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 Chen,Martin Biel

doi:10.1074/jbc.m506544200

Abstract

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.

Highlights

Identified only recently, the family of hyperpolarizationactivated cyclic nucleotide-gated (HCN)3 channels has generated great interest because it represents the molecular correlate of the hyperpolarization-activated cation current, termed Ih
Inhibition of Src Slows Down Kinetics of Expressed HCN2 Channel—We investigated the effect of Src on HCN2 channels expressed in HEK293 cells
Inhibition of Src by pharmacological blockers as well as transfection with a catalytically inactive Src mutant profoundly slowed down activation kinetics of native and expressed Ih channels indicating that the phosphorylation/dephosphorylation state determines channel kinetics

Summary

Expression Vectors

Yeast Two-hybrid (YTH) Assay—C-terminal portions of murine HCN2 (mHCN2) were subcloned into plasmid pEG202 (Clontech) inframe with the LexA DNA binding domain to yield the following bait proteins (see Fig. 4A): CT, residues 449 – 863; L, residues 449 –522; BD, residues 523– 646; LϩBD, residues 449 – 646; dC, residues 649 – 863; L-␤8, residues 449 – 607; CЈ-BD, residues 487– 646. Bacterial GST Fusion Proteins—C-terminal fragments of mHCN2 were subcloned into the EcoRI/BamHI site of pGEX2T (Amersham Biosciences) to generate the following GST-tagged proteins: CT, residues 448 – 863; L, residues 448 –520; BD, residues 521– 644; LϩBD, residues 448 – 644; dC, residues 645– 863. PEG202 and pJG4 –5 fusion plasmids, together with the lacZ reporter plasmid pSH18 –34 (BD Biosciences, Clontech) were transformed into yeast by the lithium acetate method.

Expression and Purification of GST Fusion Proteins

In Vitro Kinase Assay

Cell Culture and Heterologous Expression

RESULTS

TABLE ONE

DISCUSSION