title: State-dependent cAMP binding to functioning HCN channels studied by patch-clamp fluorometry.
abstract: One major goal of ion channel research is to delineate the molecular events from the detection of the stimuli to the movement of channel gates. For ligand-gated channels, it is challenging to separate ligand binding from channel gating. Here we studied the cyclic adenosine monophosphate (cAMP)-dependent gating in hyperpolarization-activated cAMP-regulated (HCN) channel by simultaneously recording channel opening and ligand binding, using the patch-clamp fluorometry technique with a unique fluorescent cAMP analog that fluoresces strongly in the hydrophobic binding pocket and exerts regulatory effects on HCN channels similar to those imposed by cAMP. Corresponding to voltage-dependent channel activation, we observed a robust, close-to-threefold increase in ligand binding, which was more pronounced at subsaturating ligand concentrations than higher concentrations. This observation supported the cyclic allosteric models and indicated that protein allostery can be implemented through differentiating ligand binding affinities between resting and active states. The kinetics of ligand binding largely matched channel activation. However, during channel deactivation, ligand unbinding was slower than channel closing, suggesting a delayed response to membrane potential by the ligand binding machinery. Our results provide what we believe to be new insights into the cAMP-dependent gating in HCN channel and the interpretation of protein allostery for general ligand-gated channels and receptors.
authors.affiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, Virginia, USA.
authors.forename: Shengjun
authors.initials: S
authors.lastname: Wu
authors.forename: Zhanna V
authors.initials: ZV
authors.lastname: Vysotskaya
authors.forename: Xinping
authors.initials: X
authors.lastname: Xu
authors.forename: Changan
authors.initials: C
authors.lastname: Xie
authors.forename: Qinglian
authors.initials: Q
authors.lastname: Liu
authors.forename: Lei
authors.initials: L
authors.lastname: Zhou
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism* / Q000494:pharmacology; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000737:chemistry / Q000378:metabolism*; D005470:Fluorometry; D057927:Hydrophobic and Hydrophilic Interactions; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D015640:Ion Channel Gating / Q000187:drug effects; D007700:Kinetics; D008024:Ligands; D051379:Mice; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014867:Water / Q000737:chemistry
chemical_list: D054815:Cyclic Nucleotide-Gated Cation Channels; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D008024:Ligands; D015221:Potassium Channels; D014867:Water; D000242:Cyclic AMP