Novel Methodology to Detect Physical Interaction of Inositol Phospholipid with Potassium Ion Channel Using Luminescence Resonance Energy Transfer

Abstract

Membrane proteins like ion channels are located within a lipid environment. While the bulk lipids provide a supporting architecture for proteins, some lipids also play important signaling roles in modulating protein activity. This modulation can occur through direct interaction with proteins without involving second messenger pathways. Phosphoinositides are one such lipids, which have been implicated in interactions with a diverse range of proteins and such interactions mediate a variety of cellular functions. Towards understanding how these lipids play their roles in regulating protein functions It is necessary to detect interactions between proteins and lipids and to identify binding sites on proteins. However, detection of protein-lipid interactions has been difficult especially in their native environment because only a fraction of lipids are bound to proteins while the bulk is freely diffusing. The purpose of this study is to develop an experimental method capable of identifying protein-lipid interactions in membrane environments. The strategy involves luminescence energy transfer to locally illuminate lipids around proteins thereby suppressing the large background of light from unbound lipid molecules. The approach is applied to an inward rectifier potassium ion channel for its interactions with phosphoinositide lipids. Experiments show that it can successfully detect phosphoinositides bound to proteins, and when combined with mutagenic approaches, can further identify binding site. The approach thus provides a valuable methodology for probing protein-lipid interactions in a native-like lipid environment.