Zhou Peng, Zhao Yan-Ting, Guo Yun-Bo, Xu Shi-Ming, Bai Shu-Hua, Lakatta Edward G, Cheng Heping, Hao Xue-Mei, Wang Shi-Qiang
State Key Laboratory of Biomembrane and Membrane Biotechnology, College of Life Sciences, Peking University, Beijing 100871, China.
Proc Natl Acad Sci U S A. 2009 Oct 20;106(42):18028-33. doi: 10.1073/pnas.0906560106. Epub 2009 Oct 7.
As the most prototypical G protein-coupled receptor, beta-adrenergic receptor (betaAR) regulates the pace and strength of heart beating by enhancing and synchronizing L-type channel (LCC) Ca(2+) influx, which in turn elicits greater sarcoplasmic reticulum (SR) Ca(2+) release flux via ryanodine receptors (RyRs). However, whether and how betaAR-protein kinase A (PKA) signaling directly modulates RyR function remains elusive and highly controversial. By using unique single-channel Ca(2+) imaging technology, we measured the response of a single RyR Ca(2+) release unit, in the form of a Ca(2+) spark, to its native trigger, the Ca(2+) sparklet from a single LCC. We found that acute application of the selective betaAR agonist isoproterenol (1 microM, < or = 20 min) increased triggered spark amplitude in an LCC unitary current-independent manner. The increased ratio of Ca(2+) release flux underlying a Ca(2+) spark to SR Ca(2+) content indicated that betaAR stimulation helps to recruit additional RyRs in synchrony. Quantification of sparklet-spark kinetics showed that betaAR stimulation synchronized the stochastic latency and increased the fidelity (i.e., chance of hit) of LCC-RyR intermolecular signaling. The RyR modulation was independent of the increased SR Ca(2+) content. The PKA antagonists Rp-8-CPT-cAMP (100 microM) and H89 (10 microM) both eliminated these effects, indicating that betaAR acutely modulates RyR activation via the PKA pathway. These results demonstrate unequivocally that RyR activation by a single LCC is accelerated and synchronized during betaAR stimulation. This molecular mechanism of sympathetic regulation will permit more fundamental studies of altered betaAR effects in cardiovascular diseases.
作为最典型的G蛋白偶联受体，β-肾上腺素能受体（βAR）通过增强和同步L型钙通道（LCC）的Ca2+内流来调节心跳的速率和强度，进而通过雷诺丁受体（RyRs）引发更大的肌浆网（SR）Ca2+释放通量。然而，βAR-蛋白激酶A（PKA）信号传导是否以及如何直接调节RyR功能仍然不清楚，且极具争议。通过使用独特的单通道Ca2+成像技术，我们以Ca2+火花的形式测量了单个RyR Ca2+释放单元对其天然触发因素——单个LCC产生的Ca2+小火花的反应。我们发现，急性应用选择性βAR激动剂异丙肾上腺素（1 microM，≤20分钟）以不依赖LCC单位电流的方式增加了触发火花的幅度。Ca2+火花下的Ca2+释放通量与SR Ca2+含量的增加比例表明，βAR刺激有助于同步募集更多的RyRs。对小火花-火花动力学的量化显示，βAR刺激使随机潜伏期同步，并增加了LCC-RyR分子间信号传导的保真度（即命中几率）。RyR的调节与SR Ca2+含量的增加无关。PKA拮抗剂Rp-8-CPT-cAMP（100 microM）和H89（10 microM）均消除了这些效应，表明βAR通过PKA途径急性调节RyR的激活。这些结果明确表明，在βAR刺激期间，单个LCC对RyR的激活会加速并同步。这种交感神经调节的分子机制将有助于对心血管疾病中βAR效应改变进行更深入的基础研究。
