title: Conformational Flip of Nonactivated HCN2 Channel Subunits Evoked by Cyclic Nucleotides.
abstract: Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetrameric proteins that evoke electrical rhythmicity in specialized neurons and cardiomyocytes. The channels are activated by hyperpolarizing voltage but are also receptors for the intracellular ligand adenosine-3',5'-cyclic monophosphate (cAMP) that enhances activation but is unable to activate the channels alone. Using fcAMP, a fluorescent derivative of cAMP, we analyzed the effect of ligand binding on HCN2 channels not preactivated by voltage. We identified a conformational flip of the channel as an intermediate state following the ligand binding and quantified it kinetically. Globally fitting the time courses of ligand binding and unbinding revealed modest cooperativity among the subunits in the conformational flip. The intensity of this cooperativity, however, was only moderate compared to channels preactivated by hyperpolarizing voltage. These data provide kinetic information about conformational changes proceeding in nonactivated HCN2 channels when cAMP binds. Moreover, our approach bears potential for analyzing the function of any other membrane receptor if a potent fluorescent ligand is available.
authors.affiliation: Institut für Physiologie II, Universitätsklinikum Jena, Jena, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Thon
authors.affiliation: Fachhochschule Schmalkalden, Fakultät Elektrotechnik, Blechhammer, Schmalkalden, Germany.
authors.forename: Eckhard
authors.initials: E
authors.lastname: Schulz
authors.affiliation: Institut für Physiologie II, Universitätsklinikum Jena, Jena, Germany.
authors.forename: Jana
authors.initials: J
authors.lastname: Kusch
authors.affiliation: Institut für Physiologie II, Universitätsklinikum Jena, Jena, Germany. Electronic address: klaus.benndorf@mti.uni-jena.de.
authors.forename: Klaus
authors.initials: K
authors.lastname: Benndorf
mesh_terms: D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D005260:Female; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels / Q000737:chemistry* / Q000378:metabolism; D015640:Ion Channel Gating / Q000187:drug effects; D007700:Kinetics; D009712:Nucleotides, Cyclic / Q000494:pharmacology*; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D009712:Nucleotides, Cyclic; D021122:Protein Subunits; D000242:Cyclic AMP