Methods for Studying Cholesterol-Dependent Regulation of P2X7 Receptors

Abstract

Cholesterol dynamically regulates P2X7 receptor function in both physiological and pathological conditions. Studies suggest that cholesterol suppresses P2X7 receptor activity through direct binding or through indirect effects on the biophysical properties of the membrane. Notably, the palmitoylated C-terminus seems to counteract the action of cholesterol to make it less inhibitory. However, the mechanism underlying cholesterol-dependent regulation of P2X7 receptor remains unclear. Here we describe detailed methods that facilitate the quantification of P2X7 channel activity while controlling the amount of cholesterol in the system. We will first describe the use of methyl-β-cyclodextrin (MCD), a cyclic oligosaccharide consisting of seven glucose monomers, to decrease or increase plasma membrane cholesterol levels. We will then describe protocols for the reconstitution of purified P2X7 in proteoliposomes of defined lipid composition. These methods can be combined with commonly used techniques such as dye-uptake assays or electrophysiology. We also describe a fluorescence assay to measure cholesterol-binding to P2X7. These approaches are complementary to cryo-EM or molecular dynamics simulations, which are also powerful tools for investigating cholesterol-P2X7 interactions. An improved understanding of the mechanisms of action of cholesterol on P2X7 may contribute to elucidate the roles of this receptor in ageing, inflammation, and cancer, whose progression correlates with the level of cholesterol.