Opening of rod photoreceptor CNG channels by photodynamically generated singlet oxygen

Molecular oxygen exists in three electronic states: the triplet ground state and two singlet (¹O₂) excited states. Under physiological conditions, ¹O₂ can be produced either through photodynamic processes, which require light, photosensitizer, and oxygen, or via metabolic reactions involving enzymes and other reactive oxygen species (ROS). ¹O₂ readily reacts with biomacromolecules, however, its volatile chemical nature and the lack of precise working models hamper the study of its molecular mechanism and physiological significance. Here we report that human CNG channels from rod photoreceptors are very sensitive to the process of photodynamic modification (PDM). Multiple lines of evidence indicate ¹O₂ is the major player in PDM, including the application of a genetically encoded photosensitizer, a popularly used photosensitizer to produce ¹O₂, and two known quenchers for ¹O₂. The ¹O₂-mediated modification increases the opening of hCNGA1 in the absence or under subsaturating concentrations of cyclic guanosine monophosphate (cGMP), and in conjunction with ligand gating, acts synergistically on channel opening. Mutagenesis and mass spectroscopy (MS) analysis reveal key residues affecting the PDM process. Taken together, through tackling the PDM of rod photoreceptor CNG channels, this study provides essential insights into the modification of protein molecules by ¹O₂, a ubiquitous and potentially critical signaling molecule.