Abstract Ion channels are exquisite molecular machines that regulate the flow of ions across cell membranes in response to stimuli such as voltage and small molecule ligands (e.g. second messengers, and neurotransmitters). They underlie all electrical excitability in the brain and heart, and defects in ion channels are responsible for many human disorders. Despite decades of experiments and many high-resolution molecular structures, we still do not know, for any channel, the mechanisms for voltage- or ligand-dependent gating. The missing ingredient is conformational energetics. The energetics of the different channel conformations governs the time course, voltage dependence, and ligand dependence of opening of the channel pore, and ultimately electrical excitability of the cell. In the parent grant, our goals are to determine the mechanisms of voltage-dependent gating and ligand-dependent gating and, ultimately, to understand the general themes that underlie allosteric regulation of ion channels. Our approach has been to leverage breakthrough FRET methods we developed for measuring intramolecular distance distributions and conformational energetics using fluorescence lifetimes. One limitation of this approach is that it can only measure equilibrium energetics. To truly determine the structural dynamics and energetics, we need to measure the rates of the conformational transitions in the protein. To measure conformational transition rates at steady state, we need to perform single-molecule experiments. This proposal is for a PicoQuant MicroTime 100 upgrade to our current PicoQuant system, previously purchased with the parent award, that would allow us to do time-resolved confocal microscopy to measure single-molecule FRET. The requested equipment will further accelerate our progress toward the goals of the parent grant.