GATING OF THE NATIVE AND PURIFIED CARDIAC SR CA2+-RELEASE CHANNEL WITH MONOVALENT CATIONS AS PERMEANT SPECIES

The primary aim of this study is to characterize the steady-state gating of the native and the purified cardiac sarcoplasmic reticulum Ca2+-release channel using monovalent cations (K+ in the purified, Cs+ in the native) rather than Ca2+ as the permeant ions. The improved resolution of the single-channel events under these conditions has provided a more detailed and accurate description of channel gating than was previously possible. Micromolar cytosolic Ca2+ activates the channel but in the absence of other activating ligands cannot fully open the channel. The relationship between the open probability (Po) and cytosolic free [Ca2+] in both native and purified channels indicates the binding of at least three Ca2+ ions for maximal activation. Lifetime analysis indicates a minimum of three open and five closed states for channels activated solely by Ca2+ and demonstrates that the primary mechanism for the increase in Po is an increase in the frequency of channel opening. Burst analysis also indicates that Ca2+ activates the channel by binding to closed states of the channel to increase the frequency of channel opening. Correlations between successive lifetimes suggest the existence of at least two pathways between the open and closed states. At a given activating [Ca2+](i) the Po is lower at negative than at positive holding potentials; however, we find no change in the mechanisms of Ca2+ activation at different voltages. Po measurements and lifetime analysis indicate that the gating of the purified channel when activated by Ca2+ is indistinguishable from that of the native channel and indicate that the channels are not modified by the purification procedure.