Lab of Experimental Cardiology, Catholic University of Leuven, Belgium.
J Physiol. 2011 May 15;589(Pt 10):2569-83. doi: 10.1113/jphysiol.2010.202663. Epub 2011 Mar 8.
During Ca²⁺ release from the sarcoplasmic reticulum triggered by Ca²⁺ influx through L-type Ca²⁺ channels (LTCCs), [Ca²⁺] near release sites ([Ca²⁺]nrs) temporarily exceeds global cytosolic [Ca²⁺]. [Ca²⁺]nrs can at present not be measured directly but the Na+/Ca2+ exchanger (NCX) near release sites and LTCCs also experience [Ca²⁺]nrs. We have tested the hypothesis that ICaL and INCX could be calibrated to report [Ca²⁺]nrs and would report different time course and values for local [Ca²⁺]. Experiments were performed in pig ventricular myocytes (whole-cell voltage-clamp, Fluo-3 to monitor global cytosolic [Ca²⁺], 37◦C). [Ca²⁺]nrs-dependent inactivation of ICaL during a step to +10 mV peaked around 10 ms. For INCX we computationally isolateda current fraction activated by [Ca²⁺]nrs; values were maximal at 10 ms into depolarization. The recovery of [Ca²⁺]nrs was comparable with both reporters (>90% within 50 ms). Calibration yielded maximal values for [Ca²⁺]nrs between 10 and 15 μmol l⁻¹ with both methods. When applied to a step to less positive potentials (-30 to -20 mV), the time course of [Ca²⁺]nrs was slower but peak values were not very different. In conclusion, both ICaL inactivation and INCX activation, using a subcomponent analysis, can be used to report dynamic changes of [Ca²⁺]nrs. Absolute values obtained by these different methods are within the same range, suggesting that they are reporting on a similar functional compartment near ryanodine receptors. Comparable [Ca²⁺]nrs at +10 mV and -20 mV suggests that, although the number of activated release sites differs at these potentials, local gradients at release sites can reach similar values.
在钙离子通过 L 型钙离子通道（LTCC）流入引发肌浆网钙离子释放期间，释放位点附近的钙离子浓度（[Ca²⁺]nrs）暂时超过细胞浆内总体钙离子浓度。目前，[Ca²⁺]nrs 不能直接测量，但释放位点附近的钠离子/钙离子交换器（NCX）和 LTCC 也会经历 [Ca²⁺]nrs。我们假设 ICaL 和 INCX 可以被校准来报告 [Ca²⁺]nrs，并且会报告局部钙离子的不同时间过程和值。实验在猪心室肌细胞（全细胞电压钳，Fluo-3 监测细胞浆内总体钙离子浓度，37°C）中进行。在 +10 mV 的阶跃中，ICaL 依赖于 [Ca²⁺]nrs 的失活在大约 10 ms 时达到峰值。对于 INCX，我们通过计算从 [Ca²⁺]nrs 激活的电流分数来进行隔离；在去极化 10 ms 时达到最大值。[Ca²⁺]nrs 的恢复与两种报告器相似（在 50 ms 内超过 90%）。两种方法的 [Ca²⁺]nrs 校准均产生 10 至 15 μmol l⁻¹ 的最大值。当应用于更负的去极化电位（-30 至-20 mV）时，[Ca²⁺]nrs 的时间过程较慢，但峰值值没有很大差异。总之，ICaL 失活和 INCX 激活都可以使用子组件分析来报告 [Ca²⁺]nrs 的动态变化。这些不同方法获得的绝对值处于相同范围内，这表明它们报告的是在靠近ryanodine 受体的相似功能隔室中的钙离子浓度。在 +10 mV 和 -20 mV 时的 [Ca²⁺]nrs 相似表明，尽管在这些电位下激活的释放位点数量不同，但释放位点的局部梯度可以达到相似的值。
