Direct measurement of SR release flux by tracking 'Ca2+ spikes' in rat cardiac myocytes
Direct measurement of SR release flux by tracking 'Ca2+ spikes' in rat cardiac myocytes
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DOI：
10.1111/j.1469-7793.1998.677bd.x
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发表时间：
1998-11-01
影响因子：
5.5
通讯作者：
Cheng, HP
中科院分区：
文献类型：
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作者：
Song, LS;Sham, JSK;Cheng, HP
1. 使用一种新型荧光方法研究了心脏兴奋-收缩耦合期间穿过肌浆网 (SR) 的 Ca2+ 释放通量。在全细胞电压钳条件下，用高浓度 EGTA（4.0 mM，150 nM 游离 Ca2+）透析大鼠心室肌细胞，以最大程度地缩短释放的 Ca2+ 在细胞质中的停留时间，并使用低亲和力、快速 Ca2+ 指示剂 Oregon Green 488 BAPTA-5N（OG-5N；1.0 mM，K-d 近似于 31 muM），以优化释放位点微域中局部高 [Ca2+] 的检测。采用共聚焦显微镜以高空间和时间分辨率解析细胞内[Ca2+]。2．分析和数值分析表明，在高EGTA浓度条件下，游离[Ca2+]变化是两项之和：一项主要与SR释放通量/Ca2+流入量成正比，另一项反映释放Ca2+的运行积分。3。事实上，含有 EGTA 的细胞中的 OG-5N 瞬变由一个突出的尖峰和随后的一个小基座组成。 OG-5N 尖峰与传统 Ca2+ 瞬态（无 EGTA）的一阶导数 (d[Ca2+]/dt) 非常相似，并模仿了先前报道的模型衍生的 SR Ca2+ 释放函数。在 SR Ca2+ 耗尽的细胞中，OG-5N 瞬态也密切遵循 L 型 Ca2+ 电流 (I-Ca) 的波形。使用 I-Ca 作为 Ca2+ 流入的已知来源，可以通过 I-Ca 引发的 OG-5N 信号的线性外推来校准体内 SR 通量。4 OG-5N 图像信号定位于肌节 Z 线水平的离散释放位点，表明局部 OG-5N 尖峰源自横向 (T) 肾小管 - SR 交界处的“Ca2+ 尖峰”（由于钙之间的不平衡）离子进入细胞质和缓冲分子）5。峰值 SR 释放通量和释放的 Ca2+ 总量均表现出钟形电压依赖性。 SR 释放的时间模式也随膜电压的变化而变化：Ca2+ 释放最同步，并在 0 mV 时产生最大峰值释放通量 (4.2 mM s(-1))；相反，最大总释放发生在-20 mV（0 mV 时为 71 μM 与 61 μM），但局部释放信号部分异步。由于最大常规 [Ca2+] 瞬态和收缩是在 0 mV 时引发的，因此似乎不仅 Ca2+ 释放量，而且释放位点之间的同步性也会影响全细胞 Ca2+ 瞬态和 Ca2+-肌丝相互作用。
1. Ca2+ release flux across the sarcoplasmic reticulum (SR) during cardiac excitation-contraction coupling was investigated using a novel fluorescence method. Under whole-cell voltage-clamp conditions, rat ventricular myocytes were dialysed with a high concentration of EGTA (4.0 mM, 150 nM free Ca2+), to minimize the residence time of released Ca2+ in the cytoplasm, and a low-affinity, fast Ca2+ indicator, Oregon Green 488 BAPTA-5N (OG-5N; 1.0 mM, K-d approximate to 31 mu M), to optimize the detection of localized high [Ca2+] in release site microdomains. Confocal microscopy was employed to resolve intracellular [Ca2+] at high spatial and temporal resolution.2. Analytical and numerical analyses indicated that, under conditions of high EGTA concentration, the free [Ca2+] change is the sum of two terms: one major term proportional to the SR release flux/Ca2+ influx, and the other reflecting the running integral of the released Ca2+.3. Indeed, the OG-5N transients in EGTA-containing cells consisted of a prominent spike followed by a small pedestal. The OG-5N spike closely resembled the first derivative (d[Ca2+]/dt) of the conventional Ca2+ transient (with no EGTA), and mimicked the model-derived SR Ca2+ release function reported previously. In SR Ca2+-depleted cells, the OG-5N transient also closely followed the waveform of L-type Ca2+ current (I-Ca). Using I-Ca as a known source of Ca2+ influx, SR flux can be calibrated in vivo by a linear extrapolation of the I-Ca-elicited OG-5N signal.4 The OG-5N image signal was localized to discrete release sites at the Z-line level of sarcomeres, indicating that the local OG-5N spike arises from 'Ca2+ spikes' at transverse (T) tubule-SR junctions (due to the imbalance between calcium ions entering the cytosol and the buffer molecules).5. Both peak SR release flux and total amount of released Ca2+ exhibited a bell-shaped voltage dependence. The temporal pattern of SR release also varied with membrane voltage: Ca2+ release was most synchronized and produced maximal peak release flux (4.2 mM s(-1)) at 0 mV; in contrast, maximal total release occurred at -20 mV(71 versus 61 mu M at 0 mV), but the localized release signals were partially asynchronous. Since the maximal conventional [Ca2+] transient and contraction were elicited at 0 mV, it appears that not only the amount of Ca2+ released, but also the synchronization among release sites affects the whole-cell Ca2+ transient and the Ca2+-myofilament interaction.