Phosphorylation and modulation of hyperpolarization-activated HCN4 channels by protein kinase A in the mouse sinoatrial node.
Phosphorylation and modulation of hyperpolarization-activated HCN4 channels by protein kinase A in the mouse sinoatrial node.
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DOI：
10.1085/jgp.201010488
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发表时间：
2010-09
期刊：
影响因子：
--
通讯作者：
Proenza C
中科院分区：
文献类型：
--
作者：
Liao Z;Lockhead D;Larson ED;Proenza C
交感神经系统通过激活β肾上腺素能受体和增加窦房结中肌细胞中的cAMP水平来增加心率。这种反应的分子基础尚不清楚;然而，心脏有趣电流（If）被认为是窦房肌细胞中cAMP信号传导的末端效应物之一。If由超极化激活的环核苷酸敏感性（HCN 4）通道产生，其可通过cAMP与通道C末端中的保守环核苷酸结合结构域的直接结合而增强。窦房结中If的β肾上腺素能调节被认为是通过这种直接结合机制发生的，不依赖于磷酸化。在这里，我们研究了cAMP激活的蛋白激酶（PKA）是否也可以调节窦房HCN 4通道。我们发现，PKA的抑制显着降低β肾上腺素能受体激动剂的能力，转移的电压依赖性的If在离体小鼠窦房肌细胞。PKA还将异源表达的HCN 4通道的激活电压依赖性转移到更正的电位。体外磷酸化实验和质谱分析表明PKA可以直接磷酸化HCN 4上至少13个位点，包括N端至少3个残基和C端至少10个残基。截短和丙氨酸取代的HCN 4通道的功能分析确定了PKA调节位点的远端C末端的HCN 4，这是所需的PKA调制的If。总的来说，这些数据表明，天然和表达的HCN 4通道可以通过PKA调节，并提高了这种机制可能有助于心率的交感神经调节的可能性。
The sympathetic nervous system increases heart rate by activating β adrenergic receptors and increasing cAMP levels in myocytes in the sinoatrial node. The molecular basis for this response is not well understood; however, the cardiac funny current (If) is thought to be among the end effectors for cAMP signaling in sinoatrial myocytes. If is produced by hyperpolarization-activated cyclic nucleotide–sensitive (HCN4) channels, which can be potentiated by direct binding of cAMP to a conserved cyclic nucleotide binding domain in the C terminus of the channels. β adrenergic regulation of If in the sinoatrial node is thought to occur via this direct binding mechanism, independent of phosphorylation. Here, we have investigated whether the cAMP-activated protein kinase (PKA) can also regulate sinoatrial HCN4 channels. We found that inhibition of PKA significantly reduced the ability of β adrenergic agonists to shift the voltage dependence of If in isolated sinoatrial myocytes from mice. PKA also shifted the voltage dependence of activation to more positive potentials for heterologously expressed HCN4 channels. In vitro phosphorylation assays and mass spectrometry revealed that PKA can directly phosphorylate at least 13 sites on HCN4, including at least three residues in the N terminus and at least 10 in the C terminus. Functional analysis of truncated and alanine-substituted HCN4 channels identified a PKA regulatory site in the distal C terminus of HCN4, which is required for PKA modulation of If. Collectively, these data show that native and expressed HCN4 channels can be regulated by PKA, and raise the possibility that this mechanism could contribute to sympathetic regulation of heart rate.
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