The cGMP-Dependent Protein Kinase II Is an Inhibitory Modulator of the Hyperpolarization-Activated HCN2 Channel

Verena Hammelmann,Xiangang Zong,Franz Hofmann,Stylianos Michalakis,Martin Biel

doi:10.1371/journal.pone.0017078

Abstract

Opening of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is facilitated by direct binding of cyclic nucleotides to a cyclic nucleotide-binding domain (CNBD) in the C-terminus. Here, we show for the first time that in the HCN2 channel cGMP can also exert an inhibitory effect on gating via cGMP-dependent protein kinase II (cGKII)-mediated phosphorylation. Using coimmunoprecipitation and immunohistochemistry we demonstrate that cGKII and HCN2 interact and colocalize with each other upon heterologous expression as well as in native mouse brain. We identify the proximal C-terminus of HCN2 as binding region of cGKII and show that cGKII phosphorylates HCN2 at a specific serine residue (S641) in the C-terminal end of the CNBD. The cGKII shifts the voltage-dependence of HCN2 activation to 2–5 mV more negative voltages and, hence, counteracts the stimulatory effect of cGMP on gating. The inhibitory cGMP effect can be either abolished by mutation of the phosphorylation site in HCN2 or by impairing the catalytic domain of cGKII. By contrast, the inhibitory effect is preserved in a HCN2 mutant carrying a CNBD deficient for cGMP binding. Our data suggest that bidirectional regulation of HCN2 gating by cGMP contributes to cellular fine-tuning of HCN channel activity.

Highlights

Hyperpolarization-activated cyclic nucleotide-gated channels (HCN1-4) comprise an ion channel family of four distinct members that pass a current termed Ih or If [1,2,3,4]
To further narrow down the region of HCN2 that interacts with cGMP-dependent protein kinase II (cGKII), Co-IPs with GFP-tagged cGKII and myc-proteins corresponding to the combined C-linker/cyclic-nucleotide binding domain (L+cyclic nucleotide-binding domain (CNBD), aa 443–647), the C-linker (L, aa 443–525) or the distal C-terminus of the HCN2 channel were performed (Fig. 1C, D)
We provide for the first time evidence for a bidirectional regulation of the HCN2 channel gating by cGMP (Fig. 5)

Summary

Introduction

Hyperpolarization-activated cyclic nucleotide-gated channels (HCN1-4) comprise an ion channel family of four distinct members that pass a current termed Ih or If [1,2,3,4]. HCN channels are set apart from other members of this family by their unusual activation process that includes principal gating by membrane hyperpolarization (conferred by a transmembrane voltage sensor) and modulation of the voltage-dependence of activation by binding of cyclic nucleotides to the C-terminal cyclic nucleotide-binding domain (CNBD). The latter process is of crucial relevance because it connects HCN channel activation to numerous signal transduction pathways that control cellular levels of cAMP or cGMP

Methods

Results

Conclusion