Yang Lin, Katchman Alexander, Weinberg Richard L, Abrams Jeffrey, Samad Tahmina, Wan Elaine, Pitt Geoffrey S, Marx Steven O
From the Division of Cardiology, Departments of Medicine and Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York 10032 and.
the Division of Cardiology, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
J Biol Chem. 2015 Jan 23;290(4):2166-74. doi: 10.1074/jbc.M114.602508. Epub 2014 Dec 11.
Voltage-gated Ca(2+) channels play a key role in initiating muscle excitation-contraction coupling, neurotransmitter release, gene expression, and hormone secretion. The association of CaV1.2 with a supramolecular complex impacts trafficking, localization, turnover, and, most importantly, multifaceted regulation of its function in the heart. Several studies hint at an important role for the C terminus of the α1C subunit as a hub for multidimensional regulation of CaV1.2 channel trafficking and function. Recent studies have demonstrated an important role for the four-residue PDZ binding motif at the C terminus of α1C in interacting with scaffold proteins containing PDZ domains, in the subcellular localization of CaV1.2 in neurons, and in the efficient signaling to cAMP-response element-binding protein in neurons. However, the role of the α1C PDZ ligand domain in the heart is not known. To determine whether the α1C PDZ motif is critical for CaV1.2 trafficking and function in cardiomyocytes, we generated transgenic mice with inducible expression of an N-terminal FLAG epitope-tagged dihydropyridine-resistant α1C with the PDZ motif deleted (ΔPDZ). These mice were crossed with α-myosin heavy chain reverse transcriptional transactivator transgenic mice, and the double-transgenic mice were fed doxycycline. The ΔPDZ channels expressed, trafficked to the membrane, and supported robust excitation-contraction coupling in the presence of nisoldipine, a dihydropyridine Ca(2+) channel blocker, providing functional evidence that they appropriately target to dyads. The ΔPDZ Ca(2+) channels were appropriately regulated by isoproterenol and forskolin. These data indicate that the α1C PDZ motif is not required for surface trafficking, localization to the dyad, or adrenergic stimulation of CaV1.2 in adult cardiomyocytes.
电压门控性Ca(2+)通道在引发肌肉兴奋-收缩偶联、神经递质释放、基因表达和激素分泌过程中发挥关键作用。CaV1.2与超分子复合物的结合会影响其运输、定位、更新,最重要的是影响其在心脏中功能的多方面调节。多项研究表明,α1C亚基的C末端作为CaV1.2通道运输和功能多维调节的枢纽发挥重要作用。最近的研究表明,α1C C末端的四残基PDZ结合基序在与含PDZ结构域的支架蛋白相互作用、CaV1.2在神经元中的亚细胞定位以及向神经元中cAMP反应元件结合蛋白的有效信号传导中发挥重要作用。然而,α1C PDZ配体结构域在心脏中的作用尚不清楚。为了确定α1C PDZ基序对心肌细胞中CaV1.2的运输和功能是否至关重要,我们构建了可诱导表达N末端带有FLAG表位标签且缺失PDZ基序(ΔPDZ)的抗二氢吡啶α1C的转基因小鼠。将这些小鼠与α-肌球蛋白重链逆转录反式激活因子转基因小鼠杂交,并给双转基因小鼠喂食强力霉素。在二氢吡啶Ca(2+)通道阻滞剂尼索地平存在的情况下,ΔPDZ通道表达、运输到细胞膜并支持强大的兴奋-收缩偶联,提供了它们能正确靶向二元体的功能证据。ΔPDZ Ca(2+)通道受到异丙肾上腺素和福斯可林的适当调节。这些数据表明,α1C PDZ基序对于成年心肌细胞中CaV1.2的表面运输、定位到二元体或肾上腺素能刺激并非必需。