PURIFICATION AND CHARACTERIZATION OF RYANODINE RECEPTOR-3 FROM MAMMALIAN TISSUE

The ryanodine receptors are intracellular Ca2+ release channels that p lay a key role in cell signaling via Ca2+. There are three isoforms, I soform 1 from skeletal muscle and isoform 2 from heart have been chara cterized. Isoform 3 is widely distributed in many mammalian tissues al though 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 d iaphragm sarcoplasmic reticulum fractions enriched in terminal cistern ae containing both isoforms 1 (>95%) and 3 (<5% of the ryanodine bindi ng) served as starting source, Isoform 3 was selectively immunoprecipi tated from the holamidopropyl)-dimethylammonio]-1-propanesulfonic acid (CHAPS)-solubilized fraction and eluted with peptide epitope. Isoform 3 thus prepared is highly purified as characterized by SDS-polyacryam ide gel electrophoresis, Coomassie Blue staining, and by high affinity ryanodine binding. The purified isoform 3 was incorporated into plana r lipid bilayers, and its channel properties were studied. Channel cha racteristics in common with the other two isoforms are slope conductan ce, higher selectivity to Ca2+ versus K+ (P(Ca/K)similar to 6), and re sponse to drugs and Ligands. In its response to Ca2+ and ATP, it more closely resembles isoform 2. The first two-dimensional structure of is oform 3 was obtained by cryoelectron microscopy and image enhancement techniques.