Ryanodine-affinity chromatography purifies 106 kD Ca 2+ release channels from skeletal and cardiac sarcoplasmic reticulum

Abstract

A 106 kD protein was isolated from skeletal sarcoplasmic reticulum (SR) vesicles and shown to have the properties of SR Ca 2+ release channels, including blockade by 5 nM ryanodine. In view of extensive reports that the ryanodine-receptor complex consists of four 565 kD junctional feet proteins (JFPs) and is the ‘physiological’ Ca 2+ release channel, we prepared ryanodine-affinity columns to isolate its receptor site(s). Conditions known to maximize the association and dissociation of ryanodine to SR proteins were respectively used to link, then elute, the receptor(s) from ryanodine-affinity columns. The method purified a protein at about 100 kD from both rabbit skeletal and canine cardiac SR vesicles. The skeletal and cardiac proteins isolated by ryanodine-affinity chromatography were identified as the low molecular weight Ca 2+ release channel through their antigenic reaction with an anti-106 kD monoclonal antibody. Upon reconstitution in planar bilayers, both skeletal and cardiac proteins revealed the presence of functional SR Ca 2+ release channels. Surprisingly, ryanodine-affinity columns did not retain JFPs but purified 106 kD Ca 2+ release channels which are a minor component (0.1–0.3%) of SR proteins.