Cyclic nucleotide-modulated channels play crucial roles in a myriad of physiological processes including visual and olfactory signal transduction and the pacemaker activity in the heart and brain. These channels are functional tetramers and the binding of cyclic nucleotides (cAMP and cGMP) to a specialized, conserved region of the channel modulates the opening/closing (called gating) equilibrium of the channel and the subsequent completion of the channel's role in the signaling cascade. Both the high resolution structure of these channels and the precise mechanism of ligand-mediated channel activation are unknown. The overall objective of this proposal is to understand the mechanism of ion channel modulation by cyclic nucleotides. To accomplish this goal we will utilize a prokaryotic homologue of these channels, MloK1. Prokaryotic channels lend themselves to structural and biochemical studies, while structural conclusions about eukaryotic channels are limited to inferences based on functional data. Our first major aim is to identify the linkage between cyclic nucleotide binding and channel activation. Ligand-gated channels go through a series of conformational steps from the binding of the first ligand to channel activation but the molecular details are unknown. Using direct ligand binding (the first use of this technique for cyclic nucleotide-gated channels) and functional flux assays we will quantify this process to identify the steps that lead to channel activation. Our second major aim is to identify the molecular determinants of channel response to ligands and selectivity among ligands. In order to identify the molecular determinants of ligand selectivity, we will disrupt the cyclic nucleotide binding domain with mutations of residues known to affect ligand potency and selectivity in eukaryotic channels. We will then assess the capacity of the mutated channels to directly bind cyclic nucleotides and their ability to be modulated by cyclic nucleotides and quantify these effects in terms of a model for channel activation. We will also use a modular approach in which we will construct chimeric channels with """"""""mixed and matched"""""""" domains in order to assign specific functions to each module. These experiments will identify individual residues as well as protein modules that play key roles in ligand binding and channel activation. Our third major aim is to determine the structural elements involved in channel modulation by cyclic nucleotides. Using X-ray crystallography, we will determine the structure of MloK1 and that of a functional channel chimera made of a stable core, the KcsA channel, to which we have added the cyclic nucleotide binding domain of MloK1. Direct observation of the interactions between the ligand binding and the pore domains will provide unique insights into the gating mechanism. Differences between liganded and unliganded channel structures will provide insight into the mechanism of ligand gating. The chimeric channel structures represent a novel approach in studying channel structural domains. ? ?
