Abstract
Regulation of ion channel activity occurs through various mechanisms, via small molecule regulators, membrane potential, and specific interaction with lipid molecules. Inward rectifying potassium (Kir) channels conduction is controlled both by small molecules, such as polyamines blocking ion conduction, and lipid molecules that stabilize a conductive state. Advancing molecular understanding of this lipid regulation has been difficult as many structural techniques rely on detergent solubilized channels, which removes specific channel:lipid interaction. FRET, both macroscopically and in single molecules, can help surmount this issue, providing measurements of structural and dynamic changes induced by lipid regulation in a membrane environment. We have used macroscopic FRET to measure lipid specific structural changes in Kir2.1 in liposomes of defined composition. We have optimized purification and fluorophore labeling of the human Kir2.1 channel, which are reconstituted into liposomes of various lipid composition. We have measured PIP2-dependent FRET changes at numerous sites, which indicate structural movements of the intracellular domain of Kir2.1 upon PIP2 binding. This work specifically investigates how movements of the slide helix contribute to activation of the channel. Further efforts will help to define the molecular details of bulk anionic lipid and PIP2-dependent structural dynamics of Kir2.1 and help to understand more generally how lipid-protein interactions regulate membrane protein function.
Published Version
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