Purification and characterization of ryanodine receptor 3 from mammalian tissue

The ryanodine receptors are intracellular Ca2+ release channels that play a key role in cell signaling via Ca2+. There are three isoforms, Isoform 1 from skeletal muscle and isoform 2 from heart have been characterized. Isoform 3 is widely distributed in many mammalian tissues although in minuscule amounts, Its low abundance has hampered its study. We now describe methodology to isolate mammalian isoform 3 in amounts sufficient for biochemical and biophysical characterization, Bovine diaphragm sarcoplasmic reticulum fractions enriched in terminal cisternae containing both isoforms 1 (>95%) and 3 (<5% of the ryanodine binding) served as starting source, Isoform 3 was selectively immunoprecipitated from the 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid (CHAPS)-solubilized fraction and eluted with peptide epitope. Isoform 3 thus prepared is highly purified as characterized by SDS-polyacryamide gel electrophoresis, Coomassie Blue staining, and by high affinity ryanodine binding. The purified isoform 3 was incorporated into planar lipid bilayers, and its channel properties were studied. Channel characteristics in common with the other two isoforms are slope conductance, higher selectivity to Ca2+ versus K+ (P(Ca/K)similar to 6), and response to drugs and Ligands. In its response to Ca2+ and ATP, it more closely resembles isoform 2. The first two-dimensional structure of isoform 3 was obtained by cryoelectron microscopy and image enhancement techniques.