Spooner et al 2025 Dataset (10.1073/pnas.2413308122)

Abstract The L-type Ca2+ channel (CaV1.2) is essential for cardiac excitation–contraction coupling. To contribute to the inward Ca2+ flux that drives Ca2+-induced-Ca2+-release, CaV1.2 channels must be expressed on the sarcolemma; thus the regulatory mechanisms that tune CaV1.2 expression to meet contractile demand are an emerging area of research. A ubiquitously expressed protein called 14-3-3 has been proposed to affect Ca2+ channel trafficking in nonmyocytes; however, whether 14-3-3 has similar effects on CaV1.2 in cardiomyocytes is unknown. 14-3-3 preferentially binds phospho-serine/threonine residues to affect many cellular processes and is known to regulate cardiac ion channels including NaV1.5 and the human ether-à-go-go–related gene (hERG) potassium channel. Altered 14-3-3 expression and function have been implicated in cardiac pathologies including hypertrophy. Accordingly, we tested the hypothesis that 14-3-3 interacts with CaV1.2 in a phosphorylation-dependent manner and regulates cardiac CaV1.2 trafficking and recycling. Confocal imaging, proximity ligation assays, superresolution imaging, and coimmunoprecipitation revealed a population of 14-3-3 colocalized and closely associated with CaV1.2. The degree of 14-3-3/CaV1.2 colocalization increased upon stimulation of β-adrenergic receptors with isoproterenol. Notably, only the 14-3-3-associated CaV1.2 population displayed increased cluster size with isoproterenol, revealing a role for 14-3-3 as a nucleation factor that directs CaV1.2 superclustering. Isoproterenol-stimulated augmentation of sarcolemmal CaV1.2 expression, Ca2+ currents, and Ca2+ transients in ventricular myocytes were strengthened by 14-3-3 overexpression and attenuated by 14-3-3 inhibition. These data support a model where 14-3-3 interacts with CaV1.2 in a phosphorylation-dependent manner to promote enhanced trafficking/recycling, clustering, and activity during β-adrenergic stimulation.摘要 L型钙通道（L-type Ca²+ channel, CaV1.2）是心脏兴奋-收缩耦联的关键分子。若要参与驱动钙诱导钙释放的内向钙流，CaV1.2通道必须表达于肌膜之上，因此，调控CaV1.2表达以匹配心肌收缩需求的机制是当前新兴的研究方向。此前有研究提出，一种名为14-3-3的泛在表达蛋白可影响非心肌细胞中的钙通道膜转运，但14-3-3是否对心肌细胞中的CaV1.2具有类似调控作用尚不清楚。14-3-3可优先结合磷酸化丝氨酸/苏氨酸残基，进而调控诸多细胞过程，且已知其可调控心脏离子通道，包括电压门控钠通道1.5（NaV1.5）以及人类ether-à-go-go相关基因（human ether-à-go-go–related gene, hERG）钾通道。14-3-3的表达与功能异常已被证实与心肌肥厚等心脏病理过程相关。据此，我们验证了如下假说：14-3-3以磷酸化依赖的方式与CaV1.2相互作用，并调控心肌细胞中CaV1.2的膜转运与循环再利用。共聚焦成像、邻近连接分析法、超分辨率成像以及免疫共沉淀实验结果显示，存在一部分14-3-3蛋白与CaV1.2存在共定位并紧密关联。在用异丙肾上腺素刺激β肾上腺素能受体后，14-3-3与CaV1.2的共定位程度显著升高。值得注意的是，仅与14-3-3结合的CaV1.2群体在异丙肾上腺素处理后出现簇集尺寸增大，这表明14-3-3可作为成核因子，引导CaV1.2形成超级簇集。异丙肾上腺素刺激可增强心室肌细胞的肌膜CaV1.2表达、钙电流及钙瞬变，该效应可通过14-3-3过表达进一步强化，而经14-3-3抑制后则会减弱。上述实验数据支持如下模型：14-3-3以磷酸化依赖的方式与CaV1.2相互作用，从而在β肾上腺素能刺激过程中，促进CaV1.2的膜转运/循环再利用、簇集及通道活性。