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Mapping ligand interactions with the hyperpolarization activated cyclic nucleotide modulated (HCN) ion channel binding domain using a soluble construct.
Biochemistry. 2007 Aug 21; 46(33):9417-31.B

Abstract

Hyperpolarization activated cyclic nucleotide modulated (HCN) ion channel currents are activated by hyperpolarization and modulated in response to changes in cytosolic adenosine 3',5'-cyclic monophosphate (cAMP) concentrations. A cDNA chimera combining the rat HCN2 cyclic nucleotide binding domain and the DNA binding domain of the cAMP receptor protein (CRP) from E. coli and the histidine tag (HCN2/CRP) was expressed and purified. The construct is capable of forming only non-ligand dependent dimers because the C-linker region of the channel is not present in this construct. The construct binds 8-[[2-[(fluoresceinylthioureido) amino] ethyl] thio] adenosine-3',5'-cyclic monophosphate (8-fluo cAMP) with a Kd of 0.299 microM as determined with a monomer binding model. The Ki values of 20 ligands related to cAMP were measured in order to determine the properties necessary for a ligand to bind to the HCN2 binding domain. This is the first report of cAMP and gunaosine 3',5'-cyclic monophosphate (cGMP) affinities to the HCN2 binding domain being equivalent, even though they modulate the channel with a 10-fold difference in K0.5. Furthermore, the array of ligands measured allows the preference rank order for each purine ring position to be determined: position 1, H > NH2 > O; position 2, NH2 > Cl > H > O; position 6, NH2 > Cl > H > O; and position 8, NH2 > Cl > H > O. Finally, the ability of HCN2/CRP to bind cyclic nucleotide pyrimidine rings at concentrations approximately 1.33 times greater than cAMP suggests that ribofuranose is key for binding.

Authors+Show Affiliations

Scott SP
Escuela de Medicine, Tec de Monterrey, Edificio CITES 3er piso, Area de Investigación, Av. Morones Prieto 3000 Pte., Col. Los Doctores, Monterrey, N.L. 64710, México. sscott04@msn.com
Shea PW
No affiliation info available
Dryer SE
No affiliation info available

MeSH

Amino Acid SequenceAnimalsCyclic AMPCyclic AMP Receptor ProteinCyclic GMPCyclic Nucleotide-Gated Cation ChannelsEscherichia coli ProteinsHyperpolarization-Activated Cyclic Nucleotide-Gated ChannelsLigandsModels, MolecularMolecular Sequence DataPotassium ChannelsProtein Structure, TertiaryRatsRecombinant Fusion ProteinsTranscription Factors

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

17655202

Citation

Scott, Sean-Patrick, et al. "Mapping Ligand Interactions With the Hyperpolarization Activated Cyclic Nucleotide Modulated (HCN) Ion Channel Binding Domain Using a Soluble Construct." Biochemistry, vol. 46, no. 33, 2007, pp. 9417-31.
Scott SP, Shea PW, Dryer SE. Mapping ligand interactions with the hyperpolarization activated cyclic nucleotide modulated (HCN) ion channel binding domain using a soluble construct. Biochemistry. 2007;46(33):9417-31.
Scott, S. P., Shea, P. W., & Dryer, S. E. (2007). Mapping ligand interactions with the hyperpolarization activated cyclic nucleotide modulated (HCN) ion channel binding domain using a soluble construct. Biochemistry, 46(33), 9417-31.
Scott SP, Shea PW, Dryer SE. Mapping Ligand Interactions With the Hyperpolarization Activated Cyclic Nucleotide Modulated (HCN) Ion Channel Binding Domain Using a Soluble Construct. Biochemistry. 2007 Aug 21;46(33):9417-31. PubMed PMID: 17655202.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Mapping ligand interactions with the hyperpolarization activated cyclic nucleotide modulated (HCN) ion channel binding domain using a soluble construct. AU - Scott,Sean-Patrick, AU - Shea,Patrick W, AU - Dryer,Stuart E, Y1 - 2007/07/26/ PY - 2007/7/28/pubmed PY - 2007/10/10/medline PY - 2007/7/28/entrez SP - 9417 EP - 31 JF - Biochemistry JO - Biochemistry VL - 46 IS - 33 N2 - Hyperpolarization activated cyclic nucleotide modulated (HCN) ion channel currents are activated by hyperpolarization and modulated in response to changes in cytosolic adenosine 3',5'-cyclic monophosphate (cAMP) concentrations. A cDNA chimera combining the rat HCN2 cyclic nucleotide binding domain and the DNA binding domain of the cAMP receptor protein (CRP) from E. coli and the histidine tag (HCN2/CRP) was expressed and purified. The construct is capable of forming only non-ligand dependent dimers because the C-linker region of the channel is not present in this construct. The construct binds 8-[[2-[(fluoresceinylthioureido) amino] ethyl] thio] adenosine-3',5'-cyclic monophosphate (8-fluo cAMP) with a Kd of 0.299 microM as determined with a monomer binding model. The Ki values of 20 ligands related to cAMP were measured in order to determine the properties necessary for a ligand to bind to the HCN2 binding domain. This is the first report of cAMP and gunaosine 3',5'-cyclic monophosphate (cGMP) affinities to the HCN2 binding domain being equivalent, even though they modulate the channel with a 10-fold difference in K0.5. Furthermore, the array of ligands measured allows the preference rank order for each purine ring position to be determined: position 1, H > NH2 > O; position 2, NH2 > Cl > H > O; position 6, NH2 > Cl > H > O; and position 8, NH2 > Cl > H > O. Finally, the ability of HCN2/CRP to bind cyclic nucleotide pyrimidine rings at concentrations approximately 1.33 times greater than cAMP suggests that ribofuranose is key for binding. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/17655202/Mapping_ligand_interactions_with_the_hyperpolarization_activated_cyclic_nucleotide_modulated__HCN__ion_channel_binding_domain_using_a_soluble_construct_ L2 - https://doi.org/10.1021/bi6026049 DB - PRIME DP - Unbound Medicine ER -
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