FRETting mice shed light on cardiac adrenergic signaling.
FRETting mice shed light on cardiac adrenergic signaling.
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FRET 小鼠揭示了心脏肾上腺素信号传导。
DOI：
10.1161/01.res.0000250962.61995.cf
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发表时间：
2006
影响因子：
20.1
通讯作者：
Zhang,Jin
中科院分区：
文献类型：
--
作者：
DiPilato,LisaM;Zhang,Jin
Lisa M.DiPilato，金·张，心肌细胞中的肾上腺素能受体(？AR)信号影响心脏的收缩和松弛状态。经典的做法是，激素激活后，B-AR优先与Gs偶联，Gs进而激活腺苷环化酶和cAMP的产生。CAMP的主要效应蛋白是cAMP依赖的蛋白激酶，然后使许多与心脏功能有关的蛋白质磷酸化，如L钙通道和肌浆网膜上的磷蛋白。然而，对心肌细胞的研究表明，并不是所有通过cAMP传递信号的受体都会产生同样的功能效应。1这些观察结果导致了cAMP区隔的概念，它将cAMP通路中复杂的信号分子的空间和时间控制的功能特异性和差异性调节归因于cAMP通路中复杂的空间和时间控制。在这一期的《循环研究》中，Nikolaev等人通过在成人心肌细胞中使用基于FRET的cAMP成像，再次研究了差异调节的？1和？2-肾上腺素能信号，这种方法非常适合揭示cAMP信号的时空复杂性。15年前，随着使用荧光染料标记的PKA调节和催化亚基的双分子指示剂的开发，cAMP在活细胞中的荧光比率成像首次被引入。3随着绿色荧光蛋白(GFP)变体4、5的使用，这类cAMP指示剂已经进化成可遗传编码的；近年来，通过使用单一cAMP结合结构域的蛋白质和蛋白片段，这类cAMP指示剂已经进化成单分子的。6-8 Nikolaev等人开发了另一种具有新特性的基于FRET的cAMP传感器，从而增加了我们的分子工具箱。该传感器使用超极化激活的环核苷酸门控通道2(HCN2)的cAMP结合域，而不是其他FRET传感器中使用的PKA或cAMP直接激活的交换蛋白(EPAC)的结合域。由此产生的生物传感器，HCN-cAMPS，对cAMP保持高灵敏度，但在生理cAMP浓度下似乎不饱和，因此能够报告激动剂诱导的cAMP变化。HCN2-cAMP的另一个重要特征是它在心肌细胞中的均匀分布，这使得可以测量整个细胞的cAMP动态，而不受传感器定位的影响。这种新的cAMP生物传感器的设计和应用说明了能够使用最适合特定实验、细胞系统或功能研究的定制cAMP生物传感器的重要性。在设计或选择CAMP传感器时，有几个关键特性值得考虑。首先，传感器的结合特性，如与cAMP的结合亲和力，是最重要的特征之一。不同类型的细胞具有不同的cAMP基础水平以及不同的cAMP产生和降解能力，因此需要使用具有与内源性cAMP浓度相匹配的适当检测范围的生物传感器。其次，不同传感器的表达水平和定位模式不同。例如，基于PKA的cAMP传感器由于与A激酶锚定蛋白(AKAP)的相互作用，在心肌细胞中显示出明显的亚细胞定位。5此外，生物传感器可以针对细胞中的不同亚细胞位置，以检查特定的cAMP池和信号微域，7而扩散指示器更适合于可视化全球cAMP的变化。第三，传感器的动态范围指的是荧光与…的差异
Lisa M. DiPilato, Jin Zhang ß-adrenergic receptor (ß-AR) signaling in cardiac myocytes influences contractile and relaxation states in the heart. Classically, following hormone activation, ß-AR preferentially couples with Gs, which in turn activates adenylyl cyclase and cAMP production. The predominant effector of cAMP, cAMP-dependent protein kinase (PKA), then phosphorylates many proteins important for cardiac function such as L-type calcium channels and phospholamban in the sarcoplasmic reticulum membrane. However, studies in myocytes have shown not all receptors that transduce signals via cAMP generate the same functional effects. 1 These observations have led to the concept of cAMP compartmentation, which attributes the functional specificity and differential regulation to intricate spatial and temporal control of signaling molecules in the cAMP pathway. In this issue of Circulation Research, Nikolaev et al2 take another look at the differentially regulated ß1 and ß2-adrenergic signaling by using FRET-based cAMP imaging in adult cardiomyocytes, a method that is well suited for revealing the spatiotemporal complexity in cAMP signaling. Fluorescence ratio imaging of cAMP in living cells was first introduced 15 years ago with the development of a bimolecular indicator using fluorescent dye-tagged regulatory and catalytic subunits of PKA. 3 This class of cAMP indicators has evolved to become genetically encodable with the use of green fluorescent protein (GFP) variants4, 5 and in recent years, unimolecular with the use of single cAMP binding domain-containing proteins and protein fragments. 6–8 Nikolaev et al add to our molecular toolbox with the development of yet another FRET-based cAMP sensor with new characteristics. This sensor uses the cAMP binding domain of the hyperpolarization-activated cyclic nucleotide gated channel 2 (HCN2) as opposed to the binding domains from PKA or exchange proteins directly activated by cAMP (Epac), used in other FRET sensors. The resulting biosensor, HCN2-camps, maintains a high sensitivity for cAMP but does not appear to saturate at physiological cAMP concentrations in adult cardiomyocytes, thus being able to report agonistinduced changes in cAMP. Another important characteristic of the HCN2-camps is its uniform distribution in cardiomyocytes that allows for the measurement of cAMP dynamics throughout the entire cell without bias from sensor localization.A significant point illustrated by design and application of this new cAMP biosensor is the importance of being able to use a tailored cAMP biosensor that is most suited for a particular experiment, cell system, or functional study. Several key characteristics are worth considering when designing or choosing a cAMP sensor. First, the binding property of the sensor, such as binding affinity for cAMP, is among the most important characteristics. Different cell types have varying basal levels of cAMP and different capacity of cAMP production and degradation, thus requiring use of biosensors that have appropriate detection ranges that match concentrations of endogenous cAMP. Secondly, expression levels and localization patterns vary between different sensors. For example, a PKA-based cAMP sensor showed distinct subcellular localization within cardiomyocytes because of interaction with A kinase anchoring proteins (AKAPs). 5 Furthermore, biosensors can be targeted to various subcellular sites in the cell for examining specific pools of cAMP and signaling microdomains, 7 whereas a diffusible indicator is more suited for visualizing global cAMP changes. Thirdly, the dynamic range of a sensor refers to the difference in fluorescence …