J Gen Physiol 2025 Nov 05;1581:. doi: 10.1085/jgp.202513811.

Optical control of PI(4,5)P2 sensitivity of ion channels by manipulation of single lysine residue.

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Polyphosphoinositides (PPIns) are essential components of membrane lipids and play crucial roles in cell signaling in eukaryotes. Phosphatidylinositol4,5-bisphosphate (PI(4,5)P2) is a species of PPIns enriched in the plasma membrane and regulates numerous membrane proteins, including ion channels, transporters, and receptors, primarily through direct binding to positively charged residues such as lysine and arginine. Despite recent advances in structural biology and biophysics, the specific contributions of individual amino acid residues to PI(4,5)P2 binding in membrane proteins remain unclear. These questions have been explored by functional characterization of mutant proteins with site-specific amino acid replacement and their comparison with the WT proteins. Here, we apply genetic code expansion to investigate the role of lysine residues in the PI(4,5)P2 sensitivity of ion channels. A caged lysine compound, hydroxycoumarin-lysine (HCK), was incorporated at several key lysine residues critical for PI(4,5)P2 sensitivity in the mouse inward-rectifier potassium channel Kir2.1, expressed in Xenopus oocytes. Caging of lysine by introducing HCK at K182 or K187 completely silenced Kir2.1 currents, but light-induced uncaging restored current activity. Voltage-sensing phosphatase assays revealed that this current increase was accompanied by enhanced PI(4,5)P2 sensitivity. On the other hand, introducing HCK at K219, which forms a secondary PI(4,5)P2-binding region, did not fully eliminate Kir2.1 currents, and uncaging resulted in an approximately twofold increase in current. Analysis of uncaging and PI(4,5)P2 sensitivity in Kir2.1-K219HCK revealed that the region C-terminal to residue K219 is dispensable when assembled with the full-length protein. Genetic code expansion using caged lysine provides a valuable tool for studying the mechanisms of PI(4,5)P2 regulation in ion channels, complementing existing approaches.

???displayArticle.pubmedLink??? 41251684
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