Cyclic nucleotide-gated (CNG) ion channels play important roles in vision and olfaction and have recently been identified in neurons and cardiac muscle. These channels are likely to be involved in disease, including retinal degeneration, and are potential targets for therapeutic drugs. The long-term goals of this project are to understand the molecular mechanism of channel of channel activation gating. This proposal investigates how cyclic nucleotide binding leads to the opening of the CNG channels, building upon the previous identification of two domains important for ligand gating: the C-helix of the carboxyl terminus ligand-binding domain and an amino terminus gating domain (N-S2 domain). Activation can be described by the Monod-Wyman-Changeyx (MWC) model in which the N-S2 domain participates in the concerted allosteric transition which mediates channel opening. The C-helix is important for enhanced binding of ligand to the open channel, thus stabilizing the open state. This hypothesis will be tested by investigating three specific questions: 1. What is the functional stoichiometry of ligan-gating? How many ligands must bind to activate the channel? Does the MWC model adequately describe gating? The properties of channels will be investigated in which the binding to zero to three of the channel's four subunits has been inactivated using point mutations of deletions in the binding site. These experiments will provide information as to whether ligand binding is cooperative or independent. They will revieal how much free energy for activation each binding site contributes. Finally they may suggest alternative schemes for channel activation gating other than the MWC model. 2. How does the C helix participate in activation gating? Does the C helix act to selectively stabilize cyclic mucleotide binding to the open channel? Does this stabilization require the formation of intrasubunit bonds? 3. What is the role of the N-S2 domain in subunit assembly? Does the allosteric gating transition involve a change in subunit-subunit interactions? This hypothesis is based on the fact that regions homologous to the N-S2 domain mediate subunit assembly in voltage-gated K channels. It is our hypothesis that the N-S2 domain contributes to subunit assembly of CNG channels. These experiments will thus provide a powerful means of exploring both the basic mechanism of CNG channel gating as well as a test of the role of two domains of the channel in activation gating. Such information will be important for understanding how the structure of this family of channels underlies the unique physiological roles of different types of CNG channels in sensory information processing.
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