北大程和平教授等人发文 解密纳米钙火花

　　

钙离子是所有动植物细胞内的信号物质，调控诸如神经元通讯、心脏搏动、基因表达等复杂多样的生命过程。日前，北京大学研究人员发展了一种新颖的实验方法，首次实现了对纳米尺度钙信号的高精度实时观测。研究论文“Imaging Ca2+ Nanosparks in Heart with a New Targeted Biosensor”于11月20日在线发表于Circulation Research。

早在1993年，程和平等发现了细胞钙信号的基本单位——钙火花（Calcium spark）。其后20年来超微结构研究表明，钙火花的中心点有成簇排列的钙释放通道阵列，居于由两片细胞膜构成的厚12-18纳米的空间中。这种通道阵列是如何开启的？开启后所释放的钙离子是如何扩散形成微米尺度的钙火花的？这些是钙火花研究领域的核心问题。

在这项研究中，尚维、路福建等巧妙地设计了新的实验方法。他们通过基因操作，将最新最快的钙荧光蛋白GCaMP6f与定位于纳米空间的内源性蛋白triadin或junctin相连，使得探针分子精准地集聚于通道阵列中，并且锚定在细胞膜上而限制其自由扩散。借助于新实验方法的高灵敏度和定位精度，他们成功观测到纳米空间内毫秒量级的钙信号动态，称之为“纳米钙火花”（Calcium nanospark）， 其体积仅为钙火花的1/50。

通过捕获大量的自发纳米钙火花，研究人员发现通道阵列的开启并不总是“全或无”的，促进了对钙火花形成机制的认识。在一次心肌细胞收缩过程中，细胞电生理信号触发上万个纳米钙火花，新技术可以精确测定这些事件的时间次序和空间模式，为揭示心衰和心律失常伴随的钙信号紊乱的成因提供了利器。此外，新技术可望广泛应用于各种细胞纳米尺度钙信号的研究当中。

这项实验研究在北京大学分子医学研究所程和平、陈良怡实验室和生命科学学院王世强实验室完成，并与英国Bristol大学Cannell实验室合作开展模型分析，受到国家基金委“细胞钙信号创新研究”和科技部973项目等资金支持。北京大学分子医学研究所研究生尚维、路福建同学为论文的共同第一作者。

原文摘要：
Imaging Ca2+ Nanosparks in Heart with a New Targeted Biosensor
Abstract
Rationale: In cardiac dyads, junctional Ca2+ directly controls the gating of the ryanodine receptors (RyRs), and is itself dominated by RyR-mediated Ca2+ release from the sarcoplasmic reticulum. Existing probes do not report such local Ca2+ signals due to probe diffusion, so a junction-targeted Ca2+ sensor should reveal new information on cardiac excitation-contraction coupling and its modification in disease states.

Objective: To investigate Ca2+ signaling in the nanoscopic space of cardiac dyads by targeting a new sensitive Ca2+ biosensor (GCaMP6f) to the junctional space.

Methods and Results: By fusing GCaMP6f to the N-terminus of triadin 1 or junctin, GCaMP6f-T/J was targeted to dyadic junctions, where it colocalized with t-tubules and RyRs after adenovirus-mediated gene transfer. This membrane protein-tagged biosensor displayed ~4-times faster kinetics than native GCaMP6f. Confocal imaging revealed junctional Ca2+ transients (Ca2+ nanosparks) that were ~50-times smaller in volume than conventional Ca2+ sparks (measured with diffusible indicators). The presence of the biosensor did not disrupt normal Ca2+ signaling. Because no indicator diffusion occurred, the amplitude and timing of release measurements were improved, despite the small recording volume. We could also visualize co-activation of subclusters of RyRs within a single junctional region, as well as quarky Ca2+release events.

Conclusions: This new, targeted biosensor allows selective visualization and measurement of nanodomain Ca2+ dynamics in intact cells and can be used to give mechanistic insights into dyad RyR operation in health as well as in disease states such as when RyRs become orphaned.