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+ (PCa/K approximately 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.
Jeyakumar
L H
LH
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.
Copello
J A
JA
O'Malley
A M
AM
Wu
G M
GM
Grassucci
R
R
Wagenknecht
T
T
Fleischer
S
S
D000818:Animals; D000918:Antibody Specificity; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D008274:Magnesium / Q000378:metabolism; D008854:Microscopy, Electron; D009130:Muscle, Smooth / Q000737:chemistry; D011817:Rabbits; D012430:Ruthenium Red / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism / Q000648:ultrastructure; D012519:Sarcoplasmic Reticulum / Q000737:chemistry
D019837:Ryanodine Receptor Calcium Release Channel; D012430:Ruthenium Red; D008274:Magnesium; D002118:Calcium