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William N. Zagotta, Michael Puljung, Stefan Stoll; SPECTROSCOPIC INVESTIGATION OF AGONIST-INDUCED REARRANGEMENTS OF CYCLIC NUCLEOTIDE-REGULATED ION CHANNELS. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6317.
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© ARVO (1962-2015); The Authors (2016-present)
Cyclic nucleotide-gated (CNG) channels are expressed in the outer segment of rod and cone photoreceptors and are responsible for the primary electrical response to light. Along with hyperpolarization-activated cyclic nucleotide-modulated (HCN) ion channels, CNG channels are part of a sub-family of ion channels that are activated by the direct binding of cyclic nucleotides, e.g. adenosine 3’,5’-cyclic monophosphate (cAMP) and guanosine 3’5’-cyclic monophosphate (cGMP), 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-roll followed by two helices (the B- and C-helices). Cyclic nucleotides initially bind to residues in the β-roll. 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. Knowledge of the structural mechanisms of channel activation is critical for understanding the molecular underpinnings of vision.
In this study we use transition metal ion fluorescence resonance energy transfer (tmFRET) and electron paramagnetic resonance (EPR) spectroscopy to examine cyclic nucleotide-dependent structural transitions in the purified C-terminal domain of HCN2.
We have extended our previous tmFRET studies to demonstrate that the C-helix undergoes a coiled-to-helix transition associated with agonist binding. Furthermore, we used EPR spectroscopy 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.
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