A novel mechanism of modulation of hyperpolarization-activated cyclic nucleotide-gated channels by Src kinase
A novel mechanism of modulation of hyperpolarization-activated cyclic nucleotide-gated channels by Src kinase
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DOI：
10.1074/jbc.m506544200
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发表时间：
2005-10-07
影响因子：
4.8
通讯作者：
Biel, M
中科院分区：
文献类型：
--
作者：
Zong, XG;Eckert, C;Biel, M
超极化激活的环核苷酸门控通道 (HCN1-4) 在细胞兴奋性的调节中发挥着至关重要的作用。重要的是，它们有助于大脑和心脏的自发节律活动。 HCN 通道主要由膜超极化和 cAMP 结合激活。在这里，我们将 Src 激酶的酪氨酸磷酸化确定为影响通道门控的另一种机制。特定阻断剂对 Src 的抑制减慢了天然和异源表达的 HCN 通道的激活动力学。在显性失活 Src 突变体共转染的细胞中观察到对 HCN 通道激活的相同影响。免疫沉淀证明 Src 结合天然和异源表达的 HCN2 并使其磷酸化。 Src 通过其 SH3 结构域与包含部分 C 连接子和环核苷酸结合结构域的 HCN2 序列相互作用。我们在 HCN 通道的 C 连接子（HCN2 中的 Tyr(476)）中发现了一个高度保守的酪氨酸残基，该残基可通过 Src 进行调节。在 HCN2 或 HCN4 中用苯丙氨酸替换该酪氨酸消除了对 Src 抑制剂的敏感性。质谱分析证实 Tyr476 被 Src 磷酸化。我们的结果对 HCN 通道门控具有功能意义。此外，他们表明酪氨酸磷酸化有助于体内 HCN 通道活性的微调。
Hyperpolarization-activated cyclic nucleotide-gated channels (HCN1-4) play a crucial role in the regulation of cell excitability. Importantly, they contribute to spontaneous rhythmic activity in brain and heart. HCN channels are principally activated by membrane hyperpolarization and binding of cAMP. Here, we identify tyrosine phosphorylation by Src kinase as another mechanism affecting channel gating. Inhibition of Src by specific blockers slowed down activation kinetics of native and heterologously expressed HCN channels. The same effect on HCN channel activation was observed in cells cotransfected with a dominant-negative Src mutant. Immunoprecipitation demonstrated that Src binds to and phosphorylates native and heterologously expressed HCN2. Src interacts via its SH3 domain with a sequence of HCN2 encompassing part of the C-linker and the cyclic nucleotide binding domain. We identified a highly conserved tyrosine residue in the C-linker of HCN channels ( Tyr(476) in HCN2) that confers modulation by Src. Replacement of this tyrosine by phenylalanine in HCN2 or HCN4 abolished sensitivity to Src inhibitors. Mass spectrometry confirmed that Tyr476 is phosphorylated by Src. Our results have functional implications for HCN channel gating. Furthermore, they indicate that tyrosine phosphorylation contributes in vivo to the fine tuning of HCN channel activity.