Abstract
An antiserum raised against the region of the cardiac ryanodine receptor (residues 2805-2819) containing the phosphorylation site for multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) was used to identify the brain ryanodine receptor. This antiserum, which is cardiac isoform-specific, immunoprecipitated greater than 90% of the [3H]ryanodine receptor binding sites solubilized from guinea pig brain membranes. The immunoprecipitated brain receptor exhibited the characteristic cardiac-type mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The brain ryanodine receptor, like the cardiac ryanodine receptor, was a substrate for CaM kinase. Affinity-purified, site-specific antibodies completely blocked phosphorylation of both brain and cardiac receptors by CaM kinase, and two-dimensional peptide mapping identified the same major 32P-labeled peptide in receptors from both tissues. 125I-Labeled receptors also gave the same peptide maps. These results confirm that mammalian brain expresses the cardiac isoform of the ryanodine receptor. Furthermore, the unique CaM kinase phosphorylation site, which has been shown to regulate Ca2+ channel activity, is conserved.
Highlights
An antiserum raised against the region of the cardiac tors and ryanodine receptors were shown to coexist within ryanodine receptor containing single Purkinje neurons (8).These types of studies indicate the phosphorylation site for multifunctional Ca2+/cal- that the caffeine-sensitive Ca2+pool of neurons (9) maybe modulin-dependent protein kinase (CaM kinase) was analogous to the ryanodine receptor-gated Ca2+release pool used to identify the brain ryanodine receptor
The antibody specific for the cardiac isoform of the ryanodine receptor immunoprecipitated virtually all of the [3H] ryanodine binding sites solubilized from brain membranes (Table I)
Cardiac and brain membranes were phosphorylated by added CaM kinase, solubilized with CHAPS, and immunoprecipitated with the site-specific antibody
Summary
Ca2+release from intracellular membrane organelles is an important process regulating cellular function, especially in excitable tissues such as muscle and nerve. MOPS, 3-(N-morpholino)propanesulfonicacid; SDS, sodium dodecyl conducted at 30 "C in 50 p1 of buffer containing 50-100 pg of memsulfate; PAGE, polyacrylamide gel electrophoresis For SDS-PAGE and autora- Affinity Purification of Cardiac Ryanodine Receptor Antibody-10 diography, reactions were terminated by adding 20 p1 of SDS disso- mg of cardiacryanodine receptor peptide 2805-2819 containing a ciation medium (13).SDS-PAGE was conducted according to Laem- carboxyl-terminalcysteine wascoupled to 5 ml of w-aminobutylmli (17) using 6% polyacrylamide gels (13). Immunoprecipitation of PHIRyanodine Receptor Binding Sites-3 glycine (pH 2.4), 150 mM NaC1, and the pH immediately brought to mg of canine cardiac junctional SR andguinea pig brain membrane 7.0 with concentrated MOPS.
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