DIVALENT-CATION ACTIVATION AND INHIBITION OF SINGLE CALCIUM RELEASE CHANNELS FROM SHEEP CARDIAC SARCOPLASMIC-RETICULUM

Single Ca2+ release channels from vesicles of sheep cardiac junctional sarcoplasmic reticulum have been incorporated into uncharged planar lipid bilayers. Single-channel currents were recorded from Ca2+-activated channels that had a Ca2+ conductance of ~90 pS. Channel open probability increased sublinearly as the concentration of free Ca2+ was raised at the myoplasmic face, and without additional agonists the channels could not be fully activated even by 100 µM free Ca2+. Lifetime analysis revealed a minimum of two open and three closed states, and indicates that Ca2+ activated the channels by interacting with at least one of the closed states to increase the rate of channel opening. Correlations between adjacent lifetimes suggested there were at least two pathways between the open- and closed-state aggregates. An analysis of bursting behavior also revealed correlations between successive burst lengths and the number of openings per burst. The latter had two geometric components, providing additional evidence for at least two open states. One component appeared to comprise unit bursts, and the lifetime of most of these fell within the dominant shorter open-time distribution associated with over 90% of all openings. A cyclic gating scheme is proposed, with channel activation regulated by the binding of Ca2+ to a closed conformation of the channel protein. Mg2+ may inhibit activation by competing for this binding site, but lifetime and fluctuation analysis suggested that once activated the channels continue to gate normally. © 1990, Rockefeller University Press., All rights reserved.