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J. Biol. Chem., Vol. 280, Issue 40, 34224-34232, October 7, 2005

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A Novel Mechanism of Modulation of Hyperpolarization-activated Cyclic Nucleotide-gated Channels by Src Kinase^*

Xiangang Zong, Christian Eckert, Haixin Yuan, Christian Wahl-Schott, Heike Abicht, Longfou Fang, Rongxia Li, Pavel Mistrik, Andrea Gerstner, Barbara Much, Ludwig Baumann, Stylianos Michalakis, Rong Zeng, Zhengjun Chen1, and Martin Biel2

From the Department Pharmazie, Pharmakologie für Naturwissenschaften, Ludwig-Maximilians Universität München, Butenandtstrasse 7, 81377 München and the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China

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 (Tyr⁴⁷⁶ 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.

Received for publication, June 16, 2005 , and in revised form, August 2, 2005.

^* This work was supported by grants from Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ To whom correspondence may be addressed: Inst. of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Rd., Shanghai 200031, China. Tel.: 86-21-5492-1081; E-mail: wchenzj{at}sunm.shcnc.ac.cn.

² To whom correspondence may be addressed: Dept. Pharmazie, Pharmakologie für Naturwissenschaften, Ludwig-Maximilians-Universität München, Butenandtstr. 7, 81377 München, Germany. Tel.: 49-89-2180-77327; Fax: 49-89-2180-77326; E-mail: mbiel{at}cup.uni-muenchen.de.

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