Mapping ligand interactions with the hyperpolarization activated cyclic nucleotide modulated (HCN) ion channel binding domain using a soluble construct.
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.
Scott
Sean-Patrick
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
Patrick W
PW
Dryer
Stuart E
SE
D000595:Amino Acid Sequence; D000818:Animals; D000242:Cyclic AMP / Q000737:chemistry*; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry / Q000235:genetics; D006152:Cyclic GMP / Q000737:chemistry*; D054815:Cyclic Nucleotide-Gated Cation Channels; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics
D002373:Cyclic AMP Receptor Protein; D054815:Cyclic Nucleotide-Gated Cation Channels; D029968:Escherichia coli Proteins; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D008024:Ligands; D015221:Potassium Channels; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C481662:crp protein, E coli; D000242:Cyclic AMP; D006152:Cyclic GMP