Project Summary/Abstract Membrane proteins are embedded in the biological membrane where they function and intimately interact with lipid molecules. The environment of the biological membrane is dynamic and composed of a rich chemical diversity of lipid molecules. Alongside the complexity of the biological membrane is the growing realization of the important roles of lipid molecules in the folding, structure, and function of membrane proteins. In particular, inward rectifier potassium (Kir) channels have central roles in regulating membrane potential and potassium homeostasis. It has been known for nearly 20 years that these channels require a specific lipid for function. Although many studies have provided insight into Kir channel structure and function, there remain a number of fundamental questions: What determines the selectivity of Kir channels towards lipids? What are the thermodynamic binding parameters for individual lipid binding events to Kir channels? Do lipids bind cooperatively to Kir channels? Does Kir channel conformation (open or closed) influence the selectivity towards individual lipids? How many specific lipids are required to gate Kir channels? What are the thermodynamic stabilities of Kir channel open and closed states when bound to one, two, three, or four specific lipids? Here, we propose to address these fundamental questions using native ion mobility mass spectrometry (IM-MS) technology, whereby non-covalent interactions are preserved in the mass spectrometer and capitalize on IM-MS approaches we have pioneered that, unlike other biophysical methods, allow us to resolve and interrogate individual lipid binding events to membrane protein complexes. We seek to apply novel and highly innovative IM-MS approaches that we have recently developed to deduce thermodynamic binding parameters for individual lipid binding events to membrane proteins. Moreover, we propose to investigate Kir channel gating using IM-MS that will allow us for the first time to monitor the conformational states (open and closed) for apo and lipid bound states. Our proposal will also investigate the allosteric regulation of Kir channels by lipids and other molecules using new IM-MS methods we have recently developed to resolve and interrogate heterogeneous lipid binding events at the resolution of individual lipids. Taken together, we anticipate the results from our proposed studies to provide fundamental insight into how lipids and other molecules modulate the structure and function of Kir channels.