Division of Cardiology, Department of Medicine (A.P., J.K., J.A.H., A.N.K., S.I.Z., B.-x.C., L.Y., R.L., S.L., G.L., D.R., X.L., V.T., S.O.M.), Columbia University, Vagelos College of Physicians and Surgeons, New York, NY.
Department of Physiology and Cellular Biophysics (A.P., J.D., P.J.d.R.M., H.M.C., O.C., M.B.-J.), Columbia University, Vagelos College of Physicians and Surgeons, New York, NY.
Circ Res. 2021 Jan 8;128(1):76-88. doi: 10.1161/CIRCRESAHA.120.317839. Epub 2020 Oct 22.
Changing activity of cardiac Ca1.2 channels under basal conditions, during sympathetic activation, and in heart failure is a major determinant of cardiac physiology and pathophysiology. Although cardiac Ca1.2 channels are prominently upregulated via activation of PKA (protein kinase A), essential molecular details remained stubbornly enigmatic.
The primary goal of this study was to determine how various factors converging at the Ca1.2 I-II loop interact to regulate channel activity under basal conditions, during β-adrenergic stimulation, and in heart failure.
We generated transgenic mice with expression of Ca1.2 α subunits with (1) mutations ablating interaction between α and β-subunits, (2) flexibility-inducing polyglycine substitutions in the I-II loop (GGG-α), or (3) introduction of the alternatively spliced 25-amino acid exon 9* mimicking a splice variant of α upregulated in the hypertrophied heart. Introducing 3 glycine residues that disrupt a rigid IS6-α-interaction domain helix markedly reduced basal open probability despite intact binding of Caβ to α I-II loop and eliminated β-adrenergic agonist stimulation of Ca1.2 current. In contrast, introduction of the exon 9* splice variant in the α I-II loop, which is increased in ventricles of patients with end-stage heart failure, increased basal open probability but did not attenuate stimulatory response to β-adrenergic agonists when reconstituted heterologously with β and Rad or transgenically expressed in cardiomyocytes.
Ca channel activity is dynamically modulated under basal conditions, during β-adrenergic stimulation, and in heart failure by mechanisms converging at the α I-II loop. Caβ binding to α stabilizes an increased channel open probability gating mode by a mechanism that requires an intact rigid linker between the β-subunit binding site in the I-II loop and the channel pore. Release of Rad-mediated inhibition of Ca channel activity by β-adrenergic agonists/PKA also requires this rigid linker and β-binding to α.
心脏 Ca1.2 通道在基础状态下、交感神经激活时以及心力衰竭时的活性变化是心脏生理学和病理生理学的主要决定因素。虽然心脏 Ca1.2 通道通过激活蛋白激酶 A（protein kinase A，PKA）显著上调，但关键的分子细节仍然令人费解。
本研究的主要目标是确定在基础状态下、β-肾上腺素能刺激时和心力衰竭时，各种汇聚在 Ca1.2 I-II 环的因素如何相互作用来调节通道活性。
我们生成了表达 Ca1.2α 亚基的转基因小鼠，这些 Ca1.2α 亚基具有：（1）突变消除α 和β-亚基之间相互作用；（2）I-II 环中的柔性诱导多甘氨酸取代（GGG-α）；或（3）引入 25 个氨基酸的选择性剪接外显子 9*，模拟在肥大心脏中上调的α 剪接变体。引入 3 个甘氨酸残基破坏刚性 IS6-α 相互作用结构域螺旋，尽管 Caβ 与α I-II 环的结合完整，但显著降低了基础开放概率，并消除了β-肾上腺素能激动剂对 Ca1.2 电流的刺激。相比之下，在 I-II 环中引入外显子 9*剪接变体，该变体在终末期心力衰竭患者的心室中增加，增加了基础开放概率，但当与β 和 Rad 异源重建或在心肌细胞中转基因表达时，不能减弱对β-肾上腺素能激动剂的刺激反应。
在基础状态下、β-肾上腺素能刺激时和心力衰竭时，Ca 通道活性通过汇聚在α I-II 环的机制进行动态调节。Caβ 与α 的结合通过一种机制稳定增加的通道开放概率门控模式，该机制需要 I-II 环中β 亚基结合位点和通道孔之间的完整刚性连接。β-肾上腺素能激动剂/PKA 释放 Rad 介导的对 Ca 通道活性的抑制也需要这种刚性连接和β 与α 的结合。
