Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Vlarska 5, 833 34, Bratislava, Slovak Republic.
J Membr Biol. 2010 Apr;234(3):159-69. doi: 10.1007/s00232-010-9243-8. Epub 2010 Mar 25.
The objective of this work was to identify and further characterize potential changes in the functional profile of the cardiac ryanodine receptor (RyR2) channel caused by the coupled gating phenomenon. By reconstituting an ion channel into a planar lipid membrane, we showed that coupled RyR2 channels were activated by cytosolic Ca(2+) with similar efficacy and potency as reported for the single RyR2 channel. In contrast, all examined parameters of gating kinetics were affected by the functional interaction between channels. Ignoring brief closings during main open events, the average open and closed times were considerably prolonged and the frequency of opening was reduced. Interestingly, when luminal Ca(2+) was used as a charge carrier, Ca(2+)-activated coupled RyR2 channels did not exhibit a sudden switch from slow to fast gating kinetics at an open probability of 0.5 as reported for the single RyR2 channel. Regarding flicker gating, the average closed time was significantly shorter and the frequency of closing was greatly enhanced. Furthermore, in contrast to the single RyR2 channel, both parameters for coupled channels were independent of cytosolic Ca(2+). Selected permeation properties of coupled RyR2 channels were comparable to those found for the single RyR2 channel. The Ca(2+) current amplitude-luminal Ca(2+) relationship displayed a simple saturation and the channel selectivity for Ba(2+) and Ca(2+) ions was similar. Our results suggest that the major targets influenced by coupled gating are likely the gates of individual RyR2 channels recruited into a functional complex, thus ensuring the correlation of Ca(2+) fluxes.
本工作旨在鉴定并进一步描述由耦联门控现象引起的心脏兰尼碱受体（RyR2）通道功能谱的潜在变化。通过将离子通道重新构成于平面脂质膜中，我们发现，与单通道 RyR2 报道的情况相似，耦联 RyR2 通道可被胞质 Ca2+以相似的效力和效能激活。相比之下，所有门控动力学参数均受通道之间的功能相互作用影响。忽略主开放事件期间的短暂关闭，平均开放和关闭时间明显延长，且开放频率降低。有趣的是，当使用腔 Ca2+作为电荷载体时，与单通道 RyR2 报道的情况不同，耦联 RyR2 通道在开放概率为 0.5 时，不会突然从慢门控动力学转变为快速门控动力学。关于闪烁门控，平均关闭时间明显缩短，关闭频率大大增强。此外，与单通道 RyR2 不同，耦联通道的两个参数均不依赖于胞质 Ca2+。耦联 RyR2 通道的选择渗透性与单通道 RyR2 通道的发现相当。Ca2+电流幅度-腔 Ca2+关系呈现简单的饱和，且通道对 Ba2+和 Ca2+离子的选择性相似。我们的结果表明，耦联门控主要影响的是募集到功能性复合物中的单个 RyR2 通道的门，从而确保 Ca2+流的相关性。
