title: The cGMP-dependent protein kinase II Is an inhibitory modulator of the hyperpolarization-activated HCN2 channel.
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.
authors.affiliation: Munich Center for Integrated Protein Science CIPSM, Ludwig-Maximilians-Universität München, München, Germany.
authors.forename: Verena
authors.initials: V
authors.lastname: Hammelmann
authors.forename: Xiangang
authors.initials: X
authors.lastname: Zong
authors.forename: Franz
authors.initials: F
authors.lastname: Hofmann
authors.forename: Stylianos
authors.initials: S
authors.lastname: Michalakis
authors.forename: Martin
authors.initials: M
authors.lastname: Biel
mesh_terms: D000818:Animals; D001921:Brain / Q000166:cytology / Q000378:metabolism; D062967:Cyclic GMP-Dependent Protein Kinase Type II; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D015640:Ion Channel Gating; D007473:Ion Channels / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D009474:Neurons / Q000378:metabolism; D010766:Phosphorylation; D015221:Potassium Channels; D011485:Protein Binding
chemical_list: C578710:HCN2 protein, human; C578711:Hcn2 protein, mouse; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D015221:Potassium Channels; D062967:Cyclic GMP-Dependent Protein Kinase Type II; D017869:Cyclic GMP-Dependent Protein Kinases; C569729:PRKG2 protein, human; C569730:Prkg2 protein, mouse