Ashley R H, Williams A J
Department of Cardiac Medicine, National Heart and Lung Institute, University of London, United Kingdom.
J Gen Physiol. 1990 May;95(5):981-1005. doi: 10.1085/jgp.95.5.981.
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 approximately 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 microM 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.
来自绵羊心脏连接肌浆网囊泡的单个Ca2+释放通道已被整合到不带电的平面脂质双分子层中。从Ca2(+)-激活通道记录到单通道电流，其Ca2+电导约为90 pS。随着肌质面游离Ca2+浓度升高，通道开放概率呈亚线性增加，且在没有额外激动剂的情况下，即使100 microM游离Ca2+也无法使通道完全激活。寿命分析显示至少有两个开放状态和三个关闭状态，表明Ca2+通过与至少一个关闭状态相互作用来激活通道，以增加通道开放速率。相邻寿命之间的相关性表明开放态和关闭态聚集体之间至少有两条途径。对爆发行为的分析还揭示了连续爆发长度与每次爆发开放次数之间的相关性。后者有两个几何成分，为至少两个开放状态提供了额外证据。一个成分似乎由单位爆发组成，其中大多数的寿命落在与超过90%的所有开放相关的主要较短开放时间分布范围内。提出了一种循环门控机制，通道激活由Ca2+与通道蛋白的关闭构象结合来调节。Mg2+可能通过竞争该结合位点来抑制激活，但寿命和波动分析表明，一旦激活，通道继续正常门控。
