Native ion mobility mass spectrometry studies of potassium inward rectifier channels: insight into gating and lipid binding
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Project Summary/Membrane proteins are embedded in the biological membrane where they function and intimately interact withlipid molecules. The environment of the biological membrane is dynamic and composed of a rich chemicaldiversity of lipid molecules. Alongside the complexity of the biological membrane is the growing realization of theimportant 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 potassiumhomeostasis. 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 offundamental questions: What determines the selectivity of Kir channels towards lipids? What are thethermodynamic binding parameters for individual lipid binding events to Kir channels? Do lipids bindcooperatively to Kir channels? Does Kir channel conformation (open or closed) influence the selectivity towardsindividual lipids? How many specific lipids are required to gate Kir channels? What are the thermodynamicstabilities of Kir channel open and closed states when bound to one, two, three, or four specific lipids? Here, wepropose 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-MSapproaches we have pioneered that, unlike other biophysical methods, allow us to resolve and interrogateindividual lipid binding events to membrane protein complexes. We seek to apply novel and highly innovativeIM-MS approaches that we have recently developed to deduce thermodynamic binding parameters for individuallipid binding events to membrane proteins. Moreover, we propose to investigate Kir channel gating using IM-MSthat will allow us for the first time to monitor the conformational states (open and closed) for apo and lipid boundstates. Our proposal will also investigate the allosteric regulation of Kir channels by lipids and other moleculesusing new IM-MS methods we have recently developed to resolve and interrogate heterogeneous lipid bindingevents at the resolution of individual lipids. Taken together, we anticipate the results from our proposed studiesto provide fundamental insight into how lipids and other molecules modulate the structure and function of Kirchannels.
