PROJECT SUMMARY Activation of ion channels upon binding multiple ligands at distinct subunits or domains is essential for synaptic transmission and cellular signaling. Despite recent advances in understanding their 3-dimensional structure, there remains for many channels a fundamental gap in our understanding of the sequence of events by which multiple binding sites and domains coordinate to open and close the channel pore. A major barrier to bridging this gap is that ensemble-averaged binding measures from many channels at once occlude observation of the distinct asynchronous binding steps that underlie the sequence of binding events at each individual channel. To overcome this barrier, I will use innovative single-molecule fluorescence methods developed in my lab in combination with my prior expertise with zero-mode waveguide nanophotonic arrays that enable optical tracking of each individual binding step. The objective of this proposal is to determine the energy landscape for 1) the sequence of stepwise binding events that drive activation of cyclic nucleotide gated (CNG) channels critical for visual and olfactory sensation, and 2) modulation of GABAA receptors by benzodiazepines (BZDs), one of the most widely prescribed psychotropic drugs today. The rationale is that optical tracking of individual binding events that are the chemical stimuli by which these channels operate will enable determination of the sequence of distinct energetic events that must at least partially occur prior to pore opening and thus are difficult to measure with electrophysiological approaches. The specific aims will: 1) Establish the energy landscape for sequential binding at a CNG channel; 2) Quantify the likelihood of CNG channel opening with each distinct binding step, which will test existing disparate model predictions; 3) Develop a mechanistic model for CNG channel activation that accounts for each distinct binding step; 4) Determine the energy landscape for BZD-binding or sequential agonist binding at GABAA receptors, and 5) Establish whether or not BZDs alter distinct agonist binding steps. The proposed research is significant because it will provide a necessary foundation for understanding the dynamic sequence of events governing ligand-driven behavior in these channels, which currently remain only poorly understood. The results will have an immediate positive impact as a quantitative benchmark for computational, structural, and functional studies aimed at uncovering the physical basis for the observed changes in energy. Ultimately, understanding the full sequence of events during channel activation is essential not only to advance our fundamental knowledge of ion channel mechanisms, but also to facilitate development of therapies targeting distinct steps in the activation pathway. Long-term, this knowledge will enable the rational design of new therapies to improve treatment outcomes and quality of life.