inhibits the InsP3R, the RyR is activated. Therefore, this reagent also has dual effects.

The RyR can be regulated by a number of cellular factors, including calcium, ATP, phosphorylation and a variety of associated proteins. Many sites on the cytoplasmic side of the RyR have been identified as regulatory sites. Calcium is primarily thought of as an activator of the RyR, but at elevated concentrations activity of the RyR is inhibited, as seen with the InsP3R.

In striated muscle the primary physiological activators of the RyR are calcium and intermolecular coupling. The agonist in non-muscle cells is less clear. One candidate that is very effective in oocytes is cADP ribose. Another putative agonist is leukotriene B4, a metabolite of arachadonic acid. Other metabolites are inactive, suggesting a very specific regulatory role for leukotriene B4.

In PC12 cells RyR types 2 and 3 are distributed throughout the cell. The immunolocalization of the RyR types 2 and 3 are shown in the top and bottom panels, respectively. The role of the RyR in shaping the spatio-temporal pattern in PC 12 cells was determined by comparing untreated cells (top panel) with those treated with the RyR antagonist, dantrolene (bottom panel). Calcium transients generated in the soma are shown in black and in the neurite are shown in gray. In the soma the presence of dantrolene delays and dampens the signal, suggesting that the RyR serve as amplifying molecular switches, facilitating recruitment of the InsP3R type III dependent pool. In the neurite, dantrolene inhibition of RyRs dramatically increases the probability of spiking at all given agonist concentrations suggesting that RyRs override the oscillatory responses mediated by InsP3Rs.

We are interested in how the RyR works, as a molecule, as a channel complex, and as a component of a complex signaling pathway. We hope to identify the agonist(s) used to activate these channels in non-muscle cells and to understand how these channels are regulated by processes within the cell. These data will form the background for the investigation of disease-induced changes in calcium release channel function.