title: How subunits cooperate in cAMP-induced activation of homotetrameric HCN2 channels.
abstract: Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetrameric membrane proteins that generate electrical rhythmicity in specialized neurons and cardiomyocytes. The channels are primarily activated by voltage but are receptors as well, binding the intracellular ligand cyclic AMP. The molecular mechanism of channel activation is still unknown. Here we analyze the complex activation mechanism of homotetrameric HCN2 channels by confocal patch-clamp fluorometry and kinetically quantify all ligand binding steps and closed-open isomerizations of the intermediate states. For the binding affinity of the second, third and fourth ligand, our results suggest pronounced cooperativity in the sequence positive, negative and positive, respectively. This complex interaction of the subunits leads to a preferential stabilization of states with zero, two or four ligands and suggests a dimeric organization of the activation process: within the dimers the cooperativity is positive, whereas it is negative between the dimers.
authors.affiliation: Institut für Physiologie II, Universitätsklinikum Jena, Friedrich-Schiller-Universität Jena, Jena, Germany.
authors.forename: Jana
authors.initials: J
authors.lastname: Kusch
authors.forename: Susanne
authors.initials: S
authors.lastname: Thon
authors.forename: Eckhard
authors.initials: E
authors.lastname: Schulz
authors.forename: Christoph
authors.initials: C
authors.lastname: Biskup
authors.forename: Vasilica
authors.initials: V
authors.lastname: Nache
authors.forename: Thomas
authors.initials: T
authors.lastname: Zimmer
authors.forename: Reinhard
authors.initials: R
authors.lastname: Seifert
authors.forename: Frank
authors.initials: F
authors.lastname: Schwede
authors.forename: Klaus
authors.initials: K
authors.lastname: Benndorf
mesh_terms: D000818:Animals; D000242:Cyclic AMP / Q000494:pharmacology; D006801:Humans; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D032383:Myocytes, Cardiac; D009474:Neurons; D018408:Patch-Clamp Techniques; D015221:Potassium Channels; D011485:Protein Binding; D055503:Protein Multimerization; D055550:Protein Stability; D021122:Protein Subunits
chemical_list: C578710:HCN2 protein, human; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D008024:Ligands; D015221:Potassium Channels; D021122:Protein Subunits; D000242:Cyclic AMP