title: Protein kinase C activation inhibits rat and human hyperpolarization activated cyclic nucleotide gated channel (HCN)1--mediated current in mammalian cells.
abstract: OBJECTIVE
  Hyperpolarization activated cyclic nucleotide gated 1 (HCN1) channels determine neuronal excitability in several brain regions. In contrast to HCN2 and HCN4, HCN1 is less sensitive to cAMP and the number of other known modulators is limited. One of those, the protein kinase C (PKC), showed opposing effects on mouse HCN1 channels expressed in Xenopus oocytes.
  
  
  METHODS
  In order to study PKC effects on HCN1 mediated currents in a mammalian environment we expressed rat HCN1 or human HCN1 in human embryonic kidney (HEK293) cells and rat HCN1 in murine neuroblastoma (N1E-115) cells. We recorded the resulting Ih before and during the application of the membrane permeable non-metabolizable PKC-activator 4βPMA in cell-attached mode of the patch-clamp technique, leaving the intracellular environment intact.
  
  
  RESULTS
  4βPMA reduced maximal HCN1 mediated currents to about 60-70 % and slowed its activation, but left its voltage sensitivity unchanged. The effect was neither due to species-related differences nor restricted to HEK293 cells, because it was comparable for human and rat HCN1 in HEK293 and for rat HCN1 in N1E-115 cells. However, pre-treatment with the PKC blocker GF109203X abolished 4βPMA induced Ih changes. Disrupting the intracellular environment by recording in whole-cell mode drastically reduced the 4βPMA effect.
  
  
  CONCLUSIONS
  PKC activation reduces and slows Ih in non-neuronal and neuronal mammalian cells transfected with rat or human HCN1 if the intracellular content remains intact.
authors.affiliation: Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité-Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Olivia
authors.initials: O
authors.lastname: Reetz
authors.forename: Ulf
authors.initials: U
authors.lastname: Strauss
mesh_terms: D000818:Animals; D002460:Cell Line; D057809:HEK293 Cells; D006801:Humans; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D018408:Patch-Clamp Techniques; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007211:Indoles; D008301:Maleimides; D047428:Protein Kinase Inhibitors; C020922:4-O-methyl-12-O-tetradecanoylphorbol 13-acetate; D011493:Protein Kinase C; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate