PROJECT SUMMARY Ligand-gated ion channels (LGICs) are molecular sensors that convert the chemical energy of ligand binding to electrical impulses via ion flux through the channel pore. LGICs are essential for synaptic transmission throughout the nervous system as well as cellular signaling in many other fundamental physiological processes such as vision, olfaction, motor control and heart rate to name just a few. They are also major drug targets as modulating channel behavior can be used to counteract a wide range of afflictions such as anxiety, addiction, pain, muscle impairment, etc. Gating (opening/closing) of the channel pore is initiated upon binding of ligands, often to multiple sites in distinct subunits or domains. Despite significant progress in understanding the 3- dimensional structure of LGICs, there remains a critical gap in our understanding of how these domains participate to shape the sequence of events by which chemical binding energy is transduced to gating of the ion pore. A major barrier to bridging this gap is that the single-molecule methods needed to resolve the stochastic binding and gating events only report on either the binding stimulus or the gating response, but not both as required to understand the full stimulus-response pathway. To overcome this barrier, I will use an innovative combination of micro-mirror total internal reflection fluorescence (mmTIRF) single-molecule imaging to optically track individual binding events for a fluorescently labeled ligand while simultaneously recording ion conduction through single channels in excised membrane patches with conventional patch clamp techniques. The objective of this proposal is to establish the feasibility of this combined approach for activation of cyclic nucleotide gated (CNG) channels critical for visual and olfactory sensation. The rationale is that the combination of mmTIRF and single-channel recording will enable direct experimental correlation between distinct binding events at multiple domains and the electrical gating response. Completion of this objective will 1) establish a facile approach for probing the full stimulus-response pathway in LGICs at single-molecule resolution, and 2) determine the degree to which energy from binding one or two cyclic nucleotides is transduced to opening of the pore gate. The proposed research is significant because it will enable studies that probe the dynamic sequence of events governing the transduction of chemical binding energy in multiple domains to gating of the ion pore, a process which in many cases is only poorly understood. The approach developed in this proposal will be invaluable to understanding the dynamic events by which ligands drive channel activity and which connect the dots between static structural snapshots, and thereby will constitute a major step forward for the ion channel field. Furthermore, it will have direct bearing on understanding the mechanisms of drugs that modulate channel behavior, which wil...