Properties of immunoaffinity purified 106-kDa Ca 2+ release channels from the skeletal sarcoplasmic reticulum

Abstract

The sulfhydryl-gated 106-kDa Ca 2+-release channel (SG-106) was purified by biotin-avidin chromatography from skeletal sarcoplasmic reticulum (SR) vesicles and used as an antigen to raise polyclonal anti-bodies. Western blots showed that the antisera crossreacted with the antigenic SG-106 and not with SR Ca 2+, Mg 2+-ATPase or with junctional foot proteins (JFPs) (Zaidi et al., 1989, J. Biol. Chem. 264(36), 21,725–21,736; 21,737–21,747). Polyclonal anti-body-affinity columns were used to selectively purify SG-106-kDa proteins which, upon incorporation in planar bilayers, revealed the presence of a cationic channels with properties similar to “native” Ca 2+-release channels obtained through the fusion of SR vesicles with planar bilayers. In agreement with measurements of Ca 2+ release from SR vesicles, sulfhydryl oxidizing and reducing agents (i.e., 2,2′-dithiodipyridine and dithiothreitol) respectively increased and decreased the open-time probability of 106-kDa Ca 2+-release channels. In contrast with reports on JFPs, ryanodine at 0.5–1 n m increased the open-time probability and at 2–10 n m locked 106-kDa Ca 2+-release channels in a closed state rather than an open subconductance state. The SG-106 was activated by millimolar ATP, inhibited by millimolar Mg 2+, and blocked by micromolar ruthenium red. Adriamycin (2–10 μ m) caused a transient activation of SG-106 Ca 2+-release channels, followed by closure in about 5 min, and intermittent activation to a subconductance state. Polyclonal antibodies used to purify the SG-106 also activated the channel when added to the cis side but not the trans side of the bilayer. Thus, SG-106 channels possess features that are similar to “native” SR Ca 2+-release channels, are immunologically distinct from JFPs, and interact in seconds with nanomolar ryanodine in planar bilayers.