Characterization of a Ryanodine Receptor in Periplaneta Americana

Abstract

Specific binding sites for the alkaloid ryanodine were characterized in membrane preparations from sarcoplasmatic reticulum of Periplaneta americana skeletal muscle. Binding of [³H]ryanodine was optimal at pH 8 and at CaCl₂ concentration of about 300 μmol l^-1. The Ca-chelating agents EGTA (100 μmol l^-1) and EDTA (100 μmol l^-1) abolished 95 % and 90 % of the [³H]ryanodine binding respectively. Preincubation with Ca²⁺ (100 μmol l^-1) restored the ryanodine binding in presence of up to 300 μmol l^-1 EGTA. Radioligand binding experiments showed one class of high affinity binding sites for ryanodine. Determination of rate constants revealed 7.05 × 10⁶ l mol^-1 min^-1 for associating and 3.77 × 10^-3 min^-1 for the dissociating [³H]ryandine ryanodine receptor complex. Solubility of the ryanodine receptor was examined with different anionic, non-ionic and zwitterionic detergents. Best solubilization results of “calcium release channel” of cockroach muscle membrane preparations were obtained with the detergent CHAPS in a concentration of 5 mg ml^-1.