Molecular basis of b-adrenergic regulation of cardiac calcium current

Increased cardiac contractility during fight-or-flight response is caused by b-adrenergic augmentation of CaV1.2 channels. It is assumed that this iconic regulation involves phosphorylation of CaV1.2 a1/b-subunits. In transgenic murine hearts expressing fully PKA phosphorylation-deficient mutant a1C/b, this regulation persists, however, suggesting involvement of extra-channel factors. We here show that PKA up-regulates cardiac CaV1.2 channels by phosphorylating the small G-protein Rad, relieving constitutive inhibition of CaV1.2. Peroxidase-catalyzed labeling in mice hearts expressing ascorbate peroxidase conjugated-a1C or b2b with multiplexed quantitative proteomics allowed tracking of thousands of proteins in proximity of CaV1.2.

战或逃反应（fight-or-flight response）中心肌收缩力的增强，由β肾上腺素能（b-adrenergic）对CaV1.2通道（CaV1.2 channels）的增强调控所介导。此前学界认为这一经典调控过程涉及CaV1.2通道α1/β亚基的磷酸化修饰。然而在表达完全蛋白激酶A（PKA）磷酸化缺陷型α1C/β突变体的转基因小鼠心脏中，该调控过程依然存在，这提示调控机制中存在通道以外的因子。本研究证实，PKA通过磷酸化小G蛋白Rad（small G-protein Rad），解除CaV1.2通道的组成型抑制，从而实现对心肌CaV1.2通道的上调调控。通过在表达抗坏血酸过氧化物酶（ascorbate peroxidase）融合α1C或β2b亚基的小鼠心脏中开展过氧化物酶催化标记结合多重定量蛋白质组学（multiplexed quantitative proteomics）实验，我们得以追踪CaV1.2通道邻近区域的数千种蛋白质。