title: Voltage-dependent opening of HCN channels: Facilitation or inhibition by the phytoestrogen, genistein, is determined by the activation status of the cyclic nucleotide gating ring.
abstract: Investigation of the mechanistic bases and physiological importance of cAMP regulation of HCN channels has exploited an arginine to glutamate mutation in the nucleotide-binding fold, an approach critically dependent on the mutation selectively lowering the channel's nucleotide affinity. In apparent conflict with this, in intact Xenopus oocytes, HCN and HCN-RE channels exhibit qualitatively and quantitatively distinct responses to the tyrosine kinase inhibitor, genistein -- the estrogenic isoflavonoid strongly depolarizes the activation mid-point of HCN1-R538E, but not HCN1 channels (+9.8 mV + or - 0.9 versus +2.2 mV + or - 0.6) and hyperpolarizes gating of HCN2 (-4.8 mV + or - 1.0) but depolarizes gating of HCN2-R591E (+13.2 mV + or - 2.1). However, excised patch recording, X-ray crystallography and modeling reveal that this is not due to either a fundamental effect of the mutation on channel gating per se or of genistein acting as a mutation-sensitive partial agonist at the cAMP site. Rather, we find that genistein equivalently moves both HCN and HCN-RE channels closer to the open state (rendering the channels inherently easier to open but at a cost of decreasing the coupling energy of cAMP) and that the anomaly reflects a balance of these energetic effects with the isoform-specific inhibition of activation by the nucleotide gating ring and relief of this by endogenous cAMP. These findings have specific implications with regard to findings based on HCN-RE channels and kinase antagonists and general implications with respect to interpretation of drug effects in mutant channel backgrounds.
authors.affiliation: Department of Anesthesiology, Columbia University, New York, NY 10032, USA.
authors.forename: Anjali O
authors.initials: AO
authors.lastname: Rozario
authors.forename: Harma K
authors.initials: HK
authors.lastname: Turbendian
authors.forename: Keri J
authors.initials: KJ
authors.lastname: Fogle
authors.forename: Nelson B
authors.initials: NB
authors.lastname: Olivier
authors.forename: Gareth R
authors.initials: GR
authors.lastname: Tibbs
mesh_terms: D000818:Animals; D000242:Cyclic AMP / Q000502:physiology*; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000187:drug effects* / Q000235:genetics / Q000502:physiology*; D019833:Genistein / Q000494:pharmacology*; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D015221:Potassium Channels / Q000187:drug effects* / Q000235:genetics / Q000502:physiology*; D017434:Protein Structure, Tertiary
chemical_list: D054815:Cyclic Nucleotide-Gated Cation Channels; C578666:Hcn1 protein, mouse; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D015221:Potassium Channels; D019833:Genistein; D000242:Cyclic AMP