Spectroscopic Investigation of Agonist-Induced Rearrangements of Cyclic Nucleotide-Modulated Ion Channels

Abstract

Cyclic nucleotide-gated (CNG) and hyperpolarization activated cyclic nucleotide-modulated (HCN) ion channels are activated by the direct binding of cyclic nucleotides, e.g. adenosine 3',5'-cyclic monophosphate (cAMP), to a conserved, cytoplasmic domain. The structure of the cyclic nucleotide binding domain (CNBD) is similar to those found in other cyclic nucleotide-activated proteins, including the kinases PKA and PKG, the transcription factor CAP, and the guanine nucleotide exchange factor Epac. The core of this structure contains an eight-stranded β-barrel followed by two helices (the B and C helices). Cyclic nucleotides initially bind to residues in the β-barrel. Subsequent to binding, the C helix of HCN and CNG channels undergoes a translation toward the binding pocket as well as a stabilization of its helical structure. This conformational rearrangement is coupled to opening of the ion channel pore. We have extended our previous studies using transition metal ion fluorescence resonance energy transfer (tmFRET) in the purified C-terminal domain of HCN2 to demonstrate that the B helix also reorganizes relative to the β-barrel subsequent to agonist binding. Furthermore, we have used electron paramagnetic resonance (EPR) on the spin-labeled HCN2 C- terminus to investigate the reorientation and stabilization of the CNBD induced by cAMP binding. These studies further extend our knowledge of the conformational changes in the CNBD of HCN and CNG channels and may provide a general picture of the activation of other families of cyclic nucleotide-regulated proteins.