Junctional cleft [Ca²⁺]i measurements using novel cleft-targeted Ca²⁺ sensors.
Junctional cleft [Ca²⁺]i measurements using novel cleft-targeted Ca²⁺ sensors.
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DOI：
10.1161/circresaha.115.303582
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发表时间：
2014-07-18
影响因子：
20.1
通讯作者：
Bers DM
中科院分区：
文献类型：
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作者：
Despa S;Shui B;Bossuyt J;Lang D;Kotlikoff MI;Bers DM
细胞内Ca 2+浓度（[Ca 2 +]i）在不同的亚细胞微区中受到不同的调节和信号传递，这极大地增强了其第二信使的多样性。在心脏中，肌浆网（SR）Ca 2+释放和信号传导由连接裂隙（[Ca 2 +]Cleft）中的局部[Ca 2 +]i控制，连接裂隙是肌膜和连接SR之间的小空间。构建新型传感器，允许直接测量[Ca 2 +]Cleft。我们通过将Ca 2+传感器GCaMP2.2和一种新的低Ca 2+亲和力变体GCaMP2.2Low融合到FKBP12.6来构建裂缝靶向[Ca 2 +]传感器，FKBP12.6以高亲和力和选择性结合兰尼碱受体（RyRs）。荧光模式、对RyR的亲和力和未标记的FKBP12.6的竞争表明，FKBP12.6标记的传感器被定位成测量成年大鼠肌细胞中的局部[Ca 2 +]裂隙。使用GCaMP 2. 2Low-FKBP 12.6，我们表明在兴奋-收缩耦合期间，[Ca 2 +]Cleft达到比整体[Ca 2 +]i更高的水平，具有更快的动力学。舒张期SR Ca 2+渗漏或肌膜Ca 2+内流可使局部[Ca 2 +]裂隙高于胞质[Ca 2 +]i（[Ca 2 +]Bulk），这种效应可能导致触发性心律失常甚至转录调节。我们使用GCaMP2.2-FKBP12.6与GCaMP2.2测量了舒张期站立[Ca 2 +]裂隙-[Ca 2 +]体积梯度，使用无梯度测量的[Ca 2 +]作为参考点。这种舒张期差异（[Ca 2 +]Cleft=194 nmol/L vs. [Ca 2 +]Bulk=100 nmol/L）主要由SR Ca 2+泄漏决定，而不是肌膜Ca 2+通量。我们已经开发了连接裂隙靶向传感器来测量[Ca 2 +]裂隙与[Ca 2 +]体积，并证明了电激发和站立舒张期[Ca 2 +]i梯度期间的动态差异，这可能影响连接裂隙内的局部Ca 2+依赖性信号传导。
Intracellular Ca2+ concentration ([Ca2+]i) is regulated and signals differently in various subcellular microdomains, which greatly enhances its second messenger versatility. In the heart, sarcoplasmic reticulum (SR) Ca2+ release and signaling is controlled by local [Ca2+]i in the junctional cleft ([Ca2+]Cleft), the small space between sarcolemma and junctional SR. However, methods to directly measure [Ca2+]Cleft are needed. To construct novel sensors that allow direct measurement of [Ca2+]Cleft. We constructed cleft-targeted [Ca2+] sensors by fusing Ca2+-sensor GCaMP2.2 and a new lower Ca2+-affinity variant GCaMP2.2Low to FKBP12.6, which binds with high affinity and selectivity to ryanodine receptors (RyRs). The fluorescence pattern, affinity for RyRs and competition by un-tagged FKBP12.6 demonstrated that FKBP12.6-tagged sensors are positioned to measure local [Ca2+]Cleft in adult rat myocytes. Using GCaMP2.2Low-FKBP12.6, we showed that [Ca2+]Cleft reaches higher levels with faster kinetics than global [Ca2+]i during excitation-contraction coupling. Diastolic SR Ca2+ leak or sarcolemmal Ca2+ entry may raise local [Ca2+]Cleft above bulk cytosolic [Ca2+]i ([Ca2+]Bulk), an effect that may contribute to triggered arrhythmias and even transcriptional regulation. We measured this diastolic standing [Ca2+]Cleft–[Ca2+]Bulk gradient using GCaMP2.2-FKBP12.6 vs. GCaMP2.2, using [Ca2+] measured without gradients as a reference point. This diastolic difference ([Ca2+]Cleft=194 nmol/L vs. [Ca2+]Bulk=100 nmol/L) is dictated mainly by the SR Ca2+ leak, rather than sarcolemmal Ca2+ flux. We have developed junctional cleft targeted sensors to measure [Ca2+]Cleft vs. [Ca2+]Bulk, and demonstrated dynamic differences during electrical excitation and a standing diastolic [Ca2+]i gradient which could influence local Ca2+-dependent signaling within the junctional cleft.