Institute of Molecular Physiology and Genetics, Centre of Excellence for Cardiovascular Research, Slovak Academy of Sciences, 833 34 Bratislava, Slovak Republic.
J Gen Physiol. 2012 Aug;140(2):93-108. doi: 10.1085/jgp.201110708.
The synergic effect of luminal Ca(2+), cytosolic Ca(2+), and cytosolic adenosine triphosphate (ATP) on activation of cardiac ryanodine receptor (RYR2) channels was examined in planar lipid bilayers. The dose-response of RYR2 gating activity to ATP was characterized at a diastolic cytosolic Ca(2+) concentration of 100 nM over a range of luminal Ca(2+) concentrations and, vice versa, at a diastolic luminal Ca(2+) concentration of 1 mM over a range of cytosolic Ca(2+) concentrations. Low level of luminal Ca(2+) (1 mM) significantly increased the affinity of the RYR2 channel for ATP but without substantial activation of the channel. Higher levels of luminal Ca(2+) (8-53 mM) markedly amplified the effects of ATP on the RYR2 activity by selectively increasing the maximal RYR2 activation by ATP, without affecting the affinity of the channel to ATP. Near-diastolic cytosolic Ca(2+) levels (<500 nM) greatly amplified the effects of luminal Ca(2+). Fractional inhibition by cytosolic Mg(2+) was not affected by luminal Ca(2+). In models, the effects of luminal and cytosolic Ca(2+) could be explained by modulation of the allosteric effect of ATP on the RYR2 channel. Our results suggest that luminal Ca(2+) ions potentiate the RYR2 gating activity in the presence of ATP predominantly by binding to a luminal site with an apparent affinity in the millimolar range, over which local luminal Ca(2+) likely varies in cardiac myocytes.
在平面脂质双层中研究了腔钙 (Ca(2+))、细胞质 Ca(2+) 和细胞质三磷酸腺苷 (ATP) 对心脏兰尼碱受体 (RYR2) 通道激活的协同作用。在舒张细胞质 Ca(2+)浓度为 100 nM 的范围内，研究了 RYR2 门控活性对 ATP 的剂量反应，反之亦然，在舒张腔 Ca(2+)浓度为 1 mM 的范围内，研究了细胞质 Ca(2+)浓度的范围。低水平的腔 Ca(2+)（1 mM）显著增加了 RYR2 通道对 ATP 的亲和力，但没有明显激活通道。较高水平的腔 Ca(2+)（8-53 mM）通过选择性增加 ATP 对 RYR2 活性的最大激活作用，而不影响通道对 ATP 的亲和力，显著放大了 ATP 对 RYR2 活性的影响。近舒张细胞质 Ca(2+)水平（<500 nM）极大地放大了腔 Ca(2+)的作用。细胞质 Mg(2+)的分数抑制不受腔 Ca(2+)的影响。在模型中，腔和细胞质 Ca(2+)的作用可以通过调节 ATP 对 RYR2 通道的变构效应来解释。我们的结果表明，腔 Ca(2+)离子在 ATP 存在下增强 RYR2 门控活性，主要是通过与腔位点结合来实现，腔位点的表观亲和力在毫摩尔范围内，局部腔 Ca(2+)在心肌细胞中可能在此范围内变化。
