Properties of immunoaffinity purified 106-kDa Ca2+ release channels from the skeletal sarcoplasmic reticulum.
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 antibodies. Western blots showed that the antisera crossreacted with the antigenic SG-106 and not with SR Ca2+, 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 antibody-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 Ca2+ 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 nM increased the open-time probability and at 2-10 nM 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 Mg2+, and blocked by micromolar ruthenium red. Adriamycin (2-10 microM) 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.
Hilkert
R
R
Department of Physiology, School of Medicine, University of Pittsburgh, Pennsylvania 15261.
Zaidi
N
N
Shome
K
K
Nigam
M
M
Lagenaur
C
C
Salama
G
G
D000818:Animals; D000906:Antibodies / Q000502:physiology; D015220:Calcium Channels / Q000737:chemistry* / Q000187:drug effects / Q000276:immunology; D002846:Chromatography, Affinity; D015640:Ion Channel Gating; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000737:chemistry* / Q000187:drug effects; D013438:Sulfhydryl Compounds
D000906:Antibodies; D015220:Calcium Channels; D013438:Sulfhydryl Compounds