Adenoviral-mediated expression of dihydropyridine-insensitive L-type calcium channels in cardiac ventricular myocytes and fibroblasts.
Adenoviral-mediated expression of dihydropyridine-insensitive L-type calcium channels in cardiac ventricular myocytes and fibroblasts.
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腺病毒介导的心室肌细胞和成纤维细胞中二氢吡啶不敏感的 L 型钙通道的表达。
DOI：
10.1016/j.ejphar.2007.02.049
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发表时间：
2007
影响因子：
5
通讯作者：
Hockerman,GregoryH
中科院分区：
文献类型：
--
作者：
Walsh,KennethB;Zhang,Jining;Fuseler,JohnW;Hilliard,Nathan;Hockerman,GregoryH
心脏电压门控Ca 2+通道调节细胞内Ca 2+浓度，因此对于肌肉收缩、第二信使激活、基因表达和电信号传导是必需的。作为第一步，在访问的结构与功能特性的L型钙离子通道在心脏中，我们已经表达了二氢吡啶（DHP）不敏感的CaV1.2通道在大鼠心室肌细胞和成纤维细胞。分离和培养后，用表达LacZ或含有双突变（T1039 Y和Q1043 M）的突变CaV1.2通道（CaV1.2DHPi）的腺病毒感染细胞。这种突变使通道对中性DHP化合物如尼索地平不敏感。尼索地平以浓度依赖性方式抑制对照心肌细胞的全细胞L型钙电流（伊卡），IC 50为66 nM，在250 nM时完全阻断。相反，500 nM尼索地平仅抑制AdCaV 1.2DHPi感染的ICain细胞的35%，而50 μM地尔硫卓完全阻断。为了研究CaV 1.2DHPi的分离，将用AdCaV 1.2DHPi感染的心肌细胞与尼索地平孵育。在这种条件下，细胞表达了大的伊卡（12 pA/pF），并显示Ca 2+瞬变场刺激期间。此外，加入2 μM forskolin和100 μM 3-异丁基-1-甲基黄嘌呤（IBMX）刺激蛋白激酶A，可显著增加AdCaV 1.2DHPi感染细胞中的IBin。AdCaV1.2DHPi感染的心肌成纤维细胞也可检测到Cd ~（2+）敏感的IBA。因此，CaV1.2DHPi的表达将为研究心肌细胞和成纤维细胞功能提供重要工具。
Cardiac voltage-gated Ca2+channels regulate the intracellular Ca2+concentration and are therefore essential for muscle contraction, second messenger activation, gene expression and electrical signaling. As a first step in accessing the structural versus functional properties of the L-type Ca2+channel in the heart, we have expressed a dihydropyridine (DHP)-insensitive CaV1.2 channel in rat ventricular myocytes and fibroblasts. Following isolation and culture, cells were infected with adenovirus expressing either LacZ or a mutant CaV1.2 channel (CaV1.2DHPi) containing the double mutation (T1039Y & Q1043M). This mutation renders the channel insensitive to neutral DHP compounds such as nisoldipine. The whole-cell, L-type Ca2+current (ICa) measured in control myocytes was inhibited in a concentration-dependent manner by nisoldipine with an IC50of 66 nM and complete block at 250 nM. In contrast, ICain cells infected with AdCaV1.2DHPiwas inhibited by only 35% by 500 nM nisoldipine but completely blocked by 50 μM diltiazem. In order to study CaV1.2DHPiin isolation, myocytes infected with AdCaV1.2DHPiwere incubated with nisoldipine. Under this condition the cells expressed a large ICa(12 pA/pF) and displayed Ca2+transients during field stimulation. Furthermore, addition of 2 μM forskolin and 100 μM 3-isobutyl-1-methylxanthine (IBMX), to stimulate protein kinase A, strongly increased IBain the AdCaV1.2DHPi-infected cells. A Cd2+-sensitive IBawas also recorded in cardiac fibroblasts infected with AdCaV1.2DHPi. Thus, expression of CaV1.2DHPiwill provide an important tool in studies of cardiac myocyte and fibroblast function.