Phosphorylation of serine 1928 in the distal C-terminal domain of cardiac Cav1.2 channels during β1-adrenergic regulation
Phosphorylation of serine 1928 in the distal C-terminal domain of cardiac Cav1.2 channels during β1-adrenergic regulation
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DOI：
10.1073/pnas.0607294103
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发表时间：
2006-10-31
影响因子：
11.1
通讯作者：
Catterall, William A.
中科院分区：
文献类型：
--
作者：
Hulme, Joanne T.;Westenbroek, Ruth E.;Catterall, William A.
在战斗或逃跑反应中，交感神经系统释放的肾上腺素和去甲肾上腺素增加心脏钙通道传导的L型钙电流，从而有助于增强心功能。β-肾上腺素能受体的激活通过cAMP依赖的蛋白激酶(PKA)的磷酸化来增加通道的活性，PKA通过A-激酶锚定蛋白(AKAP15)与a1亚基的远端C-末端结构域相连。在这里，我们使用一种磷酸特异性抗体来测量分离的大鼠心室肌细胞对β-肾上腺素能受体刺激的反应中S1928的磷酸化。异丙肾上腺素处理增加了S1928在远端C-末端结构域的磷酸化，腺苷环化酶的直接激活剂Forsklin也观察到类似的增加，证实cAMP和PKA是起作用的。选择性β1和β2肾上腺素能拮抗剂可使磷酸化的增加分别减少79%和42%，两种药物均可完全阻断这一作用。相反，在1，2-二(2-氨基苯氧基)乙烷-N‘，N’-四乙酸(BAPTA)-乙酰氧甲酯存在下用这些试剂缓冲细胞内钙离子的处理只会导致S1928的β1刺激的磷酸化。细胞内BAPTA的全细胞膜片钳研究表明，钙电流增加的98%可归因于β1-肾上腺素能受体。因此，β-肾上腺素能刺激通过β1-和β2-肾上腺素能受体通路导致完整的心室肌细胞Cav1.2 ALL亚基上S1928的磷酸化，但132依赖的磷酸化增加依赖于细胞内钙的升高，并不有助于基础钙水平下的全细胞钙电流的调节。我们的结果将S1928的磷酸化与BAPTA存在下完整的心室肌细胞中β1肾上腺素能功能上调的心肌钙通道联系在一起。
During the fight-or-flight response, epinephrine and norepinephrine released by the sympathetic nervous system increase L-type calcium currents conducted by Ca(V)1.2a channels in the heart, which contributes to enhanced cardiac performance. Activation of beta-adrenergic receptors increases channel activity via phosphorylation by cAMP-dependent protein kinase (PKA) tethered to the distal C-terminal domain of the a, subunit via an A-kinase anchoring protein (AKAP15). Here we measure phosphorylation of S1928 in dissociated rat ventriculair myocytes in response to beta-adrenergic receptor stimulation by using a phosphospecific antibody. Isoproterenol treatment increased phosphorylation of S1928 in the distal C-terminal domain, and a similar increase was observed with a direct activator of adenylyl cyclase, forskolin, confirming that the cAMP and PKA are responsible. Pretreatment with selective beta 1-and beta 2-adrenergic antagonists reduced the increase in phosphorylation by 79% and 42%, respectively, and pretreatment with both agents completely blocked it. In contrast, treatment with these agents in the presence of 1,2-bis(2-aminophenoxy)ethane-N', N'-tetraacetic acid (BAPTA)-acetoxymethyl ester to buffer intracellular calcium results in only beta 1-stimulated phosphorylation of S1928. Whole-cell patch clamp studies with intracellular BAPTA demonstrated that 98% of the increase in calcium current was attributable to beta 1-adrenergic receptors. Thus, beta-adrenergic stimulation results in phosphorylation of S1928 on the Cav1.2 all subunit in intact ventricular myocytes via both beta 1- and beta 2-adrenergic receptor pathways, but the 132-dependent increase in phosphorylation depends on elevated intracellular calcium and does not contribute to regulation of whole-cell calcium currents at basal calcium levels. our results correlate phosphorylation of S1928 with beta 1-adrenergic functional up-regulation of cardiac calcium channels in the presence of BAPTA in intact ventricular myocytes.